DNA methylation as an adjunct to histopathology to detect prevalent, inconspicuous dysplasia and early-stage neoplasia in Barrett's esophagus

Abstract

Purpose: Endoscopic surveillance of Barrett's esophagus is problematic because dysplasia/early-stage neoplasia is frequently invisible and likely to be missed because of sampling bias. Molecular abnormalities may be more diffuse than dysplasia. The aim was therefore to test whether DNA methylation, especially on imprinted and X-chromosome genes, is able to detect dysplasia/early-stage neoplasia.

Experimental design: 27K methylation arrays were used to find genes best able to differentiate between 22 Barrett's esophagus and 24 esophageal adenocarcinoma (EAC) samples. These were validated using pyrosequencing on a retrospective cohort (60 Barrett's esophagus, 36 dysplastic, and 90 EAC) and then in a prospective multicenter study (98 Barrett's esophagus patients, including 28 dysplastic and 9 early EAC) designed to utilize biomarkers to stratify patients according to their prevalent dysplasia/EAC status.

Results: Genes (23%) on the array, including 7% of X-linked and 69% of imprinted genes, have shown statistically significant changes in methylation in EAC versus Barrett's esophagus (Wilcoxon P < 0.05). 6/7 selected candidate genes were successfully internally (Pearson's P < 0.01) and externally validated (ANOVA P < 0.001). Four genes (SLC22A18, PIGR, GJA12, and RIN2) showed the greatest area under curve (0.988) to distinguish between Barrett's esophagus and dysplasia/EAC in the retrospective cohort. This methylation panel was able to stratify patients from the prospective cohort into three risk groups based on the number of genes methylated (low risk: <2 genes, intermediate: 2, and high: >2).

Conclusion: Widespread DNA methylation changes were observed in Barrett's carcinogenesis including ≈70% of known imprinted genes. A four-gene methylation panel stratified patients with Barrett's esophagus into three risk groups with potential clinical utility.

Figure 1

GSEA generated heat maps for the top 50 probes showing greatest differential methylation between BE and EAC (red color = high methylation, blue color = low methylation). a – all probes (22BE vs. 24EAC), b – imprinted genes probes (22BE vs. 24 EAC), c – X-chromosome probes (15BE vs. 20EAC, males only), d – X-chromosome probes (7BE vs. 4EAC, females only).

Figure 2

a: Genes selected from the array analysis showing the greatest difference in methylation between BE and EAC. Beta values from the array are plotted on the x-axis against the gene name and tissue type on y-axis. b: For genes on the X-chromosome, analyses were separated on the basis of gender to cater for the effects of X-inactivation in females. Since RGN lies on the region of X-chromosome that is inactivated, males and females have different levels of methylation. Females have higher methylation in both tissues (BE and EAC) compared to males. TCEAL7 does not appear to be affected by X-inactivation and males and females have similar levels of methylation in both BE and EAC. c: Methylation levels for RGN and TCEAL7 in the normal esophageal epithelium in males and females using pyrosequencing (N=5).

Figure 3

Internal validation. Beta values from the Illumina Infinium array (y-axis) are plotted against the % methylation from pyrosequencing (x-axis) (N=12).

Figure 4

Retrospective external validation. N(BE) = 60, N(BED) = 36, N(EAC) = 90 for SLC22A18, GJA12 and RIN2. N(BE) = 30, N(BED) = 6, N(EAC) = 70 for PIGR and TCEAL7. N(BE) = 45, N(BED) = 30, N(EAC) = 60 for RGN (Males only). Middle line = median, box = 25-75 percentile, whiskers = 10-90 percentile. *=p<0.01, **=p<0.001, ***=p<0.0001 using ANOVA (BE=Barrett’s esophagus, BED=Barrett’s esophagus with dysplasia, EAC=esophageal adenocarcinoma).

Figure 5

ROC curves for all six targets. N(BE) = 32 vs. N(BED)+ N(EAC) = 73. For RGN (Males only) N(BE) = 25 vs. N(BED)+N(EAC) = 51 (BE=Barrett’s esophagus, BED=Barrett’s esophagus with dysplasia, EAC=esophageal adenocarcinoma).

Figure 6

a: The four gene risk score (SLC22A18 + PIGR + GJA12 + RIN2) had the best AUC of 0.988 (P<0.01). b: Graphical representation of percentage of patients falling into each group. The probability of HDG/early EAC increases with an increase in the number of positive biomarkers (BE=Barrett’s esophagus, LGD=low grade dysplasia, HGD=high grade dysplasia, EAC=esophageal adenocarcinoma).