Novel DNA Methylation Biomarker Panel for Detection of Esophageal Adenocarcinoma and High-Grade Dysplasia

Abstract

Purpose: Current endoscopy-based screening and surveillance programs have not been proven effective at decreasing esophageal adenocarcinoma (EAC) mortality, creating an unmet need for effective molecular tests for early detection of this highly lethal cancer. We conducted a genome-wide methylation screen to identify novel methylation markers that distinguish EAC and high-grade dysplasia (HGD) from normal squamous epithelium (SQ) or nondysplastic Barrett's esophagus (NDBE).

Experimental design: DNA methylation profiling of samples from SQ, NDBE, HGD, and EAC was performed using HM450 methylation arrays (Illumina) and reduced-representation bisulfate sequencing. Ultrasensitive methylation-specific droplet digital PCR and next-generation sequencing (NGS)-based bisulfite-sequencing assays were developed to detect the methylation level of candidate CpGs in independent esophageal biopsy and endoscopic brushing samples.

Results: Five candidate methylation markers were significantly hypermethylated in HGD/EAC samples compared with SQ or NDBE (P < 0.01) in both esophageal biopsy and endoscopic brushing samples. In an independent set of brushing samples used to construct biomarker panels, a four-marker panel (model 1) demonstrated sensitivity of 85.0% and 90.8% for HGD and EACs respectively, with 84.2% and 97.9% specificity for NDBE and SQ respectively. In a validation set of brushing samples, the panel achieved sensitivity of 80% and 82.5% for HGD and EAC respectively, at specificity of 67.6% and 96.3% for NDBE and SQ samples.

Conclusions: A novel DNA methylation marker panel differentiates HGD/EAC from SQ/NDBE. DNA-methylation-based molecular assays hold promise for the detection of HGD/EAC using esophageal brushing samples.

Figure 1:

Development of DNA-methylation Biomarkers for HGD/EAC Early Detection (workflow)

Figure 2:

Dot-plot graphs of individual markers Cg6522, YPEL3, POU3F1, Up35–1 and Up10 (A, B, C, D, E, respectively) in DNA extracted from esophageal cytology brushing sample set 1 (training brushing sample set). Each sample was defined by histologic diagnosis, shown on x-axis, as determined by pathologist D.R. The y-axis shows the relative methylation percent when compared to the reference gene C-LESS for total DNA, as measured by MS-ddPCR assays for each marker in panels A,B,C. The fraction of methylated reads is plotted on the Y axis in panels D and E. One way ANOVA was used to determine statistical significance between histologic groups based on relative methylation percent. P-values < 0.01 when compared to SQ/BE tissue are considered significant (*).

Figure 3:

Dot-plot graphs of individual markers Cg6522, YPEL3, POU3F1, Up35–1 and Up10 (A, B, C, D, E, respectively) in DNA extracted from esophageal cytology brushing sample set 2 (validation brushing sample set). Each sample was defined by histologic diagnosis, shown on x-axis, as determined by pathologist D.R. The y-axis shows the relative methylation percent when compared to the reference gene C-LESS for total DNA, as measured by MS-ddPCR assays for each marker in panels A,B,C. The fraction of methylated reads is plotted on the Y axis in panels D and E. One way ANOVA was used to determine statistical significance between histologic groups based on relative methylation percent. P-values < 0.01 when compared to SQ/BE tissue are considered significant (*).