Discovery and validation of molecular biomarkers for differentiation of non-dysplastic Barrett esophagus from high-grade dysplasia and esophageal adenocarcinoma

Aberrant DNA methylation and copy number alterations (CNAs) drive Barrett’s esophagus (BE) progression to esophageal adenocarcinoma (EAC); however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDMs) and CNAs to distinguish EAC/high-grade dysplasia (HGD) from non-dysplastic Barrett's esophagus (NDBE). In this multi-phase, multi-center study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing data set obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N=18) and EAC (N=18) versus NDBE brushing samples (N=18) were tested in independent samples (N=146). A 52-MDM panel achieved a cross-validated area under the receiver operating characteristic curve (AUC) 0.88 (95% CI: 0.82-0.95); the addition of AS improved discrimination of HGD/EAC from NDBE to 0.91 (95% CI: 0.86-0.97) AUC. At 80% specificity, the combined model detected 93% of EAC and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI: 0.66-0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI: 0.7-0.9). MDMs and AS discerned HGD/EAC from normal esophagus (NE)/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve BE surveillance.