Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal forms of cancer, and is often diagnosed at later stages, resulting in high mortality rates. Aberrant DNA methylation (DNAme) is an epigenetic mechanism involved in many cancers, including ESCC, however critical DNA methylation events driving ESCC development are poorly understood. Here, we aimed to investigate tumor-specific DNAme in ESCC cases from nine high incidence countries spanning the Asian ESCC belt, Africa ESCC corridor and South America.

Infinium MethylationEPIC (HM850K) array was used on 108 tumor and 51 normal tissue adjacent to the tumor (NAT) for methylome analysis in the discovery phase. Replication of the selected targets was done on an independent set of 132 tumor and 36 NAT using targeted pyrosequencing.

Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMPs) and 866 DMRs with 30% Δβ difference. The majority of the identified DMPs and the DMRs were hypermethylated in tumors, particularly in the promoters and gene-body regions. The top three prioritized genes for replication, namely PAX9, SIM2 and THSD4 had similar methylation differences in discovery (PAX9: Δβ=0.41, P<1.83x10-300; SIM2: Δβ=0.40, P<1.83x10-300; THSD4: Δβ=0.39, P=1.33x10-112) and replication sets (PAX9: Δβ=0.20, P=0.0008; SIM2: Δβ=0.30, P=3.38x10-08; THSD4: Δβ=0.53, P=2.49x10-26).

Our study identified novel, robust and early tumor-specific DNAme events in ESCC tumors across several high incidence populations of the world. These identified aberrant DNAme could be potentially developed into ESCC biomarkers for early detection in minimally invasive samples.