Pyrosequencing was performed for further validation, and as expected, cg07757887 methylation was significantly lower in ESCC tumour tissues compared with corresponding non-tumour tissues (n = 50, P < 0.0001, t-test, Fig. 2a), with 92% (46/50) of tumour tissues showing CTHRC1 hypomethylation (Fig. 2b). Here, CTHRC1 is linked to esophageal squamous cell carcinoma.