This result suggests that aberrant methylation of COL14A1 may be associated with the lymph node metastasis of ESCC. Detecting cell-free Semaxanib 204005-46-9 nucleic acid in plasma or serum could be useful for numerous diagnostic applications and might prevent the need for tumor tissue biopsies. The release of nucleic acids into the blood is thought to be related to the apoptosis and necrosis of cancer cells in the tumor microenvironment. Secretion has also been suggested as a potential source of the nucleic acids. Changes in the levels of circulating nucleic acids have been associated with tumor burden and malignant progression. Several studies have revealed the presence of methylated DNA in the serum or plasma of patients with various types of malignancy, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, non-Hodgkinlymphoma, melanoma, ovarian cancer, pancreatic cancer, and prostate cancer. In our study of the methylation status of EPB41L3, GPX3 and COL14A1 in the plasma of ESCC patients and healthy individuals, the methylation frequencies of these genes are all greater than 30% in the plasma of the ESCC patients. However, absolutely no methylation was found in the plasma of healthy individuals, which may be due to several reasons and not a true case. One limitation of our study is that we used MSP as test method here, which is not quatitative and not sensitive enough to find low-level methylation DNA. There is a report of abnormal methylation detected with MethyLight method in healthy individuals’plasma. Another reason is that we only tested 50 normal individuals which is a relatively small sample size. In addition, we found that the methylation frequency of GPX3 and COL14A1 are higher in the pT3 patients compared with pT1-pT2 patients, and the methylation frequency of EPB41L3 is higher in pN2 patients than in pN0–pN1 patients. These results indicate that not only there are obvious significant difference of aberrant DNA methylation of EPB41L3, GPX3 and COL14A1 in the plasma between ESCC patients and healthy individuals, but also the methylation frequency might be associated with the advanced tumor stages. Moreover, we also evaluated the methylation status of the 3 genes in plasma for the diagnosis of ESCC using ROC curve analysis. We found that the sensitivities of the methylated genes in plasma DNA range from 31% to 40.5%. A previous study reported that diagnostic information could be increased if methylation of multiple genes in cell-free DNA were analyzed in combination. Indeed, we found that combination analysis of the three genes increased the sensitivity to 64.3%. The sensitivity of the 3 genes for use as early diagnosis tools is not high enough in this study. The combination of more methylated genes may increase the diagnostic sensitivity. These data indicated that combinatorial methylation analysis of these genes in plasma DNA has the potential to be a valuable diagnostic tool of noninvasive testing.