Future studies should consider PCR product size when designing pyrosequencing assays for plasma DNA. Using ≥5% methylation as the cutoff for positivity, the frequency of positive plasma DNA samples ranged from

37% to 63%. When any one gene positive was used to define a positive case, 87% were positive. These results, in conjunction with our previous study of plasma from controls,22 suggest that analysis of plasma DNA is feasible and may be useful for the diagnosis of HCC. However, the quality of the bisulfite-treated plasma DNA will be a key component of a successful screening assay. Among the strengths of our study is that it is the largest sample-size methylation-array study of HCC to date. Among the limitations is the lack of information on AFB1-DNA in adjacent nontumor tissue, for some cases. In addition, data on alcohol consumption and cigarette smoking were missing selleck compound for approximately 20% of the cases. These missing data limited our ability to investigate relationships between methylation profiles and these factors. In addition, almost all our cases were infected with either HBV or HCV or both. Thus, we could not investigate the role of viral infection on methylation. Cobimetinib in vivo Another limitation was the lack of healthy tissue from unaffected controls as a comparison group for

our array studies. Our tumor adjacent tissues were primarily cirrhotic. Thus, we identified genes whose methylation was increased in progression from cirrhosis to HCC. Because our aim was to identify genes whose methylation is associated

with HCC but not cirrhosis, this comparison is appropriate, but tells us nothing about progression from normal tissue. A limitation of our plasma DNA analysis is that only samples from cases were available. Thus, whereas the frequency of methylation was high, we have no data on controls. In our previous prospective study of plasma DNA analyzing three genes using methylation-specific PCR, we found 2 of 50 (4%) controls with CDKN2A methylation and comparable Demeclocycline cases positive (44% versus 48%).22 In summary, we used genome-wide methylation arrays to identify genes methylated in HCC from primarily HBV-infected Taiwanese cases. Pyrosequencing of candidate genes validated the array data, and analysis of plasma DNA suggests that these genes may be appropriate to apply as biomarkers of early HCC diagnosis. We are in the process of testing custom arrays for analyzing larger numbers of CpG sites followed by pyrosequencing that can be applied to small amounts of plasma DNA. We will then use this methodology in our prospective study that includes HCC cases and controls, as required, to further determine the utility of this approach. The authors thank Dr. Abby Siegel for careful reading of the manuscript for this article. Additional Supporting Information may be found in the online version of this article.