Hypermethylation of DNA Methylation Markers in Non-Cirrhotic Hepatocellular Carcinoma

SIMPLE SUMMARY: Hepatocellular carcinoma (HCC) remains a significant cause of cancer-related deaths worldwide. Although most HCC cases have a background of cirrhosis, up to 20–30% of patients develop HCC in a non-cirrhotic liver. The prognosis of these unusual HCC cases is poor, since surveillance is not common. Therefore, investigating sensitive and specific biomarkers to detect non-cirrhotic HCC is crucial. Aberrant DNA methylation has been reported to play an important role in the development of cirrhotic HCC, while limited information can be found on non-cirrhotic HCC. This study is the first to determine the performance of reported promising DNA methylation markers in non-cirrhotic HCC. A total of 146 liver tissues were tested for 4 methylation markers using PCR- and sequencing-related techniques. We demonstrated significant DNA methylation changes in non-cirrhotic HCC compared to non-HCC benign lesions. These findings may be highly relevant to the future application of DNA methylation markers in non-cirrhotic HCC surveillance. ABSTRACT: Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.