Aim: The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Background: The incidence of NASH, a precancerous condition for hepatocellular carcinoma (HCC), has shown an alarming increase in recent years. Although significance of DNA methylation alterations in NASH has been studies in animal models, only a limited number of papers focusing on genome-wide DNA methylation analysis in a cohort of patients with NASH and NASH-related HCC have been reported. It would be informative to understand the significance of DNA methylation alterations in NASH-related hepatocarcinogenesis, especially at the precancerous stage. Methods: Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples, i.e. 55 samples of normal liver tissue (NLT), 113 samples of non-cancerous liver tissue showing NASH (NASH-N), 22 samples of NASH-related HCC (NASH-T), 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) infection (viral-N) and 37 samples of HCC associated with HBV or HCV infection (viral-T). Results: After Bonferroni correction, 3,331 probes showed significant DNA methylation alterations in NASH-N samples as compared with NLT samples. Principal component analysis using the 3,331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and viral-N samples. Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95, again indicating that the DNA methylation status of NASH was clearly different from that of viral hepatitis and cirrhosis. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without HCC were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in NASH-T themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N and viral-T samples, were observed in tumor-related genes, such as the WHSC1 gene which encodes a histone H3 lysine 36 methyltransferase and the WDR6 gene which encodes a WD repeat family protein implicated in cell growth arrest, and were frequently associated with mRNA expression abnormalities. Conclusion: These data suggested that NASH-specific DNA methylation alterations observed at the precancerous stage were shown to be inherited by NASH-T and may continuously participate in NASH-related multistage hepatocarcingenesis, at least partly via alterations in the expression of the affected genes.

Citation Format: Junko Kuramoto, Eri Arai, Ying Tian, Nobuaki Funahashi, Masaki Hiramoto, Takao Nammo, Yuichi Nozaki, Yoriko Takahashi, Nanako Ito, Ayako Shibuya, Hidenori Ojima, Aoi Sukeda, Yosuke Seki, Kazunori Kasama, Kazuki Yasuda, Yae Kanai. Genome-wide DNA methylation analysis during nonalcoholic steatohepatitis-related multistage hepatocarcinogenesis: Comparison with hepatitis virus-related carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5320.