Abstract 5703: Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis
Authors: Kuramoto Junko , Arai Eri , Fujimoto Mao , Tian Ying , Yamada Yuriko , Yotani Takuya , Makiuchi Satomi , Tsuda Noboru , Ojima Hidenori , Fukai Moto , Seki Yosuke , Kasama Kazunori , Funahashi Nobuaki , Udagawa Haruhide , Nammo Takao , Yasuda Kazuki , Taketomi Akinobu , Kanto Tatsuya , Kanai Yae 
Published: June 2022

Abstract
Background: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH.
Results: Normal liver tissue (NLT), non-cancerous liver tissue (N) showing histological findings compatible with non-alcoholic fatty liver (NAFL) from patients without HCC (NAFL-O), N showing NASH from patients without HCC (NASH-O), N showing NAFL from patients with HCC (NAFL-W), N showing NASH from patients with HCC (NASH-W) and NASH-related HCC (T) were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples (226 samples in total) were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4,050 CpG sites showed significant alterations of DNA methylation in 22 NASH-W samples relative to 36 NLT samples in the initial cohort, indicating that DNA methylation alterations had occurred in the NASH-W samples. On 3,234 of the 4,050 CpG sites, DNA methylation alterations in NASH-W samples relative to NLT samples were inherited by or strengthened in 22 T samples (Jonckheere-Terpstra trend test). Among the 3,234 CpG sites, receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among the 415 CpG sites, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from 91 NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage (Brunt classification). The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, which will be easily introduced into clinical laboratories, and for 3 of them sufficient sensitivity and specificity was successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, of which expression alternation may affect the post-transcriptional regulation of various tumor-related genes, 10 NAFL-W and 11 NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort.
Conclusions: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.
Citation Format: Junko Kuramoto, Eri Arai, Mao Fujimoto, Ying Tian, Yuriko Yamada, Takuya Yotani, Satomi Makiuchi, Noboru Tsuda, Hidenori Ojima, Moto Fukai, Yosuke Seki, Kazunori Kasama, Nobuaki Funahashi, Haruhide Udagawa, Takao Nammo, Kazuki Yasuda, Akinobu Taketomi, Tatsuya Kanto, Yae Kanai. Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5703.