Fig. 3

High-performance liquid chromatography (HPLC) analysis using the newly developed anion-exchange column for specimens of normal liver tissue (NLT) (n = 22, black), non-cancerous liver tissue (N) showing non-alcoholic fatty liver (NAFL) derived from partial hepatectomy specimens from patients without hepatocellular carcinoma (HCC) (NAFL-O) (n = 9, blue), N samples showing non-alcoholic steatohepatitis (NASH) from patients without HCC (NASH-O) (n = 3, blue), N samples showing NAFL from patients with HCC (NAFL-W) (n = 10, red), and N samples showing NASH from patients with HCC (NASH-W) (n = 11, red) in the validation cohort. A Scattergrams of the relative DNA methylation rates obtained by HPLC analysis for cg18210511, cg09580859 and cg13719443 whose estimation ability was validated in the validation cohort. Such rates were obtained as described in “Results” section. Such rates for both NAFL-W and NASH-W samples differed significantly from both NAFL-O and NASH-O samples regardless of histological features (i.e., regardless of NAFL or NASH), whereas the rates for both NAFL-O and NASH-O samples did not differ from those of NLT samples. P values were obtained by Welch’s t test. B Histograms showing the number of CpG sites satisfying the criteria for cg18210511, cg09580859 and cg13719443 based on the cutoff values shown in Table 4. If the tissue sample was estimated to have carcinogenic risk when two marker CpG sites or more satisfied the criteria for cg18210511, cg09580859 and cg13719443, regardless of histological features (i.e., regardless of NAFL or NASH), the liver tissue specimens from which HCCs had originated (9 NAFL-W samples and all 11 NASH-W samples) were considered to possess carcinogenic risk with 95% sensitivity. The possibility that patients from whom 2 NAFL-O and 2 NASH-O samples were obtained, which satisfied the criteria for two of more markers, would develop HCCs in the future could not be ruled out