There is increasing evidence that nonalcoholic steatohepatitis (NASH) is a risk factor for hepatocellular carcinoma (HCC) in the absence of cirrhosis, a phenomenon termed noncirrhotic HCC. Early diagnosis of HCC is critical to a favorable prognosis. We tested the hypothesis that hydroxyproline content of liver biopsy samples is diagnostic for HCC in murine models of NASH induced by diet or by diet and chemicals. The training set comprised mice fed a standard diet or a fast-food diet with or without administration of thioacetamide. At harvest, livers from the modified diet cohort exhibited NASH with a subset of NASH livers exhibiting HCC. Hydroxyproline content was measured in liver biopsy samples with tissue in the NASH+HCC cohort sampled from the remote, nontumor parenchyma. Plotting the receiver operating characteristics (ROC) with hydroxyproline as the continuous variable against the absence or presence of HCC yielded an area under ROC of 0.87, a threshold of >0.18 μg hydroxyproline/mg liver and sensitivity of 91% with a specificity of 83.3%. The use of liver hydroxyproline content as a diagnostic for HCC in a test set comprising healthy, NASH and NASH+HCC livers proved 87% accurate.

Non-alcoholic steatohepatitis (NASH) is associated with an increased risk of hepatocellular carcinoma (HCC). Expression levels of hepatic oncogenes, alpha-fetoprotein (afp) and osteopontin (opn)/secreted phosphoprotein 1 (spp1), were investigated using a model of diet-induced NASH. Mice were randomized to a standard diet or a fast-food diet (FFD) for 17 months. Livers from the FFD cohort exhibited hallmark characteristics of NASH with liver fibrosis, with a subset of animals exhibiting HCC. Expression levels of hepatic afp and opn/spp1 were elevated ~2.5 and ~5-fold, respectively, in the FFD cohort. Hepatic opn/spp1 exhibited a direct (r = 0.65) and significant (p < 0.01) correlation with liver hydroxyproline content. Receiver operating characteristic (ROC) curve analysis for hepatic afp, as a diagnostic for HCC, returned an area under (AU) ROC 0.84, a sensitivity of 87.5%, a specificity of 77% and a threshold of >1.05-fold change in mRNA level. The use of hepatic opn/ssp1 as a diagnostic for HCC returned an AUROC 0.88, a sensitivity of 83.3%, a specificity of 86.7% and a threshold of >2.4-fold change in mRNA level. These data point to a transformation of NASH to an oncotype with hepatic oncogene levels as a diagnostic for NASH.