Liver Biopsy Hydroxyproline Content Is a Diagnostic for Hepatocellular Carcinoma in Murine Models of Nonalcoholic Steatohepatitis

Abstract

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.

Keywords: HCC; NASH; ROC; cancer; diagnostic; fatty; hydroxyproline; liver; nonalcoholic.

Figure 1

Nonalcoholic Fatty Liver Disease (NAFLD). Representative images (10× of H&E-stained liver sections from mice randomized to a standard diet (A, sham), fast-food diet (FFD) (B) or FFD+TAA (C). The blue arrow (B) shows one of many lipid droplets, the red arrow shows a site of inflammation and the black arrow shows hepatocyte ballooning.

Figure 2

Nonalcoholic steatohepatitis (NASH). (A) and (B), Liver function tests and (C) NAFLD Activity Score (NAS) were elevated in the FFD and FFD+TAA cohorts compared to sham. ** p < 0.01 vs. sham.

Figure 3

Liver scarring. Representative images (10×) of PSR-stained liver sections from mice randomized to a standard diet (A), FFD (B) or FFD+TAA. (C) The black arrow (B) shows bridging fibrosis. (D) Quantitation of PSR staining shows elevated scarring in the FFD and FFD+TAA cohorts. ** p < 0.01 vs. sham. # p < 0.01 vs. FFD+TAA.

Figure 4

NASH with hepatocellular carcinoma (HCC). (A–C) Several animals within the FFD (17 mo) cohort exhibited liver tumors, which manifested as one or more polyp-like (blue arrows) growths on the liver. (D–F) Representative H&E-stained section from an FFD (17 mo) liver showing a trabecular growth pattern of atypical hepatocytes and clusters of nuclei (F, 25×) with a distinct margin between the noncancerous and cancerous parenchyma (black arrows, (D) (4×) and (E) (10×)). This liver bore a large tumor at sacrifice. Steatosis is also evident (E).

Figure 5

HCC. (A–C) Representative liver sections (10×) from Ca-19-9-stained sham, FFD and FFD+TAA groups. Staining is enriched in the FFD liver indicative of HCC. This liver bore a large tumor at sacrifice. (D–F) Representative liver sections (25×) from cytokeratin-7-stained sham, FFD and FFD+TAA groups. Staining is enriched in the FFD liver, indicative of HCC. This liver bore several tumors at sacrifice.

Figure 6

Liver hydroxyproline as a diagnostic for HCC. (A) Compared to the sham cohort, biopsies from livers characterized as NASH (FFD or FFD+TAA) had increased hydroxyproline content. ** p < 0.01 vs. sham. Hydroxyproline content was highest in biopsies from NASH+HCC livers. # p < 0.01 vs. NASH. (B) Receiver operating characteristics for liver hydroxyproline as a diagnostic for HCC in NASH. The threshold was selected as further improvement in sensitivity is associated with increased false positives.