Supervised learning reveals circulating biomarker levels diagnostic of hepatocellular carcinoma in a clinically relevant model of non-alcoholic steatohepatitis; An OAD to NASH

Abstract

Although cirrhosis is a key risk factor for the development of hepatocellular carcinoma (HCC), mounting evidence indicates that in a subset of patients presenting with non-alcoholic steatohepatitis (NASH) HCC manifests in the absence of cirrhosis. Given the sheer size of the ongoing non-alcoholic fatty liver disease (NAFLD) epidemic and the dismal prognosis associated with late-stage primary liver cancer there is an urgent need for HCC surveillance in the NASH population. Using serum levels of HCC biomarkers as vectors and biopsy-proven HCC or no HCC as outputs / binary classifier, a supervised learning campaign was undertaken to develop a minimally invasive technique for making a diagnosis of HCC in a clinically relevant model of NASH. Adult mice randomized to control diet or a fast food diet (FFD) were followed for up to 14 mo and serum level of a panel of HCC-relevant biomarkers was compared with liver biopsies at 3 and 14 mo. Both NAFLD Activity Score (NAS) and hepatic hydroxyproline content were elevated at 3 and 14 mo on FFD. Picrosirius red staining of liver sections revealed a filigree pattern of fibrillar collagen deposition with no cirrhosis at 14 mo on FFD. Nevertheless, 46% of animals bore one or more tumors on their livers confirmed as HCC in hematoxylin-eosin-stained liver sections. In this training set, receiver operating characteristic (ROC) curves analysis for serum levels of the HCC biomarkers osteopontin (OPN), alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) returned concordance-statistic/area under ROC curve of ≥ 0.89. Serum levels of OPN (threshold, 218 ng/mL; sensitivity, 82%; specificity, 86%), AFP (136 ng/mL; 91%; 97%) and DKK1 (2.4 ng/mL; 82%; 81%) diagnostic for HCC were confirmed in a test set comprising mice on control diet or FFD and mice subjected to hepatic ischemia-reperfusion injury. These data suggest that levels of circulating OPN, AFP and DKK1 can be used to make a diagnosis of HCC in a clinically relevant model of NASH.

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

“…The NAFLD spectrum starts with simple steatosis or lipid deposition within the liver. Left untreated, steatosis can progress to nonalcoholic steatohepatitis (NASH), i.e., steatosis accompanied by inflammation of the liver, NASH with fibrosis, cirrhosis and finally hepatocellular carcinoma (HCC) [ 3 , 4 , 5 , 6 , 7 , 8 ]. Considering that ~30% of the population in the United States alone has some steatosis, the risk for a NASH epidemic looms large [ 1 ].…”

“…Considering that ~30% of the population in the United States alone has some steatosis, the risk for a NASH epidemic looms large [ 1 ]. In fact, it is estimated that NAFLD-related hepatic complications will emerge as the leading cause of liver transplantation in the United States over the next 2 or 3 decades [ 1 , 2 , 3 ].…”

“…Liver biopsy tissue used for diagnosing NASH could also be purposed for diagnosing HCC. Although liver biopsy followed by microscopic evaluation remains the gold standard for diagnosing HCC [ 3 , 13 ], it is possible that the presence of remote tumor is missed during biopsy, resulting in potential misdiagnosis.…”

“…The present study tested the hypothesis that liver biopsy hydroxyproline content is a diagnostic for HCC in NASH. Hydroxyproline, a major component of collagen, can be assayed biochemically, is not subject to observer bias and is routinely assayed to estimate liver fibrosis [ 3 , 14 , 15 ]. There is also evidence to suggest that excessive collagen deposition is associated with the transformation of the scarred liver an oncological phenotype [ 3 , 16 , 17 ].…”

“…Liver hydroxyproline content was evaluated in murine models of NASH with or without HCC. Disease was induced in mice with the administration of a modified diet or the modified diet with chemicals, both of which have been reported to demonstrate the entire spectrum of NAFLD culminating in HCC [ 3 , 15 , 17 , 18 , 19 , 20 ].…”