Quantitative Evaluation by Digital Pathology of Immunohistochemical Expression of CK7, CK19, and EpCAM in Advanced Stages of NASH

Introduction

Non-alcoholic steatohepatitis (NASH) is the most recurrent form of chronic liver disease. There are two patterns of NASH; cholestatic (affecting bile ducts, reducing bile flow) and hepatitic, with the cholestatic pattern associated with more severe liver damage and fibrosis and hence a worse patient prognosis.

Diagnosis is currently performed by semi-quantative histological assessment by a pathologist, along with biochemical measurements of alanine transaminase (ALT) and alkaline phosphatase (ALP) in serum.

The authors present a way of distinguishing between cholestatic and hepatic patterns of NASH using digital pathology.

Main Points

• Liver biopsies were collected from 47 patients diagnosed as having NASH with stage 3-4 fibrosis and were classified into cholestatic or hepatic NASH groups based on the ratio of ALP to ALT (increased values of ALT are present in patients as an expression of hepatic inflammation and cytolysis). An increase in ALP (an indicator of hepatobiliary disorder) is also observed in cholestatic cases.
• Sections of liver were immunohistochemically labelled for cytokeratin 7 and 19, (CK7, CK19) (expressed in bile ducts and bile ductular reactions) and Epithelial Cell Adhesion Molecule (EpCAM) (expressed in embryonic liver, in both proliferating hepatocytes and bile ducts. However, in adult liver is retained only at small bile ducts and canaliculi; adult hepatocytes are EpCAM-negative).
• Quantification of the liver sections by QuPath software corresponded well with semi-quantative assessment by a pathologist.
• It was found that EpCAM correlated with ALP, with both having higher levels in cholestatic NASH with stage 4 fibrosis.
• ALT did not correlate with any morphological features.
• The hepatitic pattern of NASH has mature ductular structures (expressing both CK7 and CK19) which are more functional / efficient in allowing bile efflux, leading to a less severe grade of fibrosis. Conversely, in cholestatic patterns it is likely that immature ducts (still expressing EpCAM other than CK7, but almost negative for CK19) determine higher retention of bile acids, leading to ALP increase and a more advanced grade of fibrosis.
• Bile acid concentration and ALP are correlated. A reduction of bile acid secretion and / or retention leads to an increase in liver ALP synthesis. Bile acid overload may be an early trigger in biliary metaplasia or dedifferentiation of hepatocytes, exerting a stimulus on parenchymal cells, which in turn activates hepatic stellate cells that proliferate with a fibrogenic effect.

Conclusion

Image analysis confirms the relation between cholestatic pattern NASH (associated with a worse prognosis) with increased ALP values, EpCAM positive biliary metaplasia and advanced fibrosis. These preliminary data could be useful for the implementation of AI algorithms for the distinction between cholestatic and hepatitic NASH.

EPISTEM SERVICES

Epistem perform in vivo murine models of  liver fibrosis, supported by in-house histology/immunohistochemistry and gene expression services.

• Carbon tetrachloride, NASH and NALFD models of liver fibrosis performed in mouse.
• Analytical services include histological quantification of tissue sections (e.g. collagen area, inflammation,) and measurement of fibrotic biomarkers in plasma and tissue (e.g. ALT, Hydroxyproline).
• The Histology and Immunohistochemistry team at EPISTEM have a wealth of experience in developing IHC protocols and troubleshooting working protocols for both automated (Ventana Discovery Ultra) and manual applications.

• Epistem also offers gene expression analysis services and RNA‑friendly laser capture microdissection of target cell populations.