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Apoptosis and Liver Injury

Hepatocyte cell death by apoptosis is emerging as a fundamental component of virtually all acute and chronic liver diseases. The ensuing responses of cell repair, inflammation regeneration and fibrosis may all be triggered by apoptosis. Of these processes, hepatic fibrosis has the potential to be the most deleterious, as progressive fibrosis can culminate in cirrhosis.

Among the main causes of liver fibrosis are viral hepatitis (i.e. hepatitis B and C virus infection), alcoholic (ASH), and non-alcoholic steatohepatitis (NASH). An increasing body of evidence from both experimental and clinical studies suggests that hepatocyte apoptosis may contribute to liver fibrogenesis. Furthermore, recent studies have demonstrated that hepatocyte apoptosis correlates with disease activity in patients with chronic HBV and HCV infection and in patients with non-alcoholic fatty liver disease (NAFLD).

Assessment of liver disease severity as well as monitoring of patients with chronic liver disease over time remains a major clinical challenge. Although liver biopsy is still considered the gold standard for assessing disease activity, it is an invasive technique with inherent risks and unsuitable for disease monitoring.
 
Noninvasive reliable tests to quantify the magnitude of hepatocyte apoptosis in humans, therefore, would be highly desirable, as serum biomarker for liver damage and for treatment response.

» Brochure: M30 Apoptosense ELISA - A Serum Apoptosis Marker for Chronic Liver Disease

Caspase-cleaved K18 ("M30") as a Liver Apoptosis Biomarker

In the final common step of apoptosis, the effector caspases (in particular caspase-3 and caspase-7) become activated. These specific intracellular proteases are known to cleave several cellular substrates, including keratin 18 (K18, also known as Cytokeratin 18 or CK18), a major intermediate filament protein expressed by hepatocytes.

The M30 monoclonal antibody detects a neo-epitope on K18 generated by caspase-cleavage at Asp396 (Leers et al., 1999; patent applications filed 1998). This antibody is used in the M30 Apoptosense® ELISA assay which was originally developed for oncology applications (Hägg et al., 2002, Ueno et al., 2003, Kramer et al., 2004).

It was subsequently found that the M30 Apoptosense® ELISA is useful for detection of hepatocyte apoptosis occuring during hepatitis C virus (HCV) infection (Bantel et al., 2004). Caspase-generated K18 fragments are released from hepatocytes and can be detected in the blood circulation. The M30 Apoptosense® ELISA test has been found to be useful both for prognosis and disease monitoring in patients with HCV (see further below). Following the initial discovery of Bantel et al. that liver apoptosis can be detected using the M30 Apoptosense® ELISA, other investigators have found that also other processes associated with hepatocyte apoptosis are associated with increased levels of circulating caspase-cleaved K18 fragments. Nonalcoholic steatohepatitis (NASH) is one such disease where the M30 Apoptosense® ELISA has been used as a diagnostic tool for determination of the extent of disease progression (Wieckowska et al., 2006). Similar findings have been reported for toxicant-associated steatohepatitis (TASH; Cave et al., 2010) and for patients with severe bile duct inflammation and severe cholestasis (Yagmur et al., 2007; reviewed by Yilmaz, 2009).

References:
»   Leers MP et al. (1999). Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol. 1999 187:567-72.  
»   Hägg M et al. (2002). A novel high-through-put assay for screening of pro-apoptotic drugs. Invest. New Drugs 20, 253-259.
» Ueno T et al. (2003). Measurements of an apoptosis product in sera of breast cancer patients. Eur J Cancer 39, 769-74.
»  Kramer G et al. (2004). Differentiation between Cell Death Modes using Measurements of Different Soluble Forms of Extracellular Cytokeratin 18. Cancer Research 64, 1751-1756.
»  Bantel H et al. (2004). Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury. Hepatology 40:1078.
»  Wieckowska A et al. (2006). In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology 44:27
»  Yagmur E et al. (2007). Elevated apopto- sis-associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation. Clin Biochem 40:651–5.
»  Cave M et al. (2010). Toxicant-associated steatohepatitis in vinyl chloride workers. Hepatology 51:474-81.
»   Yilmaz Y (2009). Systematic review: caspase-cleaved fragments of cytokeratin 18 - the promises and challenges of a biomarker for chronic liver disease. Aliment Pharmacol Ther 30:1103-9. 204:468 

Apoptosis Biomarker for the HCV clinic

An estimated 3% of the world’s population — more than 170 million people — are infected by the hepatitis C virus (HCV). About 70% of infections become chronic: a condition that is associated with developing end stage liver disease such as cirrhosis and hepatocellular carcinoma (HCC). In patients with chronic HCV infection M30 values largely correlate with conventional surrogate markers such as aminotransferase levels. However, patients with normal ALT and progressed HCV-related liver fibrosis were found to also have elevated caspase-cleaved keratin 18 products in their serum, suggesting that M30 measurements represent an even more sensitive marker suitable for the detection of early liver injury. Serological detection of caspase activity also mirrored the degree of liver steatosis in those patients. In addition, M30 is a reliable serum marker to detect liver injury in patients with chronic HBV infection.

Even with current antiviral treatment options about 50% of HCV patients do not respond to treatment, which is costly and associated with significant side effects. There is a strong need for early detection of patients that are not responding to antiviral therapy. In a recent study of 315 patients it could be demonstrated that M30 serum levels decreased in patients responding to antiviral therapy, but not in non-responding patients (Sgier et al., 2010).
 
References:
» Bantel H et al. (2004). Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury. Hepatology. (2004) 40:1078. 
» Kronenberger B et al. (2005). Apoptotic cytokeratin 18 neoepitopes in serum of patients with chronic hepatitis C. J Viral Hepat. (2005) 12:307‑14 
» Seidel N et al. (2005). The extent of liver steatosis in chronic hepatitis C virus infection is mirrored by caspase activity in serum. Hepatology. (2005) 42:113 
» Volkmann X et al. (2006). Caspase activation is required for antiviral treatment response in chronic hepatitis C virus infection. Hepatology. (2006) 43:1311 
» Yagmur E et al. (2007). Elevated apoptosis‑associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation.
Clin Biochem. (2007) 40:651-5 
»  Papatheodoridis GV et al. (2008). Serum Apoptotic Caspase Activity As A Marker Of Severity In Hbeag‑Negative Chronic Hepatitis B Virus Infection. Gut. (2008) 57:500‑6
» Sgier C et al. (2010). Effect of antiviral therapy on circulating cytokeratin-18 fragments in patients with chronic hepatitis C. J Viral Hepat. 2010, 17:845-50.

Caspase-cleaved K18 are elevated in patients with NASH

Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of liver abnormalities from benign steatosis to nonalco­holic steatohepatitis (NASH). In line with the observation that M30 serum levels correlate with liver steatosis in HCV-infected patients, a recent study has shown that the extent of caspase-cleaved K18 in serum correlates with the grade of liver steatosis in patients with NAFLD. Moreover, in this study measurement of caspase-cleaved K18 serum levels allowed the discrimination between simple NAFLD and NASH and therefore, the detection of patients with increased risk of developing progressive (end stage) liver disease. 

References:
»   Wieckowska A, et al., In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology. (2006) 44:27
»   Yilmaz Y, Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis. World J Gastroenterol. (2007) 13:837‑44.
»

Younossi Z. et al., A Novel Diagnostic Biomarker Panel for Obesity-related Nonalcoholic Steatohepatitis (NASH). Obes Surg. 2008 Nov;18(11):1430-7

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Feldstein et al., Cytokeratin-18 Fragment Levels as Noninvasive Biomarkers for Nonalcoholic Steatohepatitis: A Multicenter Validation Study. Hepatology, Vol. 50, No. 7, 2009