An apoptosis biomarker goes to the HCV clinic
Authors: Ariel E. Feldstein, Gregory J. Gores
Journal: Hepatology (2004)

Assessment of liver disease severity as well as monitoring of patients with chronic liver disease over time remains a major challenge in daily hepatology practice. Although liver biopsy is still considered the gold standard for assessing disease activity, it is an invasive technique with inherent risks that can be repeated only at infrequent intervals. Thus, there is an urgent need to develop noninvasive reliable tests to monitor hepatic disease activity, especially serum biomarkers. HCV, hepatitis C virus; ALT, alanine aminotransferase. 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.1-4 Of these processes, hepatic fibrosis has the potential to be the most deleterious, as progressive fibrosis can culminate in cirrhosis with portal hypertension and chronic liver failure. An increasing body of evidence from both experimental and clinical studies suggests that hepatocyte apoptosis may contribute to liver fibrogenesis.5 For instance, in animal models of cholestasis, attenuation of hepatocyte apoptosis also reduces fibrogenesis.6 Engulfment of apoptotic bodies by hepatic stellate cells stimulates the fibrogenic activity of these cells and may be one mechanism by which hepatocyte apoptosis promotes fibrosis.5 Furthermore, recent studies in human chronic hepatitis C virus (HCV) infection as well as nonalcoholic fatty liver disease demonstrate that hepatocyte apoptosis correlates with disease severity7 and stage of fibrosis in patients with steatosis.8, 9 Noninvasive techniques to quantify the magnitude of hepatocyte apoptosis in humans, therefore, would be highly desirable, not only as a potential biomarker for monitoring liver damage, but also as an endpoint in therapeutic trials employing antiapoptotic agents. In vivo assessment of apoptosis has been performed in small animals by radionucleotide imaging approaches. Phosphatidyl serine is "flipped" from the interior to the exterior leaflet of the plasma membrane.10 Annexin V, a protein, binds to phosphatidyl serine with a high affinity in the presence of calcium. Several studies have demonstrated that radio-labeled annexin V can be used to image apoptotic cells in small animals.11 However, background in the liver is high, precluding the use of these techniques for assessing liver cell apoptosis. Therefore, a serum test for liver cell apoptosis would be highly desirable. In this issue of HEPATOLOGY, Bantel et al. report the first assessment of a serum biomarker for hepatocyte apoptosis.12 Many forms of apoptosis involve the activation of caspases, intracellular proteases that cleave after aspartate residues. Indeed, the signature cleavage of proteins after aspartate moieties by caspases is unique to this protease family. Furthermore, the cleaved protein generates new epitope(s) for which antibodies can be developed.13 Bantel et al. took advantage of this information to measure caspase-specific cleavage products of cytokeratin 18 in serum using an ELISA assay.12, 14 The authors selected this protein because it is abundant in hepatocytes, thereby providing specificity for the source of this cleaved protein, and caspase-mediated proteolytic cleavage of cytokeratin 18 may also contribute to cell death by apoptosis.15, 16 The concepts and details of the assay are depicted in Figure 1. The authors demonstrated that HCV patients had elevated levels of serum caspase-generated cytokeratin 18 cleavage fragments as compared to healthy controls. These exciting results advance the field of clinical and experimental hepatology and provide new insights into apoptosis, HCV disease assessment and have implications for liver disease monitoring. A novel biomarker of hepatocellular apoptosis. Cytokeratin 18 is cleaved by caspases at two conserved aspartate residues (Asp238 and Asp396) during hepatocyte apoptosis. The mechanisms by which these fragments may appear in blood remain unclear. Caspase activation in hepatocyte can be indirectly quantified in serum by using the monoclonal antibody M30, which selectively recognizes the caspase cleavage–generated neoepitope of cytokeratin 18 at Asp396. N,; HRP, horseradish peroxidase; CK, cytokeratin. What does this study teach us about apoptosis? Classic, but outdated concepts regarding apoptosis suggest it is a cell-autonomous process characterized by cellular fragmentation into membrane defined apoptotic bodies. These apoptotic bodies can be eliminated via phagocytosis by neighboring epithelial cells, stellate cells and macrophages.17 The phagocytosis of apoptotic bodies is thought to prevent the release of intracellular constituents into the extracellular space and/or blood. However, the current study clearly demonstrates that intracellular proteins cleaved by the apoptotic demolition machinery do appear in blood! These observations imply that apoptotic cells either breakdown like necrotic cells, have leaky membranes, or secrete cleaved proteins, or small apoptotic bodies are swept into the bloodstream. By whichever process this occurs, it is clear that apoptotic cellular products can be measured in serum. Previous concepts regarding the fate of apoptotic bodies in pathology need to be reevaluated. What does this study teach us about HCV disease? In patients with elevated serum alanine aminotransferase (ALT) values, this assay correlated very well with the ALT value— a reassuring observation, but one that does not add to the care of this patient population. However, many patients with normal serum ALT values may have active HCV disease as assessed by histopathology,18 and many knowledgeable physicians advocate treatment for this subset of patients. Thus, their identification has clinical relevance. If HCV is truly an "apoptotic disease," then serum determination of cleaved cytokeratin 18 should identify this subset of patients. Indeed, in the current study, patients with active, normal-ALT, HCV-related liver diseases were found to have elevated caspase-cleaved cytokeratin 18 products in their serum. The caspase cleaved cytokeratin 19 subunits in serum especially identified patients with fibrosis, a finding in keeping with the emerging link between apoptosis and fibrosis described above. These results are quite important for several reasons. First, they suggest that HCV is an apoptotic liver disease, implying that antiapoptotic therapy may be efficacious for preventing HCV-related liver damage. Second, this assay, if verified and the results replicated, provides a technique to identify those patients with aggressive disease despite normal ALT values. Finally, perhaps assaying serum apoptosis products may be a clinically valuable tool and a legitimate endpoint for assessing therapy in selected HCV patients, namely those patients receiving hepatoprotective agents after failing current antiviral therapies. As mentioned above, one of the "holy grails" of clinical hepatology is the identification of biomarkers for disease severity and progression. The emerging field of proteomics may provide these markers. Bantel et al. may have identified such a biomarker based on a rationale understanding of cell biology and HCV-associated liver disease. This translational research effort, insight, and results have potential significant clinical implications not only for HCV liver disease, but also for other liver conditions such as nonalcoholic fatty liver disease in which a reliable noninvasive clinical tool to assess disease severity over time is greatly needed. Also, as antiapoptotic therapy is evaluated in the clinic, this biomarker may serve as a critical endpoint. Although time and future trials will ultimately determine the utility of this assay in assessing human liver disease, the authors are to be congratulated for their translational research efforts. The authors thank Erin Bungum for superb assistance in generating this editorial.