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Cytokeratins in hepatitis
Clinica Chimica Acta 412 (2011) 2031–2036
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Invited critical review
Cytokeratins in hepatitis Yusuf Yilmaz ⁎ Department of Gastroenterology, Marmara University, School of Medicine, Pendik, 34899 Istanbul, Turkey Institute of Gastroenterology, Marmara University, Maltepe, 34840 Istanbul, Turkey
a r t i c l e
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Article history: Received 3 August 2011 Received in revised form 30 August 2011 Accepted 2 September 2011 Available online 7 September 2011 Keywords: Hepatitis Cytokeratins Biomarkers Liver biopsy
a b s t r a c t Experimental and clinical evidence suggests that cytokeratins (CK), among other physiological functions, are expressed in hepatocytes and can be released in the bloodstream after acute or chronic inflammatory liver injury. Interest in CK in viral and nonviral hepatitis has been rapidly increasing during the last years, especially as they have been proposed as circulating biomarkers of hepatocyte necrosis and apoptosis. In the present review, we sought to summarize and discuss the alterations in circulating CK levels in different form viral and nonviral hepatitis, as well as their potential relation with liver histology. Understanding the mechanisms of hepatitis impact on CK and vice versa is a promising area of research that will positively enhance our understanding of the complexity of acute and chronic inflammatory liver injury. © 2011 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Search strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokeratin expression in hepatocytes in physiological conditions and in inflammation . Cytokeratins as biomarkers of hepatitis: biochemical assays . . . . . . . . . . . . . . Cytokeratins in viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Cytokeratins in alcoholic hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . 7. Cytokeratins in nonalcoholic steatohepatitis . . . . . . . . . . . . . . . . . . . . . 8. Cytokeratins in autoimmune hepatitis . . . . . . . . . . . . . . . . . . . . . . . . 9. Cytokeratins in toxic hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Clinical implications of CK fragments as biomarkers in hepatitis . . . . . . . . . . . . 11. Future research directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The term hepatitis indicates an inflammation of the liver parenchyma and has its origins from the ancient Greek; hepar meaning liver and the suffix -itis meaning inflammation [1–4]. Historically, the first reports of hepatitis are seen in the literature as far back as
⁎ Department of Gastroenterology, Marmara University, School of Medicine, Pendik, 34899 Istanbul, Turkey. Tel.: +90 5334403995; fax: +90 2166886681. E-mail address: [email protected]. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.09.002
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in Hippocrates writings. Inflammation of the liver can be generated by a host of different causes, including viral infections, alcohol consumption, metabolic dysfunctions, xenobiotic exposition, and metal overload. Concerning viral hepatitis, five human hepatitis viruses have been identified to date, i.e. hepatitis A, B, C, D, and E [5–7]. A sixth virus, hepatitis G virus (HGV), can replicate in hepatocytes but there is no definite evidence that it can cause hepatic inflammation and should probably not be referred to as a hepatitis virus [8]. The major hepatitis B (HBV), C (HCV), and D (HDV) viruses are most efficiently transmitted by infected blood, but also can be transmitted by exposure to
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other infectious bodily fluids [9,10]. These three viruses can cause acute or chronic hepatitis and make an important contribution to the global burden of liver disease. Nonviral causes of hepatitis include metabolic [11–13], autoimmune [14], toxic/drug-induced [15,16], and alcoholic forms [17,18] of hepatitis. Progressive liver disease in chronic hepatitis is thought to arise as a result of the chronic inflammatory response to a hepatotoxic injury, resulting in an environment that is favorable for the fibrogenic process [19,20]. From an epidemiological standpoint, HCV infection stands as a major cause of global morbidity and suffering from chronic hepatitis worldwide [21,22]. Another common cause of chronic hepatitis in Western countries is nonalcoholic steatohepatitis (NASH), which is linked to insulin resistance [3,13]. NASH is viewed as the hepatic event of the metabolic syndrome and it is characterized by hepatic triglyceride accumulation (i.e. steatosis), in combination with hepatic inflammation [12]. In all forms of hepatitis, the inflammatory process causes hepatocyte death through different mechanisms, including necrosis and apoptosis [23–25]. The development of sensitive and specific assays of serum biomarkers has allowed the noninvasive detection of small amounts of hepatocyte cell death associated with hepatic necroinflammation [26–28]. Cytocheratins (CK) are the major intermediate filament protein in the liver and any cellular damage that alters the hepatocyte membrane integrity may cause the release of CK (or their fragments) into the circulation [29,30]. At least 37 different human CK have been identified to date, of which CK8 and CK18 are the most abundant in the liver [29]. It has been observed that, when hepatocytes undergo necrosis or apoptosis as a consequence of hepatic inflammation, both uncleaved and partially degraded CK fragments are released in the bloodstream and can be measured using specific serological assays [31,32]. CK and CK fragments released from necrotic or apoptotic cells in hepatitis have proved to be useful biomarkers of hepatic inflammation [26–28]. In addition, specific motifs in certain CK make them possible substrates for caspase degradation during apoptosis [33,34]. Based on these findings, the measurement of CK and CK fragments in the serum may have value for the noninvasive detection of the necroinflammatory activity during the course of hepatitis. In the present review, we sought to summarize the expression of CK in the liver and discuss the alterations of their circulating levels in different viral and nonviral forms of hepatitis. Understanding the mechanisms of hepatitis impact on CK and vice versa is a promising area of research that will positively enhance our understanding of the complexity of acute and chronic inflammatory liver injury. 2. Search strategy In July 2011, we searched the PubMed database, without any language restriction, using the search terms ‘cytokeratin’, ‘hepatocyte’, ‘hepatitis’, ‘liver’, and ‘biomarker’, to find reports describing the alterations of CK in hepatitis and their role in the pathophysiology of liver inflammation. Additional articles were identified through the reference sections of the studies retrieved. The titles and abstracts of all identified studies were reviewed using the following inclusion criteria: use of CK or CK fragments as biomarkers of different form of hepatitis or role of CK in the pathophysiology of hepatic inflammation. 3. Cytokeratin expression in hepatocytes in physiological conditions and in inflammation In general terms, CK are the largest subgroup of phylogeneticallyconserved intermediate filament proteins and represent the most abundant proteins in epithelial cells [35,36]. The protein structures of CK consist of a central alpha helical rod domain, flanked on either side by amino terminal (head) domain and carboxy terminal (tail) domain [37]. Cytokeratins undergo several posttranslational modifications, such as phosphorylation, glycosylation, acetylation, and
transglutaminase-induced cross-linking, that may play a role in regulating their function [38]. The primary function of CK is to protecting epithelial cells from mechanical and nonmechanical forms of injury, ultimately preventing cell death [39]. Of note, other functions of CK have been described, including a key role in stress response, cell signaling, and apoptosis [40]. CK expression is the liver is a highly regulated process. Hepatocyte progenitor cells express a broad range of cytokeratins – CK8, CK18, CK19 and (transiently) CK14 [29]. In the adult normal liver, hepatocytes express only CK8 and CK18, while interlobular bile ducts, intraportal and intralobular bile ductules, and the biliary epithelial cells express primarily CK7 and CK19 [29]. Biliary epithelial cells may also express CK8 and CK18 [29]. The functional association of CK8 and CK18 with cytoprotection in hepatocytes is supported by the observation that mutations in CK8 or CK18 are associated with the development of cryptogenic and noncryptogenic forms of liver disease. Interestingly, hepatocytes isolated from CK8- [41] and CK18- [42] null mice lack any keratin filaments. Moreover, livers of CK8-null mice are highly susceptible to liver injury as compared with their heterozygous or wild-type littermates [41]. Intriguingly, adult CK18deficient mice develop mild hepatitis and increased hepatocytes fragility due to spontaneous Mallory-Denk body formation from accumulating CK8 aggregates [42]. Experimental studies have also shown that hepatocytes bearing the CK18 Ser33Ala mutation had a significant inhibition in cell cycle progression [43]. Of note, phosphorylation on serine residues of CK8 and CK18 is increased during mitosis, apoptosis, growth factor stimulation, and different forms of hepatocyte stress [44]. In human immunohistochemical studies, CK8 and CK18 expression have been shown to be useful markers of hepatocyte ballooning associated with the formation of Mallory-Denk bodies in perivenular regions in NASH [45]. As hepatocytes showing macrovesicular or microvesicular steatosis do not exhibit this marked change in CK expression, it has been suggested that immunostaining for CK8 and CK18 may be helpful in distinguishing between simple steatosis and NASH across the spectrum of nonalcoholic fatty liver disease (NAFLD) [46]. Interestingly, aberrant hepatocyte CK7 expression may occur in the portal tracts during the course of autoimmune hepatitis, albeit at a markedly lower extent than in chronic biliary tract disease [47]. 4. Cytokeratins as biomarkers of hepatitis: biochemical assays As CK8 and CK18 are abundantly expressed in the hepatocyte [29], they hold a unique position as markers of hepatic inflammation. CK released from the hepatocyte in circulation after cell death and disintregration can be detected either as partially degraded single protein fragments, as small complexes, or as large polymeric protein complexes [32–34]. The three most frequently used CK which are being evaluated as serum markers for their clinical utility in hepatitis are CK18 and CK18 fragments (M65 and M30 antigens) [31,32], tissue polypeptide antigen (TPA) [35], and tissue polypeptide specific antigen (TPS) [48]. Evidence suggests that dysregulation of hepatocyte apoptosis plays an important role in a wide range of inflammatory disorders of the liver [32,49]. A neoepitope in CK18, termed M30 antigen, becomes available at an early caspase cleavage event during hepatocyte apoptosis and is not detectable in vital or necrotic cells [32]. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30 antigen). An M30-based sandwich ELISA assay determines the circulating levels of M30 antigen, which serves as surrogate serum biomarker of hepatocyte apoptosis [27,31]. By contrast, a specific M65 ELISA assay measures the levels of both caspase-cleaved and intact CK18, the latter of which is released from cells undergoing necrosis [31,32]. During necrotic and apoptotic cell death, CK18derived M65 and M30 antigens are released into the blood in either their intact or their caspase-cleaved forms where they remain relatively stable in the circulation [27]. Both assays have now been largely
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used in the analysis of plasma and serum collected from subjects with different forms of hepatitis for the purpose of assessing their association with different clinical and histological characteristics. Assays for TPA measure an aggregate of nonepidermal cytokines 8, 18, and 19 [35], while assays for TPS are more specific and measure CK18 levels [48]. Actually TPS is considered a particular epitope of the TPA molecule and is considered as more closely related to proliferation of epithelial cells [48]. Compared with apparently healthy individuals, the level of CK markers in the circulation is higher in patients with different forms of hepatitis and it generally rises significantly in relation to the severity of histological lesions. 5. Cytokeratins in viral hepatitis 5.1. Hepatitis B The potential significance of circulating CK levels in HBV infection has been investigated in a pilot study by Papatheodoridis et al. [50]. The authors reported a significant increase in serum M30 antigen levels in patients with chronic hepatitis B (CHB), which was significantly higher in HbeAg-negative subjects than in inactive chronic HBV carriers. The marker was clinically useful for distinguishing between the two groups (area under ROC curve of 0.87), but not for determining the severity of liver histology among HBeAg-negative CHB patients [50]. To confirm and expand these findings, we subsequently measured serum M30 antigen levels in inactive HBV carriers, patients with HBeAg-negative CHB, patients with HBeAg-positive CHB, and healthy controls [26]. The results indicated that serum levels of M30 antigen were higher in both HBeAg-negative and HBeAg-positive CHB patients than in inactive HBV carriers. Interestingly, a cutoff value of 118 U/L for serum M30 antigen had a 70% sensitivity and 80% specificity for discriminating active from inactive carriers. These results suggest the usefulness of M30 antigen testing to identify acute flares of asymptomatic hepatitis in CHB patients [26]. Of interest, serum levels of CK18 fragments have been shown to decrease significantly during oral antiviral therapy in HBeAg-negative CHB in parallel with the improvements in ALT and HBV DNA levels, suggesting their usefulness to monitor treatment response [51]. Recently, Shi et al. [52] have reported that CK18 phosphorylation at Ser33 was colocalized with viral infection in the hepatocytes of inactive HBV carriers, while only basal level of Ser52 phosphorylation was detected in infected cells. These results indicate CK18 phosphorylation may serve as a progression marker for CHB. Taken together, these results suggest that CK play are involved in the molecular mechanisms of hepatitis B progression and are potentially useful as blood-borne biomarkers to distinguish among specific subgroups of patients within this clinical entity. 5.2. Hepatitis C Due to the chronic course of hepatitis C infection, there is a growing need for sensitive and noninvasive biomarkers of early liver injury [1]. Due to their abundant expression in the hepatocyte, CK have attracted considerable attention in this respect. Using site-specific antiphosphokeratin antibodies Toivola et al. [53] found keratin hyperphosphorylation on most CK8 and CK18 sites in liver explants from patients with chronic HCV infection, suggesting its usefulness as a progression marker. Concerning CK as biomarkers, Bantel et al. [54] reported in a pilot study that serum levels of CK18 fragments may be a more sensitive marker compared with aminotransferases for the detection of early liver injury in chronic HCV infection. Our research group subsequently reported a significant positive correlation between serum M30 levels and AST, the grade of steatosis on ultrasound, and the histological steatosis score in patients with chronic HCV infection [27]. Taken together, these results suggest that M30 levels may serve as a noninvasive biomarkers of liver fat accumulation in patients
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with chronic hepatitis C. Papatheodoridis et al. [55] have shown that median CK18 fragment levels were lower in HCV-positive patients with minimal/mild than patients with moderate/severe histologic lesions, offering moderate accuracy for differentiation between the two group. Valva and coworkers [56] measured serum M30 levels in serum from pediatric HCV-infected patients and assessed its utility to predict liver damage progression. The results showed that serum M30 was higher in patients than in controls and correlated both steatosis and severity grade. Different studies have reported an elevation of CK18 using the TPS assay in chronic hepatitis C [57,58], though it is clear that this increase is not a specific feature of HCV infection but simply reflect the degree of cytolysis. Recently, circulating CK fragments have also emerged as a signature of drug-induced liver injury in HCV-infected patients. In this regard, Feldstein and colleagues [59] demonstrated that HCV-796, a hepatitis C polymerase inhibitor approved by the US Food and Drug Administration for a phase 2 study of the treatment of hepatitis C in combination with PEG-Interferon and ribavirin, may cause severe hepatocellular injury and apoptosis in susceptible patients as reflected by a marked increase in serum M30 levels. Intriguingly, Sgier et al. [60] have also shown that successful antiviral therapy in patients with chronic hepatitis C results in a significant decrease in circulating levels of CK18 fragments, arguing for a reduction in hepatocellular apoptosis after clearance of the HCV. 6. Cytokeratins in alcoholic hepatitis Experimental results have suggested an association of cytokeratin hyperphosphorylation with the formation of Mallory bodies in hepatocytes, a hallmark of alcoholic hepatitis [61]. Stumptner et al. [62] have shown that Mallory bodies in human alcoholic hepatitis preferentially contain hyperphosphorylated CK8 and CK18, suggesting that CK8 and CK18 hyperphosphorylation may play a contributing role in MB pathogenesis. The potential usefulness of CK as a biomarker in alcoholic liver disease has been originally suggested by Gonzalez-Quintela et al. [48] who reported that levels of CK18 are frequently increased in alcoholics. The same researchers subsequently replicated the finding of an increased serum level of CK18 in heavy drinkers [63] and suggested a synergism between risky alcohol consumption and obesity in relation to serum concentrations of this molecule, which probably reflect liver disease [64]. Interestingly, Lavallard and coworkers [65] recently showed that CK18 and CK fragments are correlated with severe fibrosis in heavy alcohol drinkers. In addition, CK markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, and hepatic expression of proinflammatory cytokines [65]. 7. Cytokeratins in nonalcoholic steatohepatitis NASH is a clinical entity which histologically simulates alcoholic hepatitis in the absence of alcohol ingestion or intake less than 20 g/day [12,13]. There is enough data to support that NASH is a progressive disease and can lead to cirrhosis of the liver and hepatocellular carcinoma [66]. Even though the pathogenesis of NASH is not fully understood, it may be considered a two hit process [67,68]. The first hit leads to deposition of fat in the liver parenchyma for which insulin resistance has been implicated as the major pathogenetic mechanism. The exact mechanisms promoting progressive hepatic inflammation and liver injury are not well defined, although substrates derived from adipose tissue such as free fatty acid, tumor necrosis factor alpha, leptin, and adiponectin have been implicated [69]. Recent years have witnessed an increased interest in biomarkers in the spectrum of NAFLD (from simple fatty liver to NASH) and the assessment of serum CK and CK fragments is considered a promising approach for the noninvasive diagnosis of metabolic liver inflammation. In a seminal study by Wieckowska et al. [70] demonstrated that caspase-generated CK18 fragments release in NAFLD may serve as an indicator of hepatic inflammation. The increased apoptic rate
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as a consequence of the hepatic inflammatory response in NASH is reflected by an increase in serum CK18 fragments that may therefore distinguish NASH from simple steatosis [28]. These results were further confirmed even in NAFLD patients with normal aminotransferase levels [71]. The increase in CK18 fragments in NASH has been subsequently reported in specific subgroup of patients, including obese individuals [72] and children [73]. CK18 fragments are currently being incorporated in multimarker schemes to improve their diagnostic performances for detecting NASH in a noninvasive manner. Younossi et al. [74] have developed a test (NASH Diagnostics) based on four ELISA assays (cleaved and intact CK18, serum adiponectin, and serum resistin) to form a simple diagnostic biomarker for NASH, which has been recently validated by Feldstein and colleagues [75]. After allowance for potential confounders, multivariable analysis revealed that CK18 fragments remained an independent predictor of NASH. Taken together, these results suggest that CK18 fragments have sufficient-to-excellent diagnostic accuracy for the detection (or exclusion) of NASH across the spectrum of NAFLD. In keeping with these results, Malik and coworkers [76] have recently compared the clinical utility of CK18 to other biomarkers in 95 consecutive patients with NAFLD. CK18 yielded an area under the curve of 0.8 for NASH, and a cutoff value of 300 μ/L gave a positive predictive value of 81% and a negative predictive value of 85%. In addition, Papatheodoridis et al. [55] reported that CK18 fragments levels were lower in healthy subjects than patients with simple fatty liver than patients with NASH, offering excellent diagnostic accuracy for differentiation between the two latter groups. Tan et al. [77] also suggested that higher levels of caspase 3-cleaved fragment of CK18 may indicate the presence of NASH in women with the polycystic ovary syndrome. 8. Cytokeratins in autoimmune hepatitis Autoimmune hepatitis is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components [14]. In a pilot study, Murota and coworkers [78] reported that CK alterations in the hepatocytes in autoimmune hepatitis may increase the susceptibility to apoptosis and induce hepatocyte destruction. Tahiri et al. [79] demonstrated that both CK 8 and 18 represent potential candidate targets on liver membrane for autoantibodies in this clinical entity. In a case report by Inui et al. [80] demonstrated the presence of antibodies against CK8 and CK18 in a patient with de novo autoimmune hepatitis after living-donor liver transplantation. Despite the lack of systematic observations and replication studies, these preliminary findings seem to suggest that changes in CK8 and CK18 in hepatocytes might be one of the sources of pathogenesis of autoimmune hepatitis. 9. Cytokeratins in toxic hepatitis Toxic hepatitis encompasses a spectrum of conditions ranging from mild biochemical abnormalities to acute liver failure. Data on cytokeratins alterations in toxic hepatitis remain scanty. There is at present only one study available reporting the potential involvement of CK in this setting. Fortier et al. [81] have shown that griseofulvininduced toxic liver injury in the rat cause significant changes in keratin solubility in the hepatocyte with a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites was also increased and prominent in the insoluble protein fractions. There is currently a need for human studies of toxic hepatitis to establish the utility of this marker in this clinical entity. 10. Clinical implications of CK fragments as biomarkers in hepatitis The evidence reviewed above clearly suggests that CK play a crucial role in the pathophysiology of hepatic inflammation and serum CK
levels are altered in different forms of hepatitis. In general, measuring CK and CK fragments in serum/plasma provide a dynamic and powerful approach to understanding the spectrum of hepatic inflammation with potential applications in epidemiology, randomized clinical trials, and prognosis. In any case, an important prerequisites for the clinical use of CK as biomarkers for hepatitis include the elucidation of specific indications, the standardization of analytical methods, the characterization of analytical features, the assessment of performance characteristics, the incremental yield of different markers for given clinical indications, and the demonstration of cost-effectiveness [31]. Clearly, CK cannot be used to distinguish between different forms of hepatitis as they are commonly raised independently of the etiology of liver inflammation. This would ultimately lead to a low sensitivity. From a clinical standpoint, characterization of CK and CK fragments in the sera from patients with NAFLD would have value to discriminate between those with NASH from those with simple steatosis. Such a noninvasive liver test may aid clinicians in the selection of patients for liver biopsy and might also allow for noninvasive assessment of liver disease progression and therapeutic response [32,82,83]. In addition, CK may be clinically useful for distinguishing between HbeAg-negative from inactive chronic HBV carriers [26]. 11. Future research directions The previous paragraphs have documented the evidence on the potential clinical usefulness of serum CK levels in different forms of viral and nonviral hepatitis. Considering the availability and quality of evidence on this topic, we have identified several important areas for future studies. One critical direction for future research in the field of chronic viral hepatitis relates on the measurements of CK in patients with coinfection with hepatitis B virus and hepatitis D virus, human immunodeficiency virus (HIV) and HCV, and HBV/HCV/HDV coinfection. The primary concern with coinfection with hepatotropic viruses is that it can lead to more severe liver disease and an increased risk for progression to hepatocellular carcinoma [84,85]. Whether CK may serve as markers of hepatotropic virus coinfection deserves further scrutiny. Concerning subjects with NAFLD, preliminary evidence has suggested the usefulness of serum M30 values for identifying patients with NASH among patients with normal ALT values [86]. Indeed, normal ALT does not represent a valuable criterion to exclude from liver biopsy patients with persistently ultrasonographic evidence of NAFLD [87,88]. Because lifestyle intervention may result in the significant amelioration of histological derangement [89], there is a strong need for identifying individuals among those presenting with risk factors for NAFLD at risk for severe histological liver derangement despite normal ALT values. In this scenario, larger-scale studies are needed to shed more light on the potential usefulness of serum M30 levels as a biomarker of severe NAFLD in the normal ALT population. Another fruitful avenue for future research in the field of CK in NASH would be the study of whether improvements in liver histology following lifestyle interventions (or drug therapy) would be paralleled by a reduction in serum M30 levels (i.e. M30 levels may potentially serve as a “pharmacodynamic” marker in this setting). Hopefully, future studies should also investigate in more detail the interplay between CK and abnormal fatty acid metabolism in the progression of NAFLD [90]. 12. Conclusions Cytokeratin serum markers have the potential to be valuable tools for staging, prognosis, and treatment monitoring of different forms of hepatitis. Their clinical utility has been demonstrated in the spectrum of NAFLD [27,74,75] and to some extent in CHB [26,50]. It is apparent that these markers may also prove useful in identifying drug-induced or toxic liver damage [59]. However, current clinical studies must be completed with a thorough analytical evaluation to determine the
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accuracy, reliability, interpretability, and feasibility of measuring CK in patients with different forms of hepatitis. In this regard, it will be paramount to reference distributions by important variables such as age and sex, the extent of intraindividual variation, and persistence of the biomarkers. Larger clinical and epidemiological studies with a large sample size will be needed to evaluate the analytical features of CK before they can be used as noninvasive markers for prognostication, follow-up of therapy, and detection of recurrence of hepatitis.
Disclosures No conflicts of interest.
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
Cytokeratins in hepatitis Yusuf Yilmaz ⁎ Department of Gastroenterology, Marmara University, School of Medicine, Pendik, 34899 Istanbul, Turkey Institute of Gastroenterology, Marmara University, Maltepe, 34840 Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 3 August 2011 Received in revised form 30 August 2011 Accepted 2 September 2011 Available online 7 September 2011 Keywords: Hepatitis Cytokeratins Biomarkers Liver biopsy
a b s t r a c t Experimental and clinical evidence suggests that cytokeratins (CK), among other physiological functions, are expressed in hepatocytes and can be released in the bloodstream after acute or chronic inflammatory liver injury. Interest in CK in viral and nonviral hepatitis has been rapidly increasing during the last years, especially as they have been proposed as circulating biomarkers of hepatocyte necrosis and apoptosis. In the present review, we sought to summarize and discuss the alterations in circulating CK levels in different form viral and nonviral hepatitis, as well as their potential relation with liver histology. Understanding the mechanisms of hepatitis impact on CK and vice versa is a promising area of research that will positively enhance our understanding of the complexity of acute and chronic inflammatory liver injury. © 2011 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Search strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokeratin expression in hepatocytes in physiological conditions and in inflammation . Cytokeratins as biomarkers of hepatitis: biochemical assays . . . . . . . . . . . . . . Cytokeratins in viral hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Cytokeratins in alcoholic hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . 7. Cytokeratins in nonalcoholic steatohepatitis . . . . . . . . . . . . . . . . . . . . . 8. Cytokeratins in autoimmune hepatitis . . . . . . . . . . . . . . . . . . . . . . . . 9. Cytokeratins in toxic hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Clinical implications of CK fragments as biomarkers in hepatitis . . . . . . . . . . . . 11. Future research directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The term hepatitis indicates an inflammation of the liver parenchyma and has its origins from the ancient Greek; hepar meaning liver and the suffix -itis meaning inflammation [1–4]. Historically, the first reports of hepatitis are seen in the literature as far back as
⁎ Department of Gastroenterology, Marmara University, School of Medicine, Pendik, 34899 Istanbul, Turkey. Tel.: +90 5334403995; fax: +90 2166886681. E-mail address: [email protected]. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.09.002
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in Hippocrates writings. Inflammation of the liver can be generated by a host of different causes, including viral infections, alcohol consumption, metabolic dysfunctions, xenobiotic exposition, and metal overload. Concerning viral hepatitis, five human hepatitis viruses have been identified to date, i.e. hepatitis A, B, C, D, and E [5–7]. A sixth virus, hepatitis G virus (HGV), can replicate in hepatocytes but there is no definite evidence that it can cause hepatic inflammation and should probably not be referred to as a hepatitis virus [8]. The major hepatitis B (HBV), C (HCV), and D (HDV) viruses are most efficiently transmitted by infected blood, but also can be transmitted by exposure to
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other infectious bodily fluids [9,10]. These three viruses can cause acute or chronic hepatitis and make an important contribution to the global burden of liver disease. Nonviral causes of hepatitis include metabolic [11–13], autoimmune [14], toxic/drug-induced [15,16], and alcoholic forms [17,18] of hepatitis. Progressive liver disease in chronic hepatitis is thought to arise as a result of the chronic inflammatory response to a hepatotoxic injury, resulting in an environment that is favorable for the fibrogenic process [19,20]. From an epidemiological standpoint, HCV infection stands as a major cause of global morbidity and suffering from chronic hepatitis worldwide [21,22]. Another common cause of chronic hepatitis in Western countries is nonalcoholic steatohepatitis (NASH), which is linked to insulin resistance [3,13]. NASH is viewed as the hepatic event of the metabolic syndrome and it is characterized by hepatic triglyceride accumulation (i.e. steatosis), in combination with hepatic inflammation [12]. In all forms of hepatitis, the inflammatory process causes hepatocyte death through different mechanisms, including necrosis and apoptosis [23–25]. The development of sensitive and specific assays of serum biomarkers has allowed the noninvasive detection of small amounts of hepatocyte cell death associated with hepatic necroinflammation [26–28]. Cytocheratins (CK) are the major intermediate filament protein in the liver and any cellular damage that alters the hepatocyte membrane integrity may cause the release of CK (or their fragments) into the circulation [29,30]. At least 37 different human CK have been identified to date, of which CK8 and CK18 are the most abundant in the liver [29]. It has been observed that, when hepatocytes undergo necrosis or apoptosis as a consequence of hepatic inflammation, both uncleaved and partially degraded CK fragments are released in the bloodstream and can be measured using specific serological assays [31,32]. CK and CK fragments released from necrotic or apoptotic cells in hepatitis have proved to be useful biomarkers of hepatic inflammation [26–28]. In addition, specific motifs in certain CK make them possible substrates for caspase degradation during apoptosis [33,34]. Based on these findings, the measurement of CK and CK fragments in the serum may have value for the noninvasive detection of the necroinflammatory activity during the course of hepatitis. In the present review, we sought to summarize the expression of CK in the liver and discuss the alterations of their circulating levels in different viral and nonviral forms of hepatitis. Understanding the mechanisms of hepatitis impact on CK and vice versa is a promising area of research that will positively enhance our understanding of the complexity of acute and chronic inflammatory liver injury. 2. Search strategy In July 2011, we searched the PubMed database, without any language restriction, using the search terms ‘cytokeratin’, ‘hepatocyte’, ‘hepatitis’, ‘liver’, and ‘biomarker’, to find reports describing the alterations of CK in hepatitis and their role in the pathophysiology of liver inflammation. Additional articles were identified through the reference sections of the studies retrieved. The titles and abstracts of all identified studies were reviewed using the following inclusion criteria: use of CK or CK fragments as biomarkers of different form of hepatitis or role of CK in the pathophysiology of hepatic inflammation. 3. Cytokeratin expression in hepatocytes in physiological conditions and in inflammation In general terms, CK are the largest subgroup of phylogeneticallyconserved intermediate filament proteins and represent the most abundant proteins in epithelial cells [35,36]. The protein structures of CK consist of a central alpha helical rod domain, flanked on either side by amino terminal (head) domain and carboxy terminal (tail) domain [37]. Cytokeratins undergo several posttranslational modifications, such as phosphorylation, glycosylation, acetylation, and
transglutaminase-induced cross-linking, that may play a role in regulating their function [38]. The primary function of CK is to protecting epithelial cells from mechanical and nonmechanical forms of injury, ultimately preventing cell death [39]. Of note, other functions of CK have been described, including a key role in stress response, cell signaling, and apoptosis [40]. CK expression is the liver is a highly regulated process. Hepatocyte progenitor cells express a broad range of cytokeratins – CK8, CK18, CK19 and (transiently) CK14 [29]. In the adult normal liver, hepatocytes express only CK8 and CK18, while interlobular bile ducts, intraportal and intralobular bile ductules, and the biliary epithelial cells express primarily CK7 and CK19 [29]. Biliary epithelial cells may also express CK8 and CK18 [29]. The functional association of CK8 and CK18 with cytoprotection in hepatocytes is supported by the observation that mutations in CK8 or CK18 are associated with the development of cryptogenic and noncryptogenic forms of liver disease. Interestingly, hepatocytes isolated from CK8- [41] and CK18- [42] null mice lack any keratin filaments. Moreover, livers of CK8-null mice are highly susceptible to liver injury as compared with their heterozygous or wild-type littermates [41]. Intriguingly, adult CK18deficient mice develop mild hepatitis and increased hepatocytes fragility due to spontaneous Mallory-Denk body formation from accumulating CK8 aggregates [42]. Experimental studies have also shown that hepatocytes bearing the CK18 Ser33Ala mutation had a significant inhibition in cell cycle progression [43]. Of note, phosphorylation on serine residues of CK8 and CK18 is increased during mitosis, apoptosis, growth factor stimulation, and different forms of hepatocyte stress [44]. In human immunohistochemical studies, CK8 and CK18 expression have been shown to be useful markers of hepatocyte ballooning associated with the formation of Mallory-Denk bodies in perivenular regions in NASH [45]. As hepatocytes showing macrovesicular or microvesicular steatosis do not exhibit this marked change in CK expression, it has been suggested that immunostaining for CK8 and CK18 may be helpful in distinguishing between simple steatosis and NASH across the spectrum of nonalcoholic fatty liver disease (NAFLD) [46]. Interestingly, aberrant hepatocyte CK7 expression may occur in the portal tracts during the course of autoimmune hepatitis, albeit at a markedly lower extent than in chronic biliary tract disease [47]. 4. Cytokeratins as biomarkers of hepatitis: biochemical assays As CK8 and CK18 are abundantly expressed in the hepatocyte [29], they hold a unique position as markers of hepatic inflammation. CK released from the hepatocyte in circulation after cell death and disintregration can be detected either as partially degraded single protein fragments, as small complexes, or as large polymeric protein complexes [32–34]. The three most frequently used CK which are being evaluated as serum markers for their clinical utility in hepatitis are CK18 and CK18 fragments (M65 and M30 antigens) [31,32], tissue polypeptide antigen (TPA) [35], and tissue polypeptide specific antigen (TPS) [48]. Evidence suggests that dysregulation of hepatocyte apoptosis plays an important role in a wide range of inflammatory disorders of the liver [32,49]. A neoepitope in CK18, termed M30 antigen, becomes available at an early caspase cleavage event during hepatocyte apoptosis and is not detectable in vital or necrotic cells [32]. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30 antigen). An M30-based sandwich ELISA assay determines the circulating levels of M30 antigen, which serves as surrogate serum biomarker of hepatocyte apoptosis [27,31]. By contrast, a specific M65 ELISA assay measures the levels of both caspase-cleaved and intact CK18, the latter of which is released from cells undergoing necrosis [31,32]. During necrotic and apoptotic cell death, CK18derived M65 and M30 antigens are released into the blood in either their intact or their caspase-cleaved forms where they remain relatively stable in the circulation [27]. Both assays have now been largely
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used in the analysis of plasma and serum collected from subjects with different forms of hepatitis for the purpose of assessing their association with different clinical and histological characteristics. Assays for TPA measure an aggregate of nonepidermal cytokines 8, 18, and 19 [35], while assays for TPS are more specific and measure CK18 levels [48]. Actually TPS is considered a particular epitope of the TPA molecule and is considered as more closely related to proliferation of epithelial cells [48]. Compared with apparently healthy individuals, the level of CK markers in the circulation is higher in patients with different forms of hepatitis and it generally rises significantly in relation to the severity of histological lesions. 5. Cytokeratins in viral hepatitis 5.1. Hepatitis B The potential significance of circulating CK levels in HBV infection has been investigated in a pilot study by Papatheodoridis et al. [50]. The authors reported a significant increase in serum M30 antigen levels in patients with chronic hepatitis B (CHB), which was significantly higher in HbeAg-negative subjects than in inactive chronic HBV carriers. The marker was clinically useful for distinguishing between the two groups (area under ROC curve of 0.87), but not for determining the severity of liver histology among HBeAg-negative CHB patients [50]. To confirm and expand these findings, we subsequently measured serum M30 antigen levels in inactive HBV carriers, patients with HBeAg-negative CHB, patients with HBeAg-positive CHB, and healthy controls [26]. The results indicated that serum levels of M30 antigen were higher in both HBeAg-negative and HBeAg-positive CHB patients than in inactive HBV carriers. Interestingly, a cutoff value of 118 U/L for serum M30 antigen had a 70% sensitivity and 80% specificity for discriminating active from inactive carriers. These results suggest the usefulness of M30 antigen testing to identify acute flares of asymptomatic hepatitis in CHB patients [26]. Of interest, serum levels of CK18 fragments have been shown to decrease significantly during oral antiviral therapy in HBeAg-negative CHB in parallel with the improvements in ALT and HBV DNA levels, suggesting their usefulness to monitor treatment response [51]. Recently, Shi et al. [52] have reported that CK18 phosphorylation at Ser33 was colocalized with viral infection in the hepatocytes of inactive HBV carriers, while only basal level of Ser52 phosphorylation was detected in infected cells. These results indicate CK18 phosphorylation may serve as a progression marker for CHB. Taken together, these results suggest that CK play are involved in the molecular mechanisms of hepatitis B progression and are potentially useful as blood-borne biomarkers to distinguish among specific subgroups of patients within this clinical entity. 5.2. Hepatitis C Due to the chronic course of hepatitis C infection, there is a growing need for sensitive and noninvasive biomarkers of early liver injury [1]. Due to their abundant expression in the hepatocyte, CK have attracted considerable attention in this respect. Using site-specific antiphosphokeratin antibodies Toivola et al. [53] found keratin hyperphosphorylation on most CK8 and CK18 sites in liver explants from patients with chronic HCV infection, suggesting its usefulness as a progression marker. Concerning CK as biomarkers, Bantel et al. [54] reported in a pilot study that serum levels of CK18 fragments may be a more sensitive marker compared with aminotransferases for the detection of early liver injury in chronic HCV infection. Our research group subsequently reported a significant positive correlation between serum M30 levels and AST, the grade of steatosis on ultrasound, and the histological steatosis score in patients with chronic HCV infection [27]. Taken together, these results suggest that M30 levels may serve as a noninvasive biomarkers of liver fat accumulation in patients
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with chronic hepatitis C. Papatheodoridis et al. [55] have shown that median CK18 fragment levels were lower in HCV-positive patients with minimal/mild than patients with moderate/severe histologic lesions, offering moderate accuracy for differentiation between the two group. Valva and coworkers [56] measured serum M30 levels in serum from pediatric HCV-infected patients and assessed its utility to predict liver damage progression. The results showed that serum M30 was higher in patients than in controls and correlated both steatosis and severity grade. Different studies have reported an elevation of CK18 using the TPS assay in chronic hepatitis C [57,58], though it is clear that this increase is not a specific feature of HCV infection but simply reflect the degree of cytolysis. Recently, circulating CK fragments have also emerged as a signature of drug-induced liver injury in HCV-infected patients. In this regard, Feldstein and colleagues [59] demonstrated that HCV-796, a hepatitis C polymerase inhibitor approved by the US Food and Drug Administration for a phase 2 study of the treatment of hepatitis C in combination with PEG-Interferon and ribavirin, may cause severe hepatocellular injury and apoptosis in susceptible patients as reflected by a marked increase in serum M30 levels. Intriguingly, Sgier et al. [60] have also shown that successful antiviral therapy in patients with chronic hepatitis C results in a significant decrease in circulating levels of CK18 fragments, arguing for a reduction in hepatocellular apoptosis after clearance of the HCV. 6. Cytokeratins in alcoholic hepatitis Experimental results have suggested an association of cytokeratin hyperphosphorylation with the formation of Mallory bodies in hepatocytes, a hallmark of alcoholic hepatitis [61]. Stumptner et al. [62] have shown that Mallory bodies in human alcoholic hepatitis preferentially contain hyperphosphorylated CK8 and CK18, suggesting that CK8 and CK18 hyperphosphorylation may play a contributing role in MB pathogenesis. The potential usefulness of CK as a biomarker in alcoholic liver disease has been originally suggested by Gonzalez-Quintela et al. [48] who reported that levels of CK18 are frequently increased in alcoholics. The same researchers subsequently replicated the finding of an increased serum level of CK18 in heavy drinkers [63] and suggested a synergism between risky alcohol consumption and obesity in relation to serum concentrations of this molecule, which probably reflect liver disease [64]. Interestingly, Lavallard and coworkers [65] recently showed that CK18 and CK fragments are correlated with severe fibrosis in heavy alcohol drinkers. In addition, CK markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, and hepatic expression of proinflammatory cytokines [65]. 7. Cytokeratins in nonalcoholic steatohepatitis NASH is a clinical entity which histologically simulates alcoholic hepatitis in the absence of alcohol ingestion or intake less than 20 g/day [12,13]. There is enough data to support that NASH is a progressive disease and can lead to cirrhosis of the liver and hepatocellular carcinoma [66]. Even though the pathogenesis of NASH is not fully understood, it may be considered a two hit process [67,68]. The first hit leads to deposition of fat in the liver parenchyma for which insulin resistance has been implicated as the major pathogenetic mechanism. The exact mechanisms promoting progressive hepatic inflammation and liver injury are not well defined, although substrates derived from adipose tissue such as free fatty acid, tumor necrosis factor alpha, leptin, and adiponectin have been implicated [69]. Recent years have witnessed an increased interest in biomarkers in the spectrum of NAFLD (from simple fatty liver to NASH) and the assessment of serum CK and CK fragments is considered a promising approach for the noninvasive diagnosis of metabolic liver inflammation. In a seminal study by Wieckowska et al. [70] demonstrated that caspase-generated CK18 fragments release in NAFLD may serve as an indicator of hepatic inflammation. The increased apoptic rate
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as a consequence of the hepatic inflammatory response in NASH is reflected by an increase in serum CK18 fragments that may therefore distinguish NASH from simple steatosis [28]. These results were further confirmed even in NAFLD patients with normal aminotransferase levels [71]. The increase in CK18 fragments in NASH has been subsequently reported in specific subgroup of patients, including obese individuals [72] and children [73]. CK18 fragments are currently being incorporated in multimarker schemes to improve their diagnostic performances for detecting NASH in a noninvasive manner. Younossi et al. [74] have developed a test (NASH Diagnostics) based on four ELISA assays (cleaved and intact CK18, serum adiponectin, and serum resistin) to form a simple diagnostic biomarker for NASH, which has been recently validated by Feldstein and colleagues [75]. After allowance for potential confounders, multivariable analysis revealed that CK18 fragments remained an independent predictor of NASH. Taken together, these results suggest that CK18 fragments have sufficient-to-excellent diagnostic accuracy for the detection (or exclusion) of NASH across the spectrum of NAFLD. In keeping with these results, Malik and coworkers [76] have recently compared the clinical utility of CK18 to other biomarkers in 95 consecutive patients with NAFLD. CK18 yielded an area under the curve of 0.8 for NASH, and a cutoff value of 300 μ/L gave a positive predictive value of 81% and a negative predictive value of 85%. In addition, Papatheodoridis et al. [55] reported that CK18 fragments levels were lower in healthy subjects than patients with simple fatty liver than patients with NASH, offering excellent diagnostic accuracy for differentiation between the two latter groups. Tan et al. [77] also suggested that higher levels of caspase 3-cleaved fragment of CK18 may indicate the presence of NASH in women with the polycystic ovary syndrome. 8. Cytokeratins in autoimmune hepatitis Autoimmune hepatitis is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components [14]. In a pilot study, Murota and coworkers [78] reported that CK alterations in the hepatocytes in autoimmune hepatitis may increase the susceptibility to apoptosis and induce hepatocyte destruction. Tahiri et al. [79] demonstrated that both CK 8 and 18 represent potential candidate targets on liver membrane for autoantibodies in this clinical entity. In a case report by Inui et al. [80] demonstrated the presence of antibodies against CK8 and CK18 in a patient with de novo autoimmune hepatitis after living-donor liver transplantation. Despite the lack of systematic observations and replication studies, these preliminary findings seem to suggest that changes in CK8 and CK18 in hepatocytes might be one of the sources of pathogenesis of autoimmune hepatitis. 9. Cytokeratins in toxic hepatitis Toxic hepatitis encompasses a spectrum of conditions ranging from mild biochemical abnormalities to acute liver failure. Data on cytokeratins alterations in toxic hepatitis remain scanty. There is at present only one study available reporting the potential involvement of CK in this setting. Fortier et al. [81] have shown that griseofulvininduced toxic liver injury in the rat cause significant changes in keratin solubility in the hepatocyte with a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites was also increased and prominent in the insoluble protein fractions. There is currently a need for human studies of toxic hepatitis to establish the utility of this marker in this clinical entity. 10. Clinical implications of CK fragments as biomarkers in hepatitis The evidence reviewed above clearly suggests that CK play a crucial role in the pathophysiology of hepatic inflammation and serum CK
levels are altered in different forms of hepatitis. In general, measuring CK and CK fragments in serum/plasma provide a dynamic and powerful approach to understanding the spectrum of hepatic inflammation with potential applications in epidemiology, randomized clinical trials, and prognosis. In any case, an important prerequisites for the clinical use of CK as biomarkers for hepatitis include the elucidation of specific indications, the standardization of analytical methods, the characterization of analytical features, the assessment of performance characteristics, the incremental yield of different markers for given clinical indications, and the demonstration of cost-effectiveness [31]. Clearly, CK cannot be used to distinguish between different forms of hepatitis as they are commonly raised independently of the etiology of liver inflammation. This would ultimately lead to a low sensitivity. From a clinical standpoint, characterization of CK and CK fragments in the sera from patients with NAFLD would have value to discriminate between those with NASH from those with simple steatosis. Such a noninvasive liver test may aid clinicians in the selection of patients for liver biopsy and might also allow for noninvasive assessment of liver disease progression and therapeutic response [32,82,83]. In addition, CK may be clinically useful for distinguishing between HbeAg-negative from inactive chronic HBV carriers [26]. 11. Future research directions The previous paragraphs have documented the evidence on the potential clinical usefulness of serum CK levels in different forms of viral and nonviral hepatitis. Considering the availability and quality of evidence on this topic, we have identified several important areas for future studies. One critical direction for future research in the field of chronic viral hepatitis relates on the measurements of CK in patients with coinfection with hepatitis B virus and hepatitis D virus, human immunodeficiency virus (HIV) and HCV, and HBV/HCV/HDV coinfection. The primary concern with coinfection with hepatotropic viruses is that it can lead to more severe liver disease and an increased risk for progression to hepatocellular carcinoma [84,85]. Whether CK may serve as markers of hepatotropic virus coinfection deserves further scrutiny. Concerning subjects with NAFLD, preliminary evidence has suggested the usefulness of serum M30 values for identifying patients with NASH among patients with normal ALT values [86]. Indeed, normal ALT does not represent a valuable criterion to exclude from liver biopsy patients with persistently ultrasonographic evidence of NAFLD [87,88]. Because lifestyle intervention may result in the significant amelioration of histological derangement [89], there is a strong need for identifying individuals among those presenting with risk factors for NAFLD at risk for severe histological liver derangement despite normal ALT values. In this scenario, larger-scale studies are needed to shed more light on the potential usefulness of serum M30 levels as a biomarker of severe NAFLD in the normal ALT population. Another fruitful avenue for future research in the field of CK in NASH would be the study of whether improvements in liver histology following lifestyle interventions (or drug therapy) would be paralleled by a reduction in serum M30 levels (i.e. M30 levels may potentially serve as a “pharmacodynamic” marker in this setting). Hopefully, future studies should also investigate in more detail the interplay between CK and abnormal fatty acid metabolism in the progression of NAFLD [90]. 12. Conclusions Cytokeratin serum markers have the potential to be valuable tools for staging, prognosis, and treatment monitoring of different forms of hepatitis. Their clinical utility has been demonstrated in the spectrum of NAFLD [27,74,75] and to some extent in CHB [26,50]. It is apparent that these markers may also prove useful in identifying drug-induced or toxic liver damage [59]. However, current clinical studies must be completed with a thorough analytical evaluation to determine the
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accuracy, reliability, interpretability, and feasibility of measuring CK in patients with different forms of hepatitis. In this regard, it will be paramount to reference distributions by important variables such as age and sex, the extent of intraindividual variation, and persistence of the biomarkers. Larger clinical and epidemiological studies with a large sample size will be needed to evaluate the analytical features of CK before they can be used as noninvasive markers for prognostication, follow-up of therapy, and detection of recurrence of hepatitis.
Disclosures No conflicts of interest.
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