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Purpose: The liver is known to be protected from steatosis under the influence of high GH/IGF-1. Cytokeratin 18 (CK18) and insulin-like growth factor binding protein 7 (IGFBP7) increase in liver steatosis and fibrosis. The aim of this study was to use quantitative ultrasound techniques and biochemical markers to assess liver steatosis and liver fibrosis in newly diagnosed acromegaly., Methods: This single-center, cross-sectional study included 23 patients with newly diagnosed acromegaly and 46 age, sex, body mass index (BMI) and waist circumference (WC)-matched controls. Liver steatosis was assessed using tissue attenuation imaging (TAI), and stiffness, indicative of fibrosis, was assessed by shear wave elastography (SWE). Serum IGFBP7 and CK18 were studied by ELISA., Results: The acromegaly group had significantly lower liver steatosis (p = 0.006) and higher liver stiffness (p = 0.004), serum IGFBP7 (p = 0.048) and CK18 (p = 0.005) levels than the control group. The presence of fibrosis (p = 0.012) was significantly higher in the acromegaly group than in the control group. Moreover, CK18 was positively correlated with liver stiffness, WC, HOMA-IR, HbA1c, and triglyceride. In the acromegaly group, liver steatosis was negatively correlated with GH level. Stepwise multiple linear regression analysis revealed that BMI (p = 0.008) and CK18 (p = 0.015) were independent risk factors for increased liver stiffness., Conclusion: This study showed that there was an increased presence of liver fibrosis independent of liver steatosis in newly diagnosed acromegaly. Serum CK18 appears to be a potential marker of increased liver fibrosis in acromegaly., (© 2024. The Author(s).)

In this study, we aimed to explore the clinical significance of serum CK18-M65 and CK18-M30 levels in patients with chronic hepatitis B (CHB) complicated by nonalcoholic steatohepatitis (NASH) and liver fibrosis. The observation and control groups comprised 133 patients with CHB complicated by NASH and 50 healthy patients from our hospital, respectively. Liver function indices, including alanine aminotransferase, glutamic aminotransferase, γ-glutamyltransferase, total bilirubin, total protein, and total cholesterol, were determined using an automatic biochemical analyzer. Hyaluronic acid, type III procollagen, type IV collagen, laminin, and CK18-M65 and M30 levels were detected using ELISA. Serum CK18-M65 and M30 levels in patients with CHB complicated by NASH were positively correlated with the liver fibrosis stage (P < .05). While serum CK18-M65 demonstrated a low diagnostic value for liver fibrosis in the S0-1 stage, it exhibited good diagnostic value for S2-3 stage liver fibrosis. Serum CK18-M30 displayed good diagnostic value for S0-1 and S2-3 hepatic fibrosis, particularly for S2-3 hepatic fibrosis. Elevated serum CK18-M65 and CK18-M30 levels in patients with CHB complicated with NASH suggest their potential utility in evaluating the progression of liver fibrosis in this population. In particular, CK18-M30 exhibits superior diagnostic efficiency., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The transition from compensated to decompensated liver cirrhosis is a hallmark of disease progression, however, reliable predictors to assess the risk of decompensation in individual patients from routine diagnostics are lacking. Here, we characterize serum levels of cell death-associated markers and routine biochemistry from patients with chronic liver disease with and without decompensation., Methods: A post-hoc analysis was based on prospectively collected clinical data from 160 patients with chronic liver disease, stably compensated or decompensated at baseline or during follow-up, over a median period of 721 days. Serum levels of damage-associated molecular patterns (DAMPs) and routine biochemistry are quantified at baseline (for all patients) and during follow-up (for patients with acute decompensation). The panel of DAMPs assessed in this study comprises high-mobility group-box protein 1 (HMGB1), cytochrome C (cyt C), soluble Fas-ligand (sFasL), interleukin 6 (IL-6), soluble cytokeratin-18 (CK18-M65) and its caspase-cleaved fragment CK18-M30., Results: In this cohort study, 80 patients (50%) were diagnosed with alcoholic liver cirrhosis, 60 patients (37.5%) with hepatitis C virus- and 20 patients (13.5%) with hepatitis B virus-related liver cirrhosis. At baseline, 17 patients (10.6%) showed decompensated liver disease and another 28 patients (17.5%) developed acute decompensation during follow-up (within 24 months). One hundred fifteen patients showed stable liver disease (71.9%). We found DAMPs significantly elevated in patients with decompensated liver disease versus compensated liver disease. Patients with acute decompensation during follow-up showed higher baseline levels of IL-6, sFasL, CK18-M65 and-M30 (P<0.01) compared to patients with stably compensated liver disease. In multivariate analyses, we found an independent association of baseline serum levels of sFasL (P = 0.02; OR = 2.67) and gamma-glutamyl transferase (GGT) (P<0.001; OR = 2.1) with acute decompensation. Accuracy of the marker combination for predicting acute decompensation was high (AUC = 0.79). Elevated aminotransferase levels did not correlate with decompensated liver disease and acute decompensation., Conclusions: DAMPs are elevated in patients with decompensated liver disease and patients developing acute decompensation. The prognostic value of a marker combination with soluble Fas-ligand and GGT in patients with liver cirrhosis should be further evaluated., Competing Interests: The authors have declared that no competing interests exist.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP-protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well-defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2-week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in-person at study initiation (day 1) and on days 3 and 5. APAP-protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full-length keratin 18 [K18], and total high-mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP-protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP-protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short-term administration of low-dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Objectives: Nonalcoholic steatohepatitis (NASH) and fibrosis play critical roles for the prognosis of patients with nonalcoholic fatty liver disease (NAFLD). Identification of patients at risk of NASH and fibrosis is therefore critical for disease management. NAFLD Fibrosis Score (NFS) and transient elastography (TE) have been suggested to exclude advanced fibrosis. However, there is increasing evidence that also patients with NASH and early fibrosis are at risk of disease progression and complications, emphasizing the need for improved noninvasive risk stratification in NAFLD., Methods: Because hepatocyte apoptosis plays an early role in NASH pathogenesis, we evaluated whether the apoptosis biomarker M30 might identify NAFLD patients who are at risk of NASH and fibrosis despite low NFS or TE values. Serum M30 levels were assessed by enzyme-linked immunosorbent assay in combination with NFS and/or TE in an exploration (n = 103) and validation (n = 100) cohort of patients with biopsy-proven NAFLD., Results: Most patients with low NFS (cutoff value < -1.455) revealed increased M30 levels (>200 U/L) in the exploration (62%) and validation (67%) cohort, and more than 70% of them had NASH, mostly with histological fibrosis. Vice versa, most patients with NFS < -1.455 but nonelevated M30 levels showed no NASH. NASH was also detected in most patients with indeterminate NFS (-1.455 to 0.676) but elevated M30 levels, from which ∼90% showed fibrosis. Similar results were obtained when using TE instead of NFS., Discussion: The combination of the M30 biomarker with NFS or TE enables a more reliable identification of patients with an increased risk of progressed NAFLD and improves patient stratification.

Introduction: MACK-3 (combination of hoMa, Ast and CK18) was reported to be a good biomarker for the diagnosis of fibrotic non-alcoholic steatohepatitis (NASH). However, there is no external validation to date., Aim: To evaluate the accuracy of MACK-3 for the diagnosis of fibrotic NASH., Methodology: Consecutive adult non-alcoholic fatty liver disease (NAFLD) patients who had liver biopsy in a university hospital were included. MACK-3 was calculated using the online calculator using the following variables: fasting glucose, fasting insulin, aspartate aminotransferase (AST) and cytokeratin 18 (CK18). MACK-3 cut-offs ≤0.134 and ≥0.550 were used to predict absence and presence of fibrotic NASH, respectively. Histopathological examination of liver biopsy specimen was reported according to the NASH Clinical Research Network Scoring System., Results: Data for 196 subjects were analysed. MACK-3 was good for diagnosis of fibrotic NASH (area under receiver-operating characteristics curve [AUROC] 0.80), comparable to the Fibrosis-4 index (FIB4) and the NAFLD fibrosis score (NFS) and superior to the BARD score and CK18. MACK-3 was good for diagnosis of active NASH (AUROC 0.81) and was superior to other blood fibrosis tests. The overall accuracy, percentage of subjects in grey zone, sensitivity, specificity, positive predictive value and negative predictive value of MACK-3 for diagnosis of fibrotic NASH was 79.1%, 46.9%, 100%, 43.8%, 43.1% and 100%, respectively, while for diagnosis of active NASH was 90.0%, 39.3%, 84.2%, 81.4%, 88.9% and 74.5%, respectively., Conclusion: MACK-3 is promising as a non-invasive test for active NASH and fibrotic NASH and may be useful to identify patients who need more aggressive intervention., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Extensive hepatocyte death leads to hepatic inflammation and contributes to systemic inflammation in decompensated cirrhosis. We aimed to investigate the prognostic value of serum cell death markers in patients with hepatitis B virus (HBV)-related acute decompensation (AD) of cirrhosis with and without acute-on-chronic liver failure (ACLF). We studied two cohorts-cohort 1: 201 outpatients with stable chronic hepatitis B (49 cirrhosis); cohort 2: 232 inpatients with HBV-related cirrhosis admitted for AD. Cell death was determined with serum keratin-18 (K18) for total death and serum caspase-cleaved-K18 (cK18) for apoptosis. Survival analyses were performed using competing risk method. We found that serum K18 and cK18 were significantly (P < 0.001) higher in patients from cohort 2 than those from cohort 1. Among cohort 2, ACLF patients had significantly (P < 0.001) increased K18 and cK18 comparing to those without ACLF. Increased K18 and cK18 were mainly attributed to HBV flare and were associated with liver and coagulation failure. HBV-AD patients without ACLF who admitted with upper tertile of K18 or cK18 were at higher risk of developing ACLF during follow-up. Baseline serum K18 or cK18 was significantly associated with transplant-free 90-day survival independent of leucocytes, HBV DNA, bacterial infection, encephalopathy and severity scores. The combination of cell death biomarkers significantly improved the prognostic value of the currently established prognostic scores. The reduction of cell death level after standard treatment was associated with increased short-term survival. In conclusion, measurements of serum K18 or cK18 in HBV decompensated cirrhosis are a promising tool for predicting ACLF and risk stratification of short-term outcome., (© 2019 John Wiley & Sons Ltd.)

Background: Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common cause of chronic liver disease worldwide. Multiple diagnostic noninvasive methods for NAFLD were studied (both serological and imaging), either single or combined. Attention has been focused on cytokeratin-18 (CK18) as a novel serological marker for the diagnosis of steatosis/fibrosis in NAFLD and hepatitis C virus (HCV) patients., Aim: The aim of this study was to evaluate serum CK18 in NAFLD and HCV fibrosis/steatosis and also to correlate its performance with the diagnostic accuracy of transient elastography (TE) and controlled attenuation parameter (CAP) in the diagnosis of fibrosis/steatosis in these patients., Patients and Methods: Three equal groups of participants were enrolled (n=135): group I included patients with chronic HCV, group II included NAFLD patients, and group III included control participants. For all groups, TE/CAP and labs including serum CK18 were performed. Liver biopsy was performed for the NAFLD group., Results: Serum CK18 was significantly higher in the NAFLD group (19.01±3.49 ng/ml) versus the HCV group (8.95±1.06 ng/ml) and the control group (4.83±1.6 ng/ml) (P<0.001). The CK18 levels in biopsy stages (steatosis, ballooning, inflammation, and fibrosis) and FibroScan/CAP degrees showed that CK18 increased significantly with steatosis and fibrosis stages (biopsy or FibroScan/CAP), but did not reach significance with ballooning or inflammation grades. CK18 was significantly different in nonalcoholic steatohepatitis versus non-nonalcoholic steatohepatitis patients (P=0.041). The best CK18 cutoff to detect steatosis (S≥2) in NAFLD and HCV was 11.65 and 6.84 ng/ml, respectively with an overall sensitivity and specificity over 97%. The CK18 cutoff for significant fibrosis (F≥2) by FibroScan in the NAFLD/HCV groups was 9.115 ng/ml, with 62.5%/69.2% sensitivity/specificity (P=0.031). However, inflammation had a cutoff with a marginal P value (P=0.080), and a reliable cutoff for ballooning was not attained (P=0.386). There was a positive correlation between CK18 and fibrosis (by FibroScan) in the NAFLD and HCV groups (P<0.05). The correlation between CK18 and steatosis in CAP and the nonalcoholic fatty liver disease activity score was very good (P<0.001)., Conclusion: Serum CK18 is related strongly to the development/progression of NAFLD and HCV-related fibrosis/steatosis. TE was correlated highly with liver biopsy results. The combination of CK18 with other noninvasive modalities increases the diagnostic yield of these tests.

Background & Aims: Caspase-mediated apoptosis and inflammation contribute to progression of liver disease. Emricasan is a pan-caspase inhibitor that reduced serum markers of apoptosis and liver inflammation in patients with hepatitis C and non-alcoholic steatohepatitis (NASH)., Methods: We performed a multicenter study of 86 patients with cirrhosis (Child-Pugh class A or B; mean score, 6.9; 38% with alcohol-associated cirrhosis, 29% with HCV-associated cirrhosis, and 23% with NASH) and model for end-stage liver disease (MELD) scores of 11-18 (mean, 12.8). Patients were randomly assigned to groups given placebo (N = 42) or Emricasan (25 mg, N = 44), twice daily for 3 months; subjects then received open-label Emricasan (25 mg) twice-daily for 3 months. The primary endpoint was the change from baseline in serum levels of cleaved keratin 18 (CK-18) at month 3., Results: Seventy-four patients completed the 3-month study period (40 given Emricasan and 34 given placebo); 69 patients received open-label Emricasan for 3 months afterward. At the 3-month timepoint, Emricasan significantly reduced mean MELD (P = .003) and Child-Pugh (P = .003) scores in subjects with high MELD scores (15 or more), compared with placebo, with significant reductions in INR (95% CI, -0.2882 to -0.0866) and total bilirubin (95% CI, -1.5069 to -0.0823) vs placebo. There were no significant differences between Emricasan and placebo groups in mean MELD (P = .466) or Child-Pugh (P = .124) scores overall at 3 months compared to placebo. Of patients with high MELD scores, 6/9 given Emricasan (67%) had a reduction of 2 points or more at month 3, compared with 2/10 given placebo (20%). Serum levels of full-length CK-18 (P = .02) and caspase 3/7 (P < .001), but not cleaved CK-18 (P = .092), decreased significantly at 3 months in the Emricasan vs placebo group. Emricasan was well tolerated, and adverse events were balanced between groups. Emricasan's effects were generally maintained or increased after 6 months of treatment., Conclusions: In a randomized trial of patients with cirrhosis, we found 3 months treatment with Emricasan to improve liver function, compared with placebo, reducing MELD and Child-Pugh scores, INR, and total bilirubin in patients with MELD scores ≥15. ClinicalTrials.gov no: NCT02230670., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aims: Liver biopsy is currently the gold standard test for NAFLD diagnosis and staging but has many drawbacks. In addition, other tools such as transient elastography are limited to specialized research centers. To assess the usefulness of CK-18 as a non-invasive biomarker for detecting therapeutic responses in patients with liver fibrosis., Materials and Methods: Sixty overweight and obese patients with liver fibrosis were evaluated by a dietitian and given a weight-reducing diet with a calorie deficit of 500-1000 kcal/day over a 6-month period. Controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) both were performed at the beginning and at the end of the trial to determine liver steatosis and liver fibrosis, respectively. Serum CK-18 levels were measured by enzyme linked immune sorbent assay (ELISA) at baseline and at 3 and 6 months after intervention., Results: Patients experienced a rapid weight loss of -7.6 kg (8.5%) during the trial. Among all participants, liver steatosis decreased from 76.5 ± 12.2% to 51.8 ± 24.4% (baseline to end-point) (p < 0.001) and fibrosis score decreased from 9.9 ± 3.7 to 7.2 ± 2.4 (p < 0.001) (a 27.2% decrease). Serum CK-18 levels decreased from 290.2 ± 98.1 U/L to 217.6 ± 64.8 U/L (p < 0.001) (a 25.0% decrease). ΔCK-18 was found to be significantly associated with delta fibrosis score (r = 0.25, p = 0.05) CONCLUSIONS: This trial showed a significant positive association between changes in CK-18 levels and changes in liver fibrosis over a 6-month dietary intervention., (Copyright © 2019. Published by Elsevier Ltd.)

Background/aims: Noninvasive markers of liver fibrosis in alcoholic liver disease (ALD) are crucial to establish early intervention. Previous studies have suggested that plasma levels of cleaved keratin-18 (K18; M30) fragments can predict the severity of liver disease. The aim of this study was to correlate plasma M30 levels with stages of liver fibrosis in ALD., Methods: Patients with ALD (n=139, 79.1% males) and liver histology were included, and plasma samples were collected to quantify plasma M30 levels. Patients were stratified into five groups by fibrosis stage (F0=14; F1=15; F2=35; F3=17; and F4=58) according to the Kleiner score. Differences between groups were evaluated using the chi-square test or analysis of variance. Trends by fibrosis stage were calculated by logistic regression analysis, and sensitivity, specificity and positive and negative predictive values were determined., Results: There were no significant differences in M30 levels among fibrosis stages. The correlation between plasma M30 levels and fibrosis was poor (Pearson's correlation coefficient= 0.13, Spearman rho=0.20 [p=0.02]), and M30 levels did not correlate with alcohol-specific histological features. However, significant correlations of M30 levels with aspartate aminotransferase (Spearman rho=0.653, p<0.001) and alanine aminotransferase (spearman rho=0.432, p<0.001) were found. m30 levels of>200 U/L reveal a sensitivity for predicting cirrhosis of 84.5% with a negative predictive value of 73.5%., Conclusions: Plasma M30 levels are often elevated in ALD and correlate with serum transaminases but do not reflect fibrosis. The usefulness as a prognostic marker awaits evaluation in prospective studies.

Background & Aims: The non-invasive identification of steatohepatitis (NASH) in patients with Non-Alcoholic Fatty Liver Disease is an unmet need in clinical practice. Index of NASH (ION) is a new tool for the prediction of NASH. We aimed to externally validate ION and to compare it with CK-18. Since necroinflammation precedes fibrosis, we also tested ION in combination with non-invasive tools for fibrosis., Methods: We analysed data from 292 Italian patients (169 Southern cohort, and 123 Northern cohort) with an histological diagnosis of NAFLD. The ION, FIB-4 and NFS scores were calculated according to published algorithms. Serum cytokeratin18-Aspartate396 levels and liver stiffness (LS) by Fibroscan were assessed within three months from liver biopsy., Results: The diagnostic accuracy of ION for the identification of NASH was not as satisfactory as reported (area under the ROC curve, AUROC = 0.687 [95% CI = 0.62-0.75]). The proposed cut-off value ≥50 showed a poor sensitivity (Se) (28%) and a good specificity (Sp) (92%), with a positive predictive value (PPV) of 91% and a negative predictive value (NPV) of 30%. A new cut-off value >26 improved Se (73%) but decreased Sp (60%) (PPV of 84% and a NPV of 43%). ION performed slightly better in obese NAFLD (AUROC = 0.700). The combination of ION and markers of fibrosis did not improve the identification of advanced liver disease., Conclusions: ION is not feasible for the non-invasive diagnosis of NASH across different populations of NAFLD patients, mainly because its limited reproducibility in non-obese subjects., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aims of this study were to determine the role of cell death in patients with cirrhosis and acute decompensation (AD) and acute on chronic liver failure (ACLF) using plasma-based biomarkers. The patients studied were part of the CANONIC (CLIF Acute-on-Chronic Liver Failure in Cirrhosis) study (N = 337; AD, 258; ACLF, 79); additional cohorts included healthy volunteers, stable patients with cirrhosis, and a group of 16 AD patients for histological studies. Caspase-cleaved keratin 18 (cK18) and keratin 18 (K18), which reflect apoptotic and total cell death, respectively, and cK18:K18 ratio (apoptotic index) were measured in plasma by enzyme-linked immunosorbent assay. The concentrations of cK18 and K18 increased and the cK18:K18 ratio decreased with increasing severity of AD and ACLF (P < 0.001, respectively). Alcohol etiology, no previous decompensation, and alcohol abuse were associated with increased cell death markers whereas underlying infection was not. Close correlation was observed between the cell death markers and, markers of systemic inflammation, hepatic failure, alanine aminotransferase, and bilirubin, but not with markers of extrahepatic organ injury. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining confirmed evidence of greater hepatic cell death in patients with ACLF as opposed to AD. Inclusion of cK18 and K18 improved the performance of the CLIF-C AD score in prediction of progression from AD to ACLF (P < 0.05)., Conclusion: Cell death, likely hepatic, is an important feature of AD and ACLF and its magnitude correlates with clinical severity. Nonapoptotic forms of cell death predominate with increasing severity of AD and ACLF. The data suggests that ACLF is a heterogeneous entity and shows that the importance of cell death in its pathophysiology is dependent on predisposing factors, precipitating illness, response to injury, and type of organ failure. (Hepatology 2018;67:989-1002)., (© 2017 by the American Association for the Study of Liver Diseases.)

Data on cytokeratin-18 (K-18) and enhanced liver fibrosis (ELF) score in insulin-treated diabetes patients with non-alcoholic fatty liver disease (NAFLD) are limited. This study analyzed phase III data comparing basal insulin peglispro (BIL) and insulin glargine in type 1 (T1D), and type 2 diabetes (T2D) (insulin-naïve and insulin-treated). Alanine aminotransferase (ALT), K-18, ELF scores and liver fat content (LFC), measured by MRI, were obtained longitudinally. Baseline K-18 (U/L) was higher in T2D (range: 207‒247) than T1D (range: 148‒183), correlated with ALT in all populations (r (range) 0.264‒0.637, p<0.05), but with LFC only in T2D (r (range) 0.474‒0.586, p<0.05). K-18 increased significantly from baseline in BIL-treated, but not glargine-treated patients. Change from baseline (CFB) K-18 was significantly correlated with CFB in ALT in BIL-treated T2D populations. Baseline ELF scores were higher in T2D (range: 9.12‒9.20) than T1D (range: 8.24‒8.36), correlated with ALT in T1D only (0.209, p<0.05), and not correlated with LFC in any population. ELF scores increased significantly from baseline in BIL-treated but not glargine-treated patients. There were no correlations between CFB in LFC and ELF score at week 52 in any treatment group/population. In all BIL-treated populations, CFB in ALT and CFB in ELF score at week 52 were positively correlated. These data characterize associations of K-18 and ELF score with ALT and LFC in insulin-treated patients with T1D and T2D. Hepatopreferential insulins may be associated with increased K-18 and ELF scores but mechanisms and clinical significance are unknown. ClinicalTrials.gov identifiers are NCT01481779, NCT01435616, NCT01454284 and NCT01582451., Competing Interests: Competing interests: AS has stock options in Genfit, is President of Sanyal Biotechnologies, has served as a consultant to Merck, Eli Lilly and Company, Bristol Myers, Novartis, AbbVie, AstraZeneca, Gilead, Intercept, Genfit, Zafgen, Enanta, Immuron, Galmed, Nitto Denko, Durect, Ikaria, Echosens and Salix. His institution receives grant support from Intercept, Merck, AstraZeneca, Bristol Myers and Gilead. EJB was an employee and shareholder of Eli Lilly and Company during the trials and results analyses; he is currently a consultant to Viacyte. MLH, SZ, JMB-V, AMC, AH, SJJ, RJK, QZ and BJH are employees and stockholders of Eli Lilly and Company., (© American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The incidence and prevalence of nonalcoholic fatty liver disease (NAFLD) are increasing and identification of people at risk of disease progression is extremely important. The current gold standard for diagnosing NAFLD/nonalcoholic steatohepatitis (NASH) is by liver biopsy, but it has several limitations. Noninvasive tests via biomarkers and transient elastography to assess NAFLD/NASH are being used in clinical practice. The most validated diagnostic panels include the NAFLD fibrosis score, FIB-4 (Fibrosis-4), and FibroMeter. Transient elastography is very useful in evaluating advanced fibrosis and cirrhosis., (Published by Elsevier Inc.)

Cell death markers, M65 and M30, have been suggested to be sensitive markers of liver inflammation and fibrosis in nonalcoholic fatty liver disease and chronic hepatitis C. Our aim was to investigate whether these markers were useful in diagnosing liver inflammation and fibrosis in chronic hepatitis B (CHB).We examined 186 patients with CHB; 18 sex- and age-matched healthy subjects were controls. The blood samples were collected from CHB patients within 1 week before or after liver biopsy. According to METAVIR score system, liver inflammation was graded from A0 to A3, and fibrosis from F0 to F4.Serum M65 and M30 levels were in parallel with the grades of liver inflammation. M65, not M30, increased significantly in patients with severe inflammation and normal alanine aminotransferase. M65 is one of the independent predictors of severe liver inflammation (≥A2). The levels of M65 and M30 levels significantly increased in parallel with the degree of inflammation in F1 patients, whereas they showed no statistical difference between different stages of fibrosis in A1 patients.Serum M65 is a useful indicator of liver inflammation in CHB patients. Serum M65, not M30, is valuable in the grading of liver fibrosis.

Background & Aims: Macrophage activation plays a key pathogenic role in experimental non-alcoholic fatty liver disease (NAFLD) and contributes to the progression of steatohepatitis (NASH) and fibrosis. We studied macrophage activation in human NAFLD by measuring soluble (s)CD163, a specific macrophage activation marker, hypothesizing that sCD163 would be associated with the patients' morphological disease grade. Furthermore, we investigated an association between sCD163 and the apoptosis marker cytokeratin-18 (CK-18) to explore a link between macrophage activation and apoptosis., Methods: sCD163 associations with biochemical and histological measures of NAFLD were investigated in two independent cohorts of 157 Australian and 174 Italian NAFLD patients, with liver biopsies graded for NAFLD severity, steatosis and fibrosis. sCD163 and CK-18 were measured by enzyme-linked immunosorbent assay., Results: In both cohorts sCD163 increased in parallel with the patients' morphological disease grading, being independently associated with the Kleiner fibrosis score (P < 0.001). A high sCD163 predicted advanced fibrosis {F ≥ 3; Australian cohort: area under receiver-operating characteristics curve 0.77 [95% confidence interval (CI): 0.76-0.87], Italian cohort: 0.80 (95% CI: 0.72-0.88)}. In both groups, sCD163 was independently associated with CK-18 (P < 0.001)., Conclusion: Soluble CD163 reflecting macrophage activation is associated with morphological features of NAFLD suggesting their involvement in the pathogenesis of NAFLD, NASH and particularly fibrosis. An independent association between sCD163 and cytokeratin-18 suggests that apoptosis may contribute to macrophage activation in NAFLD/NASH., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Hepatocyte death is an important factor in development and progression of cirrhosis. Cytokeratin 18-based serum markers reflecting apoptotic (M30) and overall epithelial cell death (M65 and M65EpiDeath) have been used as prognostic parameters for survival in patients with acute liver failure. However, there has been no trial investigating M30, M65 and M65EpiDeath as survival parameters in patients with cirrhosis and acute-on-chronic liver failure., Methods: Patients with cirrhosis were enrolled and followed until death, liver transplantation or last contact. M30, M65 and M65EpiDeath serum levels were quantified in patient's sera., Results: Three hundred and thirty-one patients were screened and 211 patients could be included in this study. The median duration of follow-up was 322 days with a range of 1-1382 days. All three cell death parameters correlated with the extent of the severity of the disease. However, M65EpiDeath was the only of the three parameters which was associated with the severe complications of cirrhosis including ascites, spontaneous bacterial peritonitis and hepatorenal syndrome. Additionally, M65EpiDeath was the only cell death parameter which was independently from liver function and its surrogate parameter such as Child-Pugh score and the model of end-stage liver disease associated with overall survival., Conclusions: Epithelial cell death reflected by M65EpiDeath serum levels is an indicator for the severity of cirrhosis and a prognostic survival parameter in cirrhotic patients., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: A major challenge in the management of nonalcoholic fatty liver disease (NAFLD) is to identify patients with nonalcoholic steatohepatitis (NASH) and early liver fibrosis. The progression of NAFLD is accompanied by distinctive changes in very low density lipoprotein (VLDL), a lipoprotein particle produced exclusively in the liver. Herein, we sought to determine the characteristics of VLDL profiles associated with NASH and liver fibrosis., Methods: We evaluated VLDL profiles of 128 patients from a single centre NAFLD registry, and examined VLDL size, total and subclass VLDL concentrations in relation to NAFLD activity score (NAS), steatohepatitis and liver fibrosis as determined by liver biopsy., Results: A near linear relationship was observed between mean VLDL particle size and NAFLD activity score (NAS). In multivariate models, VLDL particle size was significantly associated with both NAS and NASH, after adjustment for BMI and diabetes. A decrease in small VLDL particle concentration was associated with more advanced liver fibrosis. In receiver operative characteristic analyses, mean VLDL size performed similarly to cytokeratin 18 in predicting NASH, whereas small VLDL particle concentration had similar performance to NAFLD fibrosis score in predicting stage 2 or above liver fibrosis., Conclusions: The increase in mean VLDL size in NASH and decrease in small VLDL particle concentration in liver fibrosis likely reflect changes in the number and state of hepatocytes associated with NASH and fibrosis. In addition to its value in risk stratification of cardiovascular diseases, circulating VLDL profile may provide information for the staging of NAFLD disease severity., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the obesity epidemic and affects approximately 10% of children in the US. The presence of hepatic fibrosis may be the most important factor in determining the prognosis of NAFLD. Noninvasive methods to identify the presence of fibrosis in children with NAFLD are greatly needed. Hepatocyte apoptosis activates hepatic stellate cells and plays a central role in fibrosis progression in NAFLD. The aim of the present study was to evaluate the use of plasma cytokeratin-18 (CK18) fragment levels, a marker of hepatocyte apoptosis, as a noninvasive biomarker in detecting liver fibrosis in pediatric NAFLD., Methods: Consecutive children with biopsy-proven NAFLD were included and blood samples and anthropometric measurements were collected at the time of the biopsy. NAFLD activity score was calculated (0-8) and fibrosis stage was scored (0-4). We measured plasma CK18 levels using the M30-Apoptosense enzyme-linked immunosorbent assay kit., Results: A total of 201 subjects were enrolled in the study. The mean age was 10.7 ± 2.5 years, and 37 % were boys. Sixty-eight percent of the patients had any fibrosis, with 56% having F1, 6% having F2, and 6 % having F3. CK18 levels were found to be significantly higher in subjects with any fibrosis compared with those without fibrosis (304.6 ± 124.8 vs 210.4 ± 70.9, P < 0.001). CK18 level revealed good accuracy for prediction of any fibrosis (F1-F3) with AUROC of 0.75. Multivariate logistic regression was performed to assess whether CK18 in combination with another clinical factor could improve accuracy of prediction of fibrosis. Together, CK18 with waist circumference percentile generated an area under the receiver operating characteristics curve of 0.842 for prediction of any fibrosis., Conclusions: CK18 is a promising noninvasive biomarker for fibrosis in NAFLD in children. A fibrosis prediction model that includes CK18 and waist circumference percentile should be validated in other populations.

Background: Little is known about the importance of liver fibrosis and fatty liver in HIV-monoinfected individuals without hepatitis virus co-infection, particularly among the Asian population., Aim: To evaluate prevalence and risk factors for liver fibrosis and fatty liver in Asian HIV-monoinfected individuals., Methods: Eighty asymptomatic HIV-monoinfected individuals (tested negative for HBV/HCV) were compared with 160 matched HIV-uninfected healthy controls. Transient elastography and proton-magnetic resonance spectroscopy ((1) H-MRS) were performed to measure liver stiffness and hepatic steatosis respectively. Blood samples were analysed for metabolic profiles and markers of steatohepatitis (e.g. cytokeratin-18)., Results: All HIV-infected individuals (mean ± s.d. age 54 ± 11 years, male 93%, Chinese 94%; diagnosis median duration 8 (IQR 4-13 years) were stable on anti-retrovirals (PI-based 58.7%, NNRTI-based 25.0% integrase-inhibitors 16.3%); diabetes, dyslipidaemia, and metabolic syndrome were common. Fatty liver disease was detected in 28.7%. There was significantly higher degree of liver stiffness [4.9 (IQR 4.1-6.2) kPa vs. 4.2 (IQR 3.6-5.0) kPa, P < 0.001], and greater proportions developed significant fibrosis (7.0 kPa, 14.3% vs. 3.1%, P = 0.001) and cirrhosis (10.3 kPa, 5.2% vs. 0.6%, P = 0.040) compared with controls. HIV infection was an independent risk factor for significant fibrosis (adjusted OR 4.00, 95% CI 1.29-12.41, P = 0.016). HIV-infected individuals with fatty liver had excessive liver stiffness and fibrosis. Two cases of asymptomatic hepatocellular carcinoma were detected., Conclusions: HIV-monoinfected patients are at risk for liver fibrosis and cirrhosis. HIV-related mechanisms and fatty liver disease may play important roles. Screening and intervention to prevent severe outcomes should be considered., (© 2016 John Wiley & Sons Ltd.)

Background: The combination of non-invasive markers for the detection of fibrosis in patients with chronic liver diseases is still a matter of debate., Aims: To test the performance of cytokeratin18-Aspartate396 alone or in combination with transient elastography as a marker of fibrosis, compared to liver biopsy as gold standard., Methods: In 259 prospectively enrolled patients with chronic liver diseases, clinical, biochemical, and histological features were assessed. Serum cytokeratin18-Aspartate396 and Fibroscan were performed within 6 months prior to liver biopsy., Results: Cytokeratin18-Aspartate396 levels predicted both significant and advanced fibrosis in non-alcoholic fatty liver disease group, correctly identifying 83.7% and 80.8% of cases, respectively. Liver stiffness performed best in predicting severe fibrosis in patients with chronic viral infection, correctly identifying 78.7% of chronic hepatitis B and 88.6% of chronic hepatitis C subjects. The combination of cytokeratin18-Aspartate396 and liver stiffness improved their diagnostic performance for the detection of significant and advanced fibrosis in non-alcoholic fatty liver disease group, only (sensitivity=78.3%, specificity=90.7%; sensitivity=91.7%, specificity=71.6%, respectively)., Conclusion: Cytokeratin18-Aspartate396 and liver stiffness can improve the non-invasive prediction of significant and advanced fibrosis in patients with non-alcoholic fatty liver disease, while in hepatitis B and C virus infected patients their combined use had no advantage over the diagnostic accuracy of transient elastography alone., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Intrahepatic expression of dipeptidyl peptidase-4 (DPP4), and circulating DPP4 (cDPP4) levels and its enzymatic activity, are increased in non-alcoholic fatty liver disease (NAFLD) and in type 2 diabetes mellitus and/or obesity. DPP4 has been implicated as a causative factor in NAFLD progression but few studies have examined associations between cDPP4 activity and NAFLD severity in humans. This study aimed to examine the relationship of cDPP4 activity with measures of liver disease severity in NAFLD in subjects with diabetes and/or obesity., Methods: cDPP4 was measured in 106 individuals with type 2 diabetes who had transient elastography (Cohort 1) and 145 individuals with morbid obesity who had liver biopsy (Cohort 2). Both cohorts had caspase-cleaved keratin-18 (ccK18) measured as a marker of apoptosis., Results: Natural log increases in cDPP4 activity were associated with increasing quartiles of ccK18 (Cohorts 1 and 2) and with median liver stiffness ≥10.3 kPa (Cohort 1) and significant fibrosis (F ≥ 2) on liver biopsy (Cohort 2)., Conclusions: In diabetes and/or obesity, cDPP4 activity is associated with current apoptosis and liver fibrosis. Given the pathogenic mechanisms by which DPP4 may progress NAFLD, measurement of cDPP4 activity may have utility to predict disease progression and DPP4 inhibition may improve liver histology over time., (© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

The development of simple, noninvasive markers of liver fibrosis is urgently needed for primary biliary cirrhosis (PBC). This study examined the ability of several serum biomarkers of cell death to estimate fibrosis and prognosis in PBC. A cohort of 130 patients with biopsy-proven PBC and 90 healthy subjects were enrolled. We assessed the utility of the M30 ELISA, which detects caspase-cleaved cytokeratin-18 (CK-18) fragments and is representative of apoptotic cell death, as well as the M65 and newly developed M65 Epideath (M65ED) ELISAs, which detect total CK-18 as indicators of overall cell death, in predicting clinically relevant fibrosis stage. All 3 cell death biomarkers were significantly higher in patients with PBC than in healthy controls and were significantly correlated with fibrosis stage. The areas under the receiver operating characteristic curve for the M65 and M65ED assays for differentiation among significant fibrosis, severe fibrosis, and cirrhosis were 0.66 and 0.76, 0.66 and 0.73, and 0.74 and 0.82, respectively. In multivariate analysis, high M65ED (hazard ratio 6.13; 95% confidence interval 1.18-31.69; P = 0.031) and severe fibrosis (hazard ratio 7.45; 95% confidence interval 1.82-30.51; P = 0.005) were independently associated with liver-related death, transplantation, or decompensation. High serum M65ED was also significantly associated with poor outcome in PBC (log-rank test; P = 0.001). Noninvasive cell death biomarkers appear to be clinically useful in predicting fibrosis in PBC. Moreover, the M65ED assay may represent a new surrogate marker of adverse disease outcome.

Background: Transient elastography (TE) has recently emerged as an accurate noninvasive imaging method for the diagnosis of liver fibrosis in nonalcoholic fatty liver disease (NAFLD). Here, we tested whether adding serum measures of serum procollagen-III peptide (PIIIP) and caspase-cleaved cytokeratin 18 fragment (M30) to TE could improve its diagnostic accuracy in patients with biopsy-proven NAFLD., Patients and Methods: TE was performed in 87 patients with NAFLD. Serum PIIIP and M30 levels were determined by enzyme-linked immunosorbent assay. Liver histology was considered the gold standard. The diagnostic accuracies were assessed by measuring the area under the receiver operating curve., Results: At histopathological examination, 34 patients (39.1%) had significant fibrosis (F2-F4) and 19 patients (21.8%) had advanced fibrosis (F3-F4). Both TE and serum M30 levels were independent predictors of fibrosis, whereas no association was found with PIIIP. No significant differences in terms of sensitivity and specificity for both significant and advanced fibrosis were evident for TE, serum M30, or their combination. Moreover, the area under the receiver operating curves of serum M30 combined with TE did not differ significantly from those of either test alone., Conclusion: Both TE and serum M30 levels are accurate for the noninvasive diagnosis of fibrosis in NAFLD. However, their combination did not improve the overall diagnostic accuracy.

Background & Aims: Liver biopsy is the only reliable way of diagnosing and staging NASH but its invasive nature limits its use. Plasma caspase-generated cytokeratin-18 fragments (CK-18) have been proposed as a non-invasive alternative. We studied its clinical value in a large multiethnic NAFLD population and examined its relationship to clinical/metabolic/histological parameters., Methods: 424 middle-aged subjects in whom we measured adipose tissue, liver and muscle insulin resistance (IR), liver fat by MRS (n=275) and histology (n=318)., Results: Median CK-18 were elevated in patients with vs. without NAFLD by MRS (209 [IQR: 137-329] vs. 122 [IQR: 98-155]U/L) or with vs. without NASH (232 [IQR: 151-387] vs. 170 [IQR: 135-234]U/L, both p<0.001). Plasma CK-18 raised significantly with any increase in steatosis, inflammation and fibrosis, but there was a significant overlap across disease severity. The CK-18 AUROC to predict NAFLD, NASH or fibrosis were 0.77 (95% CI=0.71-0.84), 0.65 (95% CI=0.59-0.71) and 0.68 (95% CI=0.61-0.75), respectively. The overall sensitivity/specificity for NAFLD, NASH and fibrosis were 63% (57-70%)/83% (69-92%), 58% (51-65%)/68% (59-76%) and 54% (44-63%)/85% (75-92%), respectively. CK-18 correlated most strongly with ALT (r=0.57, p<0.0001) and adipose tissue IR (insulin-suppression of FFA: r=-0.43; p<0.001), less with steatosis, lobular inflammation and fibrosis (r=0.28-0.34, all p<0.001), but not with ballooning, BMI, metabolic syndrome or T2DM., Conclusions: Plasma CK-18 has a high specificity for NAFLD and fibrosis, but its limited sensitivity makes it inadequate as a screening test for staging NASH. Whether combined as a diagnostic panel with other biomarkers or clinical/laboratory tests may prove useful requires further study., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Liver biopsy represents the gold standard for evaluating damage and progression in patients with chronic hepatitis C (CHC); however, developing noninvasive tests that can predict liver injury represents a growing medical need. Considering that hepatocyte apoptosis plays a role in CHC pathogenesis; the aim of our study was to evaluate the presence of different apoptosis markers that correlate with liver injury in a cohort of pediatric and adult patients with CHC., Methods: Liver biopsies and concomitant serum samples from 22 pediatric and 22 adult patients with CHC were analyzed. Histological parameters were evaluated. In serum samples soluble Fas (sFas), caspase activity and caspase-generated neoepitope of the CK-18 proteolytic fragment (M30) were measured., Results: sFas was associated with fibrosis severity in pediatric (significant fibrosis p = 0.03, advanced fibrosis p = 0.01) and adult patients (advanced fibrosis p = 0.02). M30 levels were elevated in pediatric patients with severe steatosis (p = 0.01) while in adults no relation with any histological variable was observed. Caspase activity levels were higher in pediatric samples with significant fibrosis (p = 0.03) and they were associated with hepatitis severity (p = 0.04) in adult patients. The diagnostic accuracy evaluation demonstrated only a good performance for sFas to evaluate advanced fibrosis both in children (AUROC: 0.812) and adults (AUROC: 0.800) as well as for M30 to determine steatosis severity in children (AUROC: 0.833)., Conclusions: Serum sFas could be considered a possible marker of advanced fibrosis both in pediatric and adult patient with CHC as well as M30 might be a good predictor of steatosis severity in children.

BACKGROUND. In the management of chronic hepatitis C (CHC) patients, liver biopsy is the gold standard for liver fibrosis assessment despite some technical limits and risks. Non-invasive approaches have been proposed as alternative methods to evaluate structural liver damage. AIM. To investigate the diagnostic accuracy of transient elastography, 13C-aminopyrine breath test (13C-ABT), serum hyaluronic acid (HA) and cytokeratin 18 Asp396 fragment (CK-18) as non-invasive methods of liver fibrosis assessment ad their correlation to METAVIR score. MATERIAL AND METHODS. In a cohort of 57 CHC patients, liver stiffness, cumulative percentage of administered dose of 13C-aminopyrine at 120 min, serum HA and serum CK-18 concentration were determined. Diagnostic accuracy in detecting significant fibrosis (F ≥ 2), severe fibrosis (F ≥ 3) and cirrhosis (F = 4) was assessed by the area under the receiver operating characteristic curve. RESULTS. Liver fibrosis score showed a strong correlation with liver stiffness (r = 0.667; p < 0.0001) and a significant inverse correlation with 13C-ABT results (r = -0.418; p = 0.0012). A weaker correlation was found with CK18 (r = 0.329; p = 0.0126) and no correlation with HA. Areas under the curve of elastography, 13C-ABT, HA and CK18 were: 0.98, 0.75, 0.69, 0.64, respectively, for F ≥ 2; 0.97, 0.69, 0.80, 0.66, respectively, for F ≥ 3; 0.95, 0.64, 0.70, 0.56, respectively, for F = 4. CONCLUSION. Elastography has the best diagnostic accuracy for the assessment of the degree of liver fibrosis in CHC patients. Its application can provide an alternative useful tool for monitoring the disease evolution.

High prevalence of non-alcoholic fatty liver disease (NAFLD) and very diverse outcomes that are related to disease form and severity at presentation have made the search for noninvasive diagnostic tools in NAFLD one of the areas with most intense development in hepatology today. Various methods have been investigated in the recent years, including imaging methods like ultrasound and magnetic resonance imaging, different forms of liver stiffness measurement, various biomarkers of necroinflammatory processes (acute phase reactants, cytokines, markers of apoptosis), hyaluronic acid and other biomarkers of liver fibrosis. Multicomponent tests, scoring systems and diagnostic panels were also developed with the purposes of differentiating non-alcoholic steatohepatitis from simple steatosis or discriminating between various fibrosis stages. In all of the cases, performance of noninvasive methods was compared with liver biopsy, which is still considered to be a gold standard in diagnosis, but is by itself far from a perfect comparative measure. We present here the overview of the published data on various noninvasive diagnostic tools, some of which appear to be very promising, and we address as well some of still unresolved issues in this interesting field.

Unlabelled: Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme-linked immunosorbent assay (ELISA), which detects a caspase-cleaved cytokeratin-18 (CK-18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase-cleaved and uncleaved CK-18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls., Conclusion: Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Background: Patients with biopsy-proven NASH and especially those with fibrosis are at risk for progressive liver disease, emphasizing the clinical importance of developing non-invasive biomarkers for NASH and NASH-related fibrosis., Aim: This study examines the performance of a new biomarker panel for NASH and NASH-related fibrosis with a combination of clinical and laboratory variables., Methods: Enrolled patients had biopsy-proven NAFLD. Clinical data, laboratory data, and serum samples were collected at the time of biopsy. Fasting serum was assayed for adiponectin, resistin, glucose, M30, M65, Tissue inhibitor of metalloproteinases-1 (Timp-1), ProCollagen 3 N-terminal peptide (PIIINP), and hyaluronic acid (HA). Regression models predictive of NASH, NASH-related fibrosis, and NASH-related advanced fibrosis were designed and cross-validated., Results: Of the 79 enrolled NAFLD patients, 40 had biopsy-proven NASH and 39 had non-NASH NAFLD. Clinical and laboratory data were from this cohort were used to develop a NAFLD Diagnostic Panel that includes three models (models for NASH, NASH-related fibrosis, and NASH-related advanced fibrosis). The model for predicting NASH includes diabetes, gender, BMI, triglycerides, M30 (apoptosis), and M65-M30 (necrosis) [AUC: 0.81, 95% CI, 0.70-0.89, 300 p value <9E 301 (-06)]. The NASH-related fibrosis prediction model includes the same predictors [AUC: 0.80, 95% CI 0.68-0.88, 307 p value <0.00014]. Finally, the NASH-related advanced fibrosis model includes type 2 diabetes, serum triglycerides, Timp-1, and AST [AUC: 0.81, 95% CI, 0.70-0.89; p value, 0.000062]., Conclusions: This NAFLD Diagnostic Panel based on a clinical and laboratory data has good performance characteristics and is easy to use. This biomarker panel could become useful in the management of patients with NAFLD.

Objectives: There is a need to replace liver biopsy with non-invasive markers that predict the degree of liver fibrosis in fatty liver disease related to obesity. Therefore, we studied four potential serum markers of liver fibrosis and compared them with histopathological findings in liver biopsy in children with non-alcoholic fatty liver disease (NAFLD)., Methods: We determined fasting serum level of hyaluronic acid (HA), laminin, YKL-40 and cytokeratin-18 M30 in 52 children (age range 4-19, mean 12 years, 80 % of them were overweight or obese) with biopsy-verified NAFLD. Viral hepatitis, autoimmune and metabolic liver diseases (Wilson's disease, alpha-1-antitrypsin deficiency, cystic fibrosis) were excluded. Fibrosis stage was assessed in a blinded fashion by one pathologist according to Kleiner. Receiver operating characteristics (ROC) analysis was used to calculate the power of the assays to detect liver fibrosis (AccuROC, Canada)., Results: Liver fibrosis was diagnosed in 19 children (37 %). The levels of HA and CK18M30 were significantly higher in children with fibrosis compared to children without fibrosis (p=0.04 and 0.05 respectively). The ability of serum HA (cut-off 19.1 ng/ml, Se=84 %, Sp=55 %, PPV=52 %, NPV=86 %) and CK18M30 (cut-off 210 u/l, Se=79 %, Sp=60 %, PPV=56 %, NPV=82 %) to differentiate children with fibrosis from those without fibrosis was significant (AUC=0.672 and 0.666, respectively). The combination of both markers was superior (AUC=0.73, p=0.002). Laminin and YKL-40 levels did not allow a useful prediction., Conclusions: Cytokeratin-18 and hyaluronic acid are suitable serum markers predicting liver fibrosis in children with NAFLD. Studying these markers may identify patients at risk of disease progression.

Background and Aims: We identified patients with nonalcoholic fatty liver disease (NAFLD) to determine the predictive value of serum markers to diagnose histological steatohepatitis (NASH)., Methods: Demographic, serological, radiological and histological variables on 95 consecutive patients with NAFLD were recorded. The serum markers studied were CK18, Hyaluronic acid, TIMP 1 and YKL 40. The NAS score and the metavir score were the histological scoring systems used., Results: CK18 levels were higher in the NASH group compared to the simple steatosis group (394 +/- 53 micro/L vs 194 +/- 26 micro/L; P < 0.05). In assessing clinical effectiveness, CK18 yielded an AUC of 0.8 for NASH (cut-off value 300 micro/L gives PPV 81% and NPV 85%).The fibrosis markers showed no differences between groups. We stratified the same cohort according to liver fibrosis (F0 vs F1-F4). Fibrosis was associated with advanced age, high body mass index and type 2 diabetes. The biomarkers performed relatively poorly at identifying liver fibrosis (F1-F4), with HA performing the best (AUC 0.73); performance improved for advanced fibrosis (F3/F4) - (HA: AUC 0.77). The NAS score performed the best overall at identifying liver fibrosis (AUC 0.79)., Discussion: CK18 is the only biomarker studied that can identify NASH. Additionally, liver biopsy should be performed in all high risk patients to determine the standardised NAS score to identify patients at high risk of disease progression.

Apoptosis of epithelial hepatocytes plays a pivotal role in acute as well as in chronic liver diseases. The cleavage of cytokeratin-18 (CK-18) by caspases is an early event in the apoptotic process. We therefore sought to investigate serum levels of CK-18 and 20S proteasome in patients with liver cirrhosis, primary graft dysfunction (PDF), and acute liver failure (ALF), and in healthy volunteers. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure the concentration of M30, a fragment of CK-18 cleaved at Asp396 (M30 neoantigen), and the concentration of 20S proteasome. Serum levels of the CK-18 neoepitope M30 were significantly increased in ALF, primary graft dysfunction, and liver cirrhosis vs. healthy controls (1,993.6+/-124.7 U/L, 2,238.1+/-235.9 U/L, and 673.6+/-86.5 U/L vs. 66.8+/-29.1 U/L, respectively, P<0.001). Similar results were detected with the evaluation of 20S proteasome (124,014.5+/-13,235.6 ng/mL, 76,993.2+/-15,720.1 ng/mL, and 2,395.9+/-1,098.2 ng/mL vs. 1,074.5+/-259.4 ng/mL, respectively; P<0.001). Detection of CK-18 neoepitope M30 and 20S proteasome may represent a novel marker of tracing apoptotic epithelium, respectively mirroring degenerative liver processes in affected patient population., ((c) 2007 Wiley-Liss, Inc.)