Alastair D. Burt

The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.

Patients with nonalcoholic fatty liver disease (NAFLD) and advanced liver fibrosis are at the highest risk for progressing to end‐stage liver disease. We constructed and validated a scoring system consisting of routinely measured and readily available clinical and laboratory data to separate NAFLD patients with and without advanced fibrosis. A total of 733 patients with NAFLD confirmed by liver biopsy were divided into 2 groups to construct (n = 480) and validate (n = 253) a scoring system. Routine demographic, clinical, and laboratory variables were analyzed by multivariate modeling to predict presence or absence of advanced fibrosis. Age, hyperglycemia, body mass index, platelet count, albumin, and AST/ALT ratio were independent indicators of advanced liver fibrosis. A scoring system with these 6 variables had an area under the receiver operating characteristic curve of 0.88 and 0.82 in the estimation and validation groups, respectively. By applying the low cutoff score (−1.455), advanced fibrosis could be excluded with high accuracy (negative predictive value of 93% and 88% in the estimation and validation groups, respectively). By applying the high cutoff score (0.676), the presence of advanced fibrosis could be diagnosed with high accuracy (positive predictive value of 90% and 82% in the estimation and validation groups, respectively). By applying this model, a liver biopsy would have been avoided in 549 (75%) of the 733 patients, with correct prediction in 496 (90%). Conclusion: a simple scoring system accurately separates patients with NAFLD with and without advanced fibrosis, rendering liver biopsy for identification of advanced fibrosis unnecessary in a substantial proportion of patients. (HEPATOLOGY 2007;45:846–854.)

Pathologic diagnosis of early hepatocellular carcinoma : a report of the international consensus group for hepatocellular neoplasia

Advances in imaging techniques and establishment of surveillance protocols for high-risk populations have led to the detection of small hepatic nodules in patients with chronic liver diseases, particularly those with cirrhosis or chronic hepatitis caused by hepatitis B or C viruses. These nodules, comprising a broad range of diagnostic entities—some benign and some with malignant potential—are currently defined histologically, and their clinical management often depends on the ability to make a reliable histologic diagnosis. Evidence accumulated in the last two decades strongly favors the existence of a sequence of events in hepatic nodules that precedes the emergence of hepatocellular carcinoma (HCC),1-10 and these lesions are recognized as precursors of HCC. However, from the beginning of their recognition, there has been considerable confusion concerning nomenclature and diagnostic approaches to these hepatic nodules. To clarify these issues, an International Working Party (IWP) of the World Congresses of Gastroenterology proposed a consensus nomenclature and diagnostic criteria for hepatocellular nodular lesions in 1995.11 The IWP classified nodular lesions found in chronic liver disease into large regenerative nodule, lowgrade dysplastic nodule (L-DN), high-grade dysplastic nodule (H-DN), and HCC; this nomenclature has been widely adopted. In addition, the IWP introduced the concept of dysplastic focus as a cluster of hepatocytes with features of early neoplasia (in particular small cell change or iron-free foci in a siderotic background) measuring less than 0.1 cm, and defined small HCC as a tumor measuring less than 2 cm. More recent studies support the division of small HCC into two clinico-pathological groups that have been termed early HCC and progressed HCC. Early HCC has a vaguely nodular appearance and is well differentiated. Progressed HCC has a distinctly nodular pattern and is mostly moderately differentiated, often with evidence of microvascular invasion.12 Early HCC has a longer time to recurrence and a higher 5-year survival rate compared with progressed HCC.13 Small lesions with malignant potential have only subtle differences from the surrounding parenchyma, making them difficult to assess reproducibly. Differences in the application of diagnostic criteria between Western and Eastern pathologists has been a persistent difficulty in research and clinical management of these lesions.14 In order to obtain a refined and up-to-date international consensus on the histopathologic diagnosis of nodular lesions, such as dysplastic nodules and early HCC, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in April 2002 in Kurume, Japan. The group has met several times up to July 2007 under the auspices of the Laennec Liver Pathology Society. The ICGHN is currently comprised of 34 pathologists and two clinicians from 13 countries. It includes most members of the original IWP who are still active and all the participants from the first ICGHN meeting. This consensus document summarizes the results of our meetings.

Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: Validating the European Liver Fibrosis Panel and exploring simple markers

The detection of fibrosis within nonalcoholic fatty liver disease (NAFLD) is important for ascertaining prognosis and the stratification of patients for emerging therapeutic intervention. We validated the Original European Liver Fibrosis panel (OELF) and a simplified algorithm not containing age, the Enhanced Liver fibrosis panel (ELF), in an independent cohort of patients with NAFLD. Furthermore, we explored whether the addition of simple markers to the existing panel test could improve diagnostic performance. One hundred ninety‐six consecutively recruited patients from 2 centers were included in the validation study. The diagnostic accuracy of the discriminant scores of the ELF panel, simple markers, and a combined panel were compared using receiver operator curves, predictive values, and a clinical utility model. The ELF panel had an area under the curve (AUC) of 0.90 for distinguishing severe fibrosis, 0.82 for moderate fibrosis, and 0.76 for no fibrosis. Simplification of the algorithm by removing age did not alter diagnostic performance. Addition of simple markers to the panel improved diagnostic performance with AUCs of 0.98, 0.93, and 0.84 for the detection of severe fibrosis, moderate fibrosis, and no fibrosis, respectively. The clinical utility model showed that 82% and 88% of liver biopsies could be potentially avoided for the diagnosis of severe fibrosis using ELF and the combined panel, respectively. The ELF panel has good diagnostic accuracy in an independent validation cohort of patients with NAFLD. The addition of established simple markers augments the diagnostic performance across different stages of fibrosis, which will potentially allow superior stratification of patients with NAFLD for emerging therapeutic strategies. (HEPATOLOGY 2007.)

Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients

ABSTRACT Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, ≤1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.

Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease

Background Accurate evaluation of liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) is important to identify patients who may develop complications. The aim of this study was to compare the diagnostic performance of simple non-invasive tests in identifying advanced fibrosis among patients with biopsy-proven NAFLD. Methods Consecutive patients with biopsy proven NAFLD were recruited from the Newcastle Hospitals Fatty Liver Clinic from 2003 to 2009. The AST/ALT ratio, AST to platelet ratio index, BARD (weighted sum of BMI>28=1 point, AST/ALT ratio>0.8=2 points, diabetes=1 point), FIB-4 (age×AST (IU/l)/platelet count (×109/litre)×√ALT (IU/l)) and NAFLD fibrosis scores were calculated from blood tests taken at time of biopsy. Results 145 patients (82 male (61%), mean age 51±12 years) were included. The mean body mass index was 35±5 kg/m2. 73 subjects (50%) had diabetes. 93 patients (64%) had non-alcoholic steatohepatitis. 27 (19%) had advanced fibrosis (Kleiner stage 3–4). The FIB-4 score had the best diagnostic accuracy for advanced fibrosis (area under receiver operator characteristic curve (AUROC) 0.86), followed by AST/ALT ratio (AUROC 0.83), NAFLD fibrosis score (AUROC 0.81), BARD (AUROC 0.77) and AST to platelet ratio index (AUROC 0.67). The AST/ALT ratio, BARD score, FIB-4 and NAFLD fibrosis scores had negative predictive values greater than 90% (93%, 95%, 95% and 92% respectively). Positive predictive values were modest. In order to exclude advanced fibrosis liver biopsy could potentially be avoided in 69% with AST/ALT ratio, 62% with FIB-4, 52% with NAFLD fibrosis score and 38% with BARD. Conclusions The ALT/AST ratio, FIB-4 and NAFLD fibrosis scores can reliably exclude advanced fibrosis in a high proportion of patients with NAFLD, allowing liver biopsy to be used in a more directed manner.

Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications for prognosis and clinical management

BACKGROUND AND AIMS There remains uncertainty about the natural history of non-alcoholic fatty liver disease (NAFLD). The spectrum of NAFLD includes non-alcoholic fatty liver (NAFL; steatosis without hepatocellular injury) and steatohepatitis (NASH; steatosis with hepatocyte ballooning degeneration±fibrosis). Our aim was to assess the histological severity of NAFLD in a cohort with serial biopsy data, and determine factors predicting progression. METHODS Patients with two liver biopsies more than a year apart were identified. Clinical and laboratory data were collected from the time of liver biopsy. RESULTS 108 patients had serial biopsies (median interval 6.6years, range 1.3-22.6). 81 (75%) patients had NASH and 27 had NAFL. Overall, 45 (42%) patients had fibrosis progression, 43 (40%) had no change in fibrosis, while 20 (18%) had fibrosis regression. Importantly, no significant difference in the proportion exhibiting fibrosis progression was found between those with NAFL or NASH at index biopsy (37% vs. 43%, p=0.65). Progression to NASH was seen in 44% of patients with baseline NAFL. Of 10 patients with NAFL who had fibrosis progression, 3 progressed by 1 stage, 5 by 2 stages and 2 by 3 stages; all had NASH on follow-up biopsy. Of concern, 6 of 27 (22%) patients with baseline NAFL, reached stage 3 fibrosis at follow-up biopsy. Among the patients with NAFL, 80% of those having fibrosis progression were diabetic at the follow-up liver biopsy compared with 25% of non-progressors (p=0.005). CONCLUSIONS Contrary to current dogma, this study suggests that steatosis can progress to NASH and clinically significant fibrosis.

Elevated endotoxin levels in non-alcoholic fatty liver disease

BackgroundEmerging data indicate that gut-derived endotoxin may contribute to low-grade systemic inflammation in insulin resistant states. This study aimed to examine the importance of serum endotoxin and inflammatory markers in non-alcoholic fatty liver disease (NAFLD) patients, with and without type 2 diabetes mellitus (T2DM), and to explore the effect of treatment with a lipase inhibitor, Orlistat, on their inflammatory status.MethodsFasted serum from 155 patients with biopsy proven NAFLD and 23 control subjects were analysed for endotoxin, soluble CD14 (sCD14), soluble tumour necrosis factor receptor II (sTNFRII) and various metabolic parameters. A subgroup of NAFLD patients were re-assessed 6 and 12 months after treatment with diet alone (n = 6) or diet plus Orlistat (n = 8).ResultsEndotoxin levels were significantly higher in patients with NAFLD compared with controls (NAFLD: 10.6(7.8, 14.8) EU/mL; controls: 3.9(3.2, 5.2) EU/mL, p < 0.001); NAFLD alone produced comparable endotoxin levels to T2DM (NAFLD: T2DM: 10.6(5.6, 14.2) EU/mL; non-diabetic: 10.6(8.5, 15.2) EU/mL), whilst a significant correlation between insulin resistance and serum endotoxin was observed (r = 0.27, p = 0.008). Both sCD14 (p < 0.01) and sTNFRII (p < 0.001) increased with severity of fibrosis. A positive correlation was also noted between sTNFRII and sCD14 in the NAFLD subjects (r = 0.29, p = 0.004).Sub-cohort treatment with Orlistat in patients with NAFLD showed significant decreases in ALT (p = 0.006), weight (p = 0.005) and endotoxin (p = 0.004) compared with the NAFLD, non-Orlistat treated control cohort at 6 and 12 months post therapy, respectively.ConclusionsEndotoxin levels were considerably increased in NAFLD patients, with marked increases noted in early stage fibrosis compared with controls. These results suggest elevated endotoxin may serve as an early indicator of potential liver damage, perhaps negating the need for invasive liver biopsy. As endotoxin may promote insulin resistance and inflammation, interventions aimed at reducing endotoxin levels in NAFLD patients may prove beneficial in reducing inflammatory burden.

Enhanced liver fibrosis test can predict clinical outcomes in patients with chronic liver disease

Background Clinicians use fibrosis in a liver biopsy to predict clinical outcomes of chronic liver disease. The performance of non-invasive tests has been evaluated against histological assessment of fibrosis but use of clinical outcomes as the reference standard would be ideal. The enhanced liver fibrosis (ELF) test was derived and validated in a large cohort of patients and shown to have high diagnostic accuracy (area under the curve (AUC)=0.80 95% CI 0.76 to 0.85) in identification of significant fibrosis on biopsy. Objective To evaluate ELF performance in predicting clinical outcomes by following up the original ELF cohort. Methods Patients recruited to the ELF study at seven English centres were followed up for liver morbidity and mortality by examination of clinical data. Defaulting/discharged patients were followed up by family practitioner questionnaires. Primary outcome measure was liver-related morbidity/liver-related death. Results 457 patients were followed up (median 7 years), with ascertainment of clinical status in 92%. There were 61 liver-related outcomes (39 deaths). Survival analysis showed that the ELF score predicts liver outcomes, with people having the highest ELF scores being significantly more likely to have clinical outcomes than those in lower-score groups. A Cox proportional hazards model showed fully adjusted HRs of 75 (ELF score 12.52–16.67), 20 (10.426–12.51) and 5 (8.34–10.425) compared with patients with ELF <8.34. A unit change in ELF is associated with a doubling of risk of liver-related outcome. Conclusions An ELF test can predict clinical outcomes in patients with chronic liver disease and may be a useful prognostic tool in clinical practice.

TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition, strongly associated with the metabolic syndrome, that can lead to progressive hepatic fibrosis, cirrhosis and hepatic failure. Subtle inter-patient genetic variation and environmental factors combine to determine variation in disease progression. A common non-synonymous polymorphism in TM6SF2 (rs58542926 c.449 C>T, p.Glu167Lys) was recently associated with increased hepatic triglyceride content, but whether this variant promotes clinically relevant hepatic fibrosis is unknown. Here we confirm that TM6SF2 minor allele carriage is associated with NAFLD and is causally related to a previously reported chromosome 19 GWAS signal that was ascribed to the gene NCAN. Furthermore, using two histologically characterized cohorts encompassing steatosis, steatohepatitis, fibrosis and cirrhosis (combined n=1,074), we demonstrate a new association, independent of potential confounding factors (age, BMI, type 2 diabetes mellitus and PNPLA3 rs738409 genotype), with advanced hepatic fibrosis/cirrhosis. These findings establish new and important clinical relevance to TM6SF2 in NAFLD.

Epithelial–mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease

The relationship between bile duct damage and portal fibrosis in chronic liver diseases remains unclear. This study was designed to show whether human intrahepatic biliary epithelial cells can undergo epithelial–mesenchymal cell transition, thereby directly contributing to fibrogenesis. Primary human cholangiocytes were stimulated with transforming growth factor-β (TGFβ) or TGFβ-presenting T cells and examined for evidence of transition to a mesenchymal phenotype. Liver sections were labelled to detect antigens associated with biliary epithelial cells (cytokeratin 7 and 19 and E-cadherin), T cells (CD8), epithelial–mesenchymal transition (S100A4, vimentin and matrix metalloproteinase-2 (MMP-2)), myofibroblasts (α-smooth muscle actin) and intracellular signal-transduction mediated by phosphorylated (p)Smad 2/3; in situ hybridisation was performed to detect mRNA encoding TGFβ and S100A4. Stimulation of cultured cells with TGFβ induced the expression of pSmad2/3, S100A4 and α-smooth muscle actin; these cells became highly motile. Although normal bile ducts expressed ALK5 (TGFβ RI), low levels of TGFβ mRNA and nuclear pSmad2/3, they did not express S100A4, vimentin or MMP-2. However, TGFβ mRNA and nuclear pSmad2/3 were strongly expressed in damaged ducts, which also expressed S100A4, vimentin and MMP-2. Fibroblast-like cells which expressed S100A4 were present around many damaged bile ducts. Cells in the ‘ductular reaction’ expressed both epithelial and mesenchymal markers together with high levels of TGFβ mRNA and pSmad2/3. In conclusion, the cells forming small- and medium-sized bile ducts and the ductular reaction undergo EMT during chronic liver diseases, resulting in the formation of invasive fibroblasts; this process may be driven by a response to local TGFβ, possibly presented by infiltrating T cells.