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Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease

Liver biopsy is the reference standard for the detection of nonalcoholic steatohepatitis (NASH) within nonalcoholic fatty liver disease (NAFLD). The aim of this study was to identify a biomarker of NASH in patients without significant fibrosis. In all, 172 patients from two centers with biopsy‐proven NAFLD were included in this study. Eighty‐four patients from a single center were included as a derivation cohort and 88 patients from a second center were included as a validation cohort. Serum samples were tested for candidate markers of fibrosis and inflammation alongside hematological and biochemical markers. Among patients without advanced fibrosis, terminal peptide of procollagen III (PIIINP) was the only marker found to be associated with a histological diagnosis of NASH in both cohorts. PIIINP also correlated with the total NAFLD activity score (NAS) and its constituent components (P < 0.001). Area under receiver operating characteristic curve (AUROC) for PIIINP in discriminating between NASH and simple steatosis (SS) was 0.77‐0.82 in patients with F0‐2 fibrosis and 0.82‐0.84 in patients with F0‐3 fibrosis. PIIINP was elevated in patients with advanced fibrosis, the overwhelming majority of whom had NASH. When incorporating patients with all degrees of fibrosis from both cohorts, PIIINP was able to discriminate between patients with SS and those with NASH or advanced fibrosis with AUROC 0.85‐0.87. Conclusion: PIIINP discriminates between SS and NASH or advanced fibrosis. The use of a single biomarker in this context will be of clinical utility in detecting the minority of patients with NAFLD who have NASH or advanced fibrosis related to NASH. (HEPATOLOGY 2013)

Performance of Enhanced Liver Fibrosis test and comparison with transient elastography in the identification of liver fibrosis in patients with chronic hepatitis B infection.

Assessment of liver fibrosis is important in determining prognosis, disease progression and need for treatment in patients with chronic hepatitis B (CHB). Limitations to the use of liver biopsy in assessing fibrosis are well recognized, and noninvasive tests are being increasingly evaluated including transient elastography (TE) and serum markers such as the Enhanced Liver Fibrosis (ELF) test. We assessed performance of ELF and TE in detecting liver fibrosis with reference to liver histology in a cohort of patients with CHB (n = 182), and compared the performance of these modalities. Median age was 46 and mean AST 70 IU/L. Cirrhosis was reported in 20% of liver biopsies. Both modalities performed well in assessing fibrosis at all stages. Area under receiver operator characteristic (AUROC) curves for detecting METAVIR fibrosis stages F ≥ 1, F ≥ 2, F ≥ 3 and F4 were 0.77, 0.82, 0.80 and 0.83 for ELF and 0.86, 0.86, 0.90 and 0.95 for TE. TE performed significantly better in the assessment of severe fibrosis (AUROC 0.80 for ELF and 0.90 for TE, P < 0.01) and cirrhosis (0.83 for ELF and 0.95 for TE, P < 0.01). This study demonstrates that ELF has good performance in detection of liver fibrosis in patients with CHB, and when compared, TE performs better in detection of severe fibrosis/cirrhosis.

Treatment decisions and contemporary versus pending treatments for hepatitis C

The primary aim of antiviral therapy for chronic hepatitis C (CHC) is the prevention of progressive disease. A response to interferon (IFN) treatment is associated with an improvement in all-cause mortality and liver-related mortality from hepatitis C. Unless contraindicated, patients with CHC are thus potential candidates for treatment. Improved response rates are observed in patients with HCV genotype 1 infection treated with first-generation protease inhibitors. However, treatment with current first-generation protease inhibitors and IFN is complex and can result in appreciable adverse effects. The advent of potent, pan-genotypic all-oral direct-acting antiviral (DAA) regimens necessitates a critical examination of the immediate application of PEG-IFN, ribavirin and DAA regimens in patients with CHC. Current guidelines and position statements do not make clear recommendations, and are behind the emerging data. Some aspects of the conundrums facing physicians and patients are summarized in this Review. Cirrhosis presents an immediate threat of disease, and ideally treatment should be targeted at those patients who have advancing or advanced disease; unfortunately, a disparity exists, as response rates are reduced in patients with cirrhosis and the risks of adverse events are increased. On balance, patients with mild disease could consider deferring treatment.

Telaprevir or boceprevir based therapy for chronic hepatitis C infection: Development of resistance-associated variants in treatment failure

The use of triple-therapy, pegylated-interferon, ribavirin and either of the first generation hepatitis C virus (HCV) protease inhibitors telaprevir or boceprevir, is the new standard of care for treating genotype 1 chronic HCV. Clinical trials have shown response rates of around 70-80%, but there is limited data from the use of this combination outside this setting. Through an expanded access programme, we treated 59 patients, treatment naïve and experienced, with triple therapy. Baseline factors predicting treatment response or failure during triple therapy phase were identified in 58 patients. Thirty seven (63.8%) of 58 patients had undetectable HCV RNA 12weeks after the end of treatment. Genotype 1a (p=0.053), null-response to previous treatment (p=0.034), the rate of viral load decline after 12weeks of previous interferon-based treatment (p=0.033) were all associated with triple-therapy failure. The most common cause of on-treatment failure for telaprevir-based regimens was the development of resistance-associated variants (RAVs) at amino acids 36 and/or 155 of HCV protease (p=0.027) whereas in boceprevir-based regimens mutations at amino acid 54 were significant (p=0.015). SVR12 rates approaching 64% were achieved using triple therapy outside the clinical trial setting, in a patient cohort that included cirrhotics.

Biomarkers of Hepatic Fibrosis in Chronic Hepatitis C: A Comparison of 10 Biomarkers Using 2 Different Assays for Hyaluronic Acid.

Background: Advancing fibrosis is regarded as the most important factor when stratifying patients with chronic hepatitis C for retreatment. Goals: (1) To compare the performance of 10 biomarkers of fibrosis, including patented tests, among patients with chronic hepatitis C and treatment failure; and (2) to assess the impact on biomarker performance of using 2 different assays of hyaluronic acid (HA). Study: For 80 patients, liver histology (Metavir) was compared with biomarker scores using sera obtained within 6 months of liver biopsy (indirect biomarkers: AST:ALT ratio, APRI, Forns index, FIB-4, Fibrometer V3G; direct biomarkers: ELF, Fibrospect II, Hyaluronic acid-HA, Fibrometer V2G, Hepascore). Direct biomarker scores were calculated using 2 validated assays for HA (ELISA and radiometric). Results: Using the ELISA assay for HA to calculate the direct panels, all 10 of the biomarkers exhibited comparable overall discriminatory performance (unweighted Obuchowski measure, ordROC 0.92-0.94, P-value>0.05) except AST:ALT ratio and APRI (ordROC 0.86-0.88, P-value<0.05). For the detection of moderate (F2-4) and advanced (F3-4) fibrosis, the AUROC of Fibrometer 2G were significantly higher than AST:ALT ratio and APRI but none of the other biomarkers. Good correlation was observed between the 2 HA assays (intraclass correlation coefficient=0.873) with the ELISA assay exhibiting superior diagnostic performance (ordROC 0.92 vs. 0.88, P-value=0.003). Importantly, the performance of many of the direct biomarkers at their diagnostic thresholds was heavily influenced by the choice of HA assay. Conclusions: Although many biomarkers exhibited good diagnostic performance for the detection of advancing fibrosis, our results indicate that diagnostic performance may be significantly affected by the selection of individual component assays.

Complexities of HCV management in the new era of direct-acting antiviral agents.

The availability of the direct-acting antiviral agents (DAAs) boceprevir and telaprevir provides improved treatment outcomes for many patients infected with hepatitis C virus (HCV) genotype 1. However, HCV infection must first be identified before a decision on treatment can be made and currently many patients remain unaware that they have the virus. Given the lack of prompt diagnosis, disease severity should be determined as a baseline reference for treatment, and novel non-invasive techniques for evaluating fibrosis are now available. For patients receiving a DAA regimen, response-guided therapy based on the detection, absence or level of HCV RNA at specified time points is required to achieve an optimal treatment outcome. Knowledge of the test used to measure HCV RNA and its analytical sensitivity, as well as how to interpret the results correctly, are therefore required to administer therapy appropriately. Furthermore, effective treatment management includes appropriate handling of side effects. This increased complexity associated with DAA regimens has resulted in confusion over many aspects of care, including treatment monitoring, viral load result interpretation and the optimal duration of therapy. These issues are discussed here in addition to the benefits of referring patients infected with HCV to a specialist centre.

Noninvasive markers of liver fibrosis: on-treatment changes of serum markers predict the outcome of antifibrotic therapy.

Aim The utility of noninvasive serum markers to longitudinally monitor liver fibrosis is not established. Methods A total of 70 patients with chronic hepatitis C who had previously failed antiviral therapy were randomized to receive pegylated interferon with or without silymarin for 24 months. Enhanced Liver Fibrosis (ELF) tests (hyularonic acid, terminal peptide of procollagen III, tissue inhibitor of matrix metaloproteinase-1) were performed on patient sera obtained before, during and at the end of the study (0, 12, 24 months) and liver histology obtained before and at the end of the study. Results At 24 months, absolute changes in Ishak fibrosis stage and ELF ranged from −4 to +4 and from −2.41 to +2.68, respectively. Absolute changes in ELF at 12 months were significantly associated with changes in both ELF and histology at 24 months. A model combining both baseline ELF and change of ELF at 12 months could predict the 24-month ELF (R2=0.609, P<1×10–11), a decrease in ELF at 24 months [area under the curve (AUC): 0.80–0.85] and an increase in ELF at 24 months (AUC: 0.81–0.85). Furthermore, a model combining both baseline histologic stage and ELF together with the change of ELF at 12 months could predict 24-month histology (R2=0.601, P<1×10–11, AUC: 0.88–0.92), histologic fibrosis regression (AUC: 0.81–0.84) and progression (AUC: 0.86–0.91). Conclusion Our observations suggest that a change in the serum marker ELF predicts changes in liver fibrosis over a longer period. These data support the use of ELF as a surrogate marker of liver fibrosis evolution in monitoring antifibrotic treatments, thus permitting ‘response-guided’ therapy by the early identification of patients who will benefit from prolonged treatment.

OC-006 Influence of BMI and Alcohol on Liver-Related Morbidity and Mortality in a Cohort of 108,000 Women from the General Population from Ukctocs

Introduction Alcohol and fat are major causes of chronic liver disease (CLD), however their relative influences are not well understood. We aimed to determine liver-related morbidity and mortality attributable to fat and alcohol by stratifying a cohort of 202,638 women according to BMI and alcohol intake. Methods 107,742 women participating in the UK Collaborative Trial of Ovarian Cancer Screening where self-reported height, weight and alcohol intake were available were included. First episode related to cirrhosis (ICD-10 codes K70, K73, K74) either from inpatient Hospital Episode Statistics or death certificate was recorded following trial entry. Participants were stratified by low or high BMI ( < 25 or ≥25 kg/m2), low or high alcohol intake (0–15 or over 15 units/week) and combinations of these parameters. Results Median age at recruitment was 60 years (50–75). Mean BMI was 26.4 kg/m2. There were 90 events (54 inpatient episodes and 36 deaths). There was no difference in risk of event between the BMI groups, however there was a significant increase in risk in the high alcohol group (Log-Rank < 0.001). Cox proportional hazards regression analysis (covariates age, smoking, BMI and alcohol intake), found that compared to the low BMI/low alcohol group, there was an incremental increase in risk of event in the high BMI/low alcohol, low BMI/high alcohol and high BMI/high alcohol groups respectively. However, only the combination of high BMI and high alcohol reached significance (table and figure). Abstract OC-006 Table Group Number of participants (%) Exp(B)/HR (p) 95% Confidence intervals Low BMI/Low Alcohol 46,011 (41.9) 1.0 High BMI/Low Alcohol 58,432 (53.2) 1.1 (0.65) 0.7 – 1.8 Low BMI/High Alcohol 2,683 (2.4) 2.4 (0.11) 0.8 – 6.9 High BMI/High Alcohol 2,616 (2.4) 5.3 (0.002) 2.5 – 11.4 Abstract OC-006 Figure Conclusion These data indicate that the combination of high BMI and alcohol intake is associated with a synergistically increased risk of CLD. Alcohol may be the more significant contributing factor. Further work will define thresholds for each risk factor that independently and in combination increase CLD risk. Disclosure of Interest None Declared

PMO-172 Direct non-invasive serum markers of liver fibrosis predict fibrosis evolution in chronic hepatitis C but are increased by interferon-based therapy: Abstract PMO-172 Figure 1

Introduction Liver biopsy remains the reference standard for the detection of liver fibrosis. While non-invasive markers of liver fibrosis have been validated in chronic hepatitis C, their performance during interferon-based therapy is not established. Methods 70 previous non-responders to interferon based therapy for chronic hepatitis C (40 male, age range 24–67, mean 48.7) were recruited from five centres. Patients were randomised to receive pegylated interferon with or without silymarin for 24 months as an exploratory antifibrotic therapy. All patients underwent a liver biopsy and ELF tests (HA, P3NP, TIMP-1) prior to, and after treatment (month 0 and 24). Changes in histological fibrosis stages before and after therapy (0 to 24 months) were compared with changes in marker scores before, during and after therapy (0 to 24 months). Results Mean ELF score prior to therapy was 9.32 (SD 1.10), during therapy 9.91 (SD1.18) and after therapy 9.53 (SD 1.27). ELF scores were significantly higher during therapy than when compared to both before therapy (p<0.00001) and after therapy (p=0.0002) but levels before therapy were not significantly different from post therapy. Elevated scores on therapy were attributable to an increase in mean HA (p<0.0002) and P3NP (p<0.01) levels on therapy, whereas a significant change in mean TIMP-1 during therapy was not seen. These elevations were seen in all patients regardless of changes in histological fibrosis after therapy (n=20 decrease, n=25 no change, n=35 increase in Ishak stage). However, individual changes in TIMP1 (r=0.239, p=0.04) and changes in ELF (r=0.315, p=0.004) from pre- to post-therapy levels were found to correlate with the change of Ishak fibrosis stage before and during treatment. Conclusion During interferon-based therapy, levels of HA and P3NP and the ELF score rise globally and subsequently fall to values similar to those seen prior to therapy regardless of fibrosis evolution, while TIMP1 levels remained unaffected. However, ELF scores pre and post therapy did accurately reflect changes in histology. This suggests that ELF scores and non-invasive panels incorporating HA and P3NP should be interpreted with caution during interferon-based therapy.Abstract PMO-172 Figure 1 Fibrosis evolution compared to changes in mean ELF score during interferon therapy. Competing interests None declared.

PWE-140 Comparison Of 4 Serum Markers Panels of Fibrosis in Chc: Variants of the Hyaluronic Acid (HA) Assay Significantly Affect Their Diagnostic Performance

Introduction The detection of advancing fibrosis in patients with CHC and prior treatment failure is important for ascertaining prognosis. HA has been used alone and as a constituent component of fibrosis marker panels. The aim of this study was to compare the performance of 4 marker panels in the detection of moderate-to-severe fibrosis (Metavir F2–4) and to assess the influence on diagnostic performance of using 2 different validated assays for HA. Methods 80 patients with CHC, all non-responders or relapsers to IFN-based treatment, were included in this study. Sera obtained within 6 months of liver biopsy were used to measure 4 biomarker panels incorporating HA (ELF, Fibrospect-II, Hepascore, Fibrometer-2G) using 2 validated assays for HA (ELISA-Siemens, radiometric-Pharmacia). Diagnostic performance for the detection of moderate-to-severe fibrosis was assessed by AUROC and by evaluating biomarker performance at published thresholds. Results The prevalence of moderate-to-severe fibrosis was 63% (F0–8%, F1–29%, F2–24%, F3–30%, F4–9%). The AUROC of the Siemens HA assay was higher than the Pharmacia assay (0.80 Vs. 0.69, P = 0.005). Incorporating the Siemens assay for HA, the performance of the panels were not statistically significantly different but Fibrometer 2G generated the highest AUROC. Using the Siemens assay, ELF and Fibrometer 2G had the highest PPV and NPV respectively (88% and 100% using published thresholds). The use of the Pharmacia assay for HA to calculate the biomarkers did not reduce the discriminatory ability of the 4 panels (p = NS). However whereas the other panels were largely unaffected, the use of the Pharmacia assay resulted in a dramatic reduction in the performance of published thresholds of the ELF test with the a prioriand a posteriori probability of fibrosis being equal. Abstract PWE-140 Table HA-SiemensAUROC(PPV/NPV) HA-PharmaciaAUROC(PPV/NPV) HEPASCORE 0.85 (79/67) 0.81 (73/72) FIBROMETER2G 0.88 (71/100) 0.87 (70/100) ELF 0.84 (88/62) 0.79 (63/37) FIBROSPECTII 0.84 (86/59) 0.81 (84/55) Conclusion In this study the performance of the 4 biomarker panels to detect moderate-to-severe fibrosis was comparable. The diagnostic performance of biomarker panels may be significantly effected by the selection of the individual component assays as demonstrated by comparison of the results obtained with different HA assays. Disclosure of Interest None Declared.