Infection with the hepatitis C virus causes viral genotype-specific differences in cholesterol metabolism and hepatic steatosis

Lipids play essential roles in the hepatitis C virus (HCV) life cycle and patients with chronic HCV infection display disordered lipid metabolism which resolves following successful anti-viral therapy. It has been proposed that HCV genotype 3 (HCV-G3) infection is an independent risk factor for hepatocellular carcinoma and evidence suggests lipogenic proteins are involved in hepatocarcinogenesis. We aimed to characterise variation in host lipid metabolism between participants chronically infected with HCV genotype 1 (HCV-G1) and HCV-G3 to identify likely genotype-specific differences in lipid metabolism. We combined several lipidomic approaches: analysis was performed between participants infected with HCV-G1 and HCV-G3, both in the fasting and non-fasting states, and after sustained virological response (SVR) to treatment. Sera were obtained from 112 fasting patients (25% with cirrhosis). Serum lipids were measured using standard enzymatic methods. Lathosterol and desmosterol were measured by gas-chromatography mass spectrometry (MS). For further metabolic insight on lipid metabolism, ultra-performance liquid chromatography MS was performed on all samples. A subgroup of 13 participants had whole body fat distribution determined using in vivo magnetic resonance imaging and spectroscopy. A second cohort of (non-fasting) sera were obtained from HCV Research UK for comparative analyses: 150 treatment naïve patients and 100 non-viraemic patients post-SVR. HCV-G3 patients had significantly decreased serum apoB, non-HDL cholesterol concentrations, and more hepatic steatosis than those with HCV-G1. HCV-G3 patients also had significantly decreased serum levels of lathosterol, without significant reductions in desmosterol. Lipidomic analysis showed lipid species associated with reverse cholesterol transport pathway in HCV-G3. We demonstrated that compared to HCV-G1, HCV-G3 infection is characterised by low LDL cholesterol levels, with preferential suppression of cholesterol synthesis via lathosterol, associated with increasing hepatic steatosis. The genotype-specific lipid disturbances may shed light on genotypic variations in liver disease progression and promotion of hepatocellular cancer in HCV-G3

Accuracy of FibroScan Controlled Attenuation Parameter and Liver Stiffness Measurement in Assessing Steatosis and Fibrosis in Patients With Non-alcoholic Fatty Liver Disease.

BACKGROUND & AIMS: We estimated the accuracy of FibroScan vibration-controlled transient elastography controlled attenuation parameter (CAP) and liver stiffness measurements (LSMs) in assessing steatosis and fibrosis in patients with suspected NAFLD. METHODS: We collected data from 450 consecutive adults who underwent liver biopsy analysis for suspected NAFLD at 7 centers in the United Kingdom from March 2014 through January 2017. FibroScan examinations with M or XL probe were completed within the 2 weeks of the biopsy analysis (404 had a valid examination). The biopsies were scored by 2 blinded expert pathologists according to non-alcoholic steatohepatitis clinical research network criteria. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROC) for the categories of steatosis and fibrosis. We assessed effects of disease prevalence on positive and negative predictive values. For LSMs, the effects of histological parameters and probe type were appraised using multivariable analysis. RESULTS: Using biopsy analysis as the reference standard, we found that CAP identified patients with steatosis with an AUROCs of 0.87 (95% CI, 0.82-0.92) for S≥S1, 0.77 (95% CI, 0.71-0.82) for S≥S2, and 0.70 (95% CI, 0.64-0.75) for S=S3. Youden cut-off values for S≥S1, S≥S2 and S≥S3 were 302 dB/m, 331 dB/m, and 337 dB/m respectively. LSM identified patients with fibrosis with AUROCs of 0.77 (95% CI, 0.72-0.82) for F≥F2, 0.80 (95% CI, 0.75-0.84) for F≥F3, and 0.89 (95% CI, 0.84-0.93) for F=F4. Youden cut-off values for F≥F2, F≥F3 and F=F4 were 8.2 kPa, 9.7 kPa, and 13.6 kPa respectively. Applying the optimal cut-off values, determined from this cohort, to populations of lower fibrosis prevalence increased negative predictive values and reduced positive predictive values. Multivariable analysis found that the only parameter that significantly affect LSMs was fibrosis stage (P<10-16); we found no association with steatosis or probe type. CONCLUSIONS: In a prospective analysis of patients with NAFLD, we found CAP and LSMs by FibroScan to assess liver steatosis and fibrosis, respectively, with AUROC values ranging from 0.7 to 0.89. Probe type and steatosis did not affect LSMs

Current considerations for clinical management and care of non-alcoholic fatty liver disease: Insights from the 1st International Workshop of the Canadian NASH Network (CanNASH).

Non-alcoholic fatty liver disease (NAFLD) affects approximately 8 million Canadians. NAFLD refers to a disease spectrum ranging from bland steatosis to non-alcoholic steatohepatitis (NASH). Nearly 25% of patients with NAFLD develop NASH, which can progress to liver cirrhosis and related end-stage complications. Type 2 diabetes and obesity represent the main risk factors for the disease. The Canadian NASH Network is a national collaborative organization of health care professionals and researchers with a primary interest in enhancing understanding, care, education, and research around NAFLD, with a vision of best practices for this disease state. At the 1st International Workshop of the CanNASH network in April 2021, a joint event with the single topic conference of the Canadian Association for the Study of the Liver (CASL), clinicians, epidemiologists, basic scientists, and community members came together to share their work under the theme of NASH. This symposium also marked the initiation of collaborations between Canadian and other key opinion leaders in the field representative of international liver associations. The main objective is to develop a policy framework that outlines specific targets, suggested activities, and evidence-based best practices to guide provincial, territorial, and federal organizations in developing multidisciplinary models of care and strategies to address this epidemic

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis

Background & Aims: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota. Methods: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy. Results: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of Faecalibacterium prausnitzii, a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels. Conclusion: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation. Lay summary: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis

Biomarkers for staging fibrosis and non-alcoholic steatohepatitis in non-alcoholic fatty liver disease (the LITMUS project): a comparative diagnostic accuracy study.

BACKGROUND The reference standard for detecting non-alcoholic steatohepatitis (NASH) and staging fibrosis-liver biopsy-is invasive and resource intensive. Non-invasive biomarkers are urgently needed, but few studies have compared these biomarkers in a single cohort. As part of the Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) project, we aimed to evaluate the diagnostic accuracy of 17 biomarkers and multimarker scores in detecting NASH and clinically significant fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) and identify their optimal cutoffs as screening tests in clinical trial recruitment. METHODS This was a comparative diagnostic accuracy study in people with biopsy-confirmed NAFLD from 13 countries across Europe, recruited between Jan 6, 2010, and Dec 29, 2017, from the LITMUS metacohort of the prospective European NAFLD Registry. Adults (aged ≥18 years) with paired liver biopsy and serum samples were eligible; those with excessive alcohol consumption or evidence of other chronic liver diseases were excluded. The diagnostic accuracy of the biomarkers was expressed as the area under the receiver operating characteristic curve (AUC) with liver histology as the reference standard and compared with the Fibrosis-4 index for liver fibrosis (FIB-4) in the same subgroup. Target conditions were the presence of NASH with clinically significant fibrosis (ie, at-risk NASH; NAFLD Activity Score ≥4 and F≥2) or the presence of advanced fibrosis (F≥3), analysed in all participants with complete data. We identified thres holds for each biomarker for reducing the number of biopsy-based screen failures when recruiting people with both NASH and clinically significant fibrosis for future trials. FINDINGS Of 1430 participants with NAFLD in the LITMUS metacohort with serum samples, 966 (403 women and 563 men) were included after all exclusion criteria had been applied. 335 (35%) of 966 participants had biopsy-confirmed NASH and clinically significant fibrosis and 271 (28%) had advanced fibrosis. For people with NASH and clinically significant fibrosis, no single biomarker or multimarker score significantly reached the predefined AUC 0·80 acceptability threshold (AUCs ranging from 0·61 [95% CI 0·54-0·67] for FibroScan controlled attenuation parameter to 0·81 [0·75-0·86] for SomaSignal), with accuracy mostly similar to FIB-4. Regarding detection of advanced fibrosis, SomaSignal (AUC 0·90 [95% CI 0·86-0·94]), ADAPT (0·85 [0·81-0·89]), and FibroScan liver stiffness measurement (0·83 [0·80-0·86]) reached acceptable accuracy. With 11 of 17 markers, histological screen failure rates could be reduced to 33% in trials if only people who were marker positive had a biopsy for evaluating eligibility. The best screening performance for NASH and clinically significant fibrosis was observed for SomaSignal (number needed to test [NNT] to find one true positive was four [95% CI 4-5]), then ADAPT (six [5-7]), MACK-3 (seven [6-8]), and PRO-C3 (nine [7-11]). INTERPRETATION None of the single markers or multimarker scores achieved the predefined acceptable AUC for replacing biopsy in detecting people with both NASH and clinically significant fibrosis. However, several biomarkers could be applied in a prescreening strategy in clinical trial recruitment. The performance of promising markers will be further evaluated in the ongoing prospective LITMUS study cohort. FUNDING The Innovative Medicines Initiative 2 Joint Undertaking

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis

Objective: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM- VCTE), Fibrosis-4 index (FIB-4) and NAFLD Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies.Design: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individu-ally and in sequential combinations.Results: Data were included from 37 primary studies (n=5735; 45% female; median age: 54 years; median BMI: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: diagnostic and mechanistic relevance

Background & Aims: Serum microRNAs (miRNAs) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages.Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 NAFLD cases representing the complete NAFLD spectrum and 10 population controls). MiRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional NAFLD cases and 15 population controls by quantitative reverse transcriptase-polymerase chain reaction.Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages but miR-193a-5p consistently the showed increased levels in all comparisons. Relative to NAFL/NASH with mild fibrosis (stage 0/1), three miRNAs (miR-193a-5p, miR-378d and miR378d) were increased in cases with NASH and clinically significant fibrosis (stage 2-4), seven (miR193a-5p, miR-378d, miR-378e, miR-320b, c, d & e) increased in cases with NAFLD Activity Score (NAS) 5-8 compared with lower NAS, and three (miR-193a-5p, miR-378d, miR-378e) increased but one (miR-19b-3p) decreased in steatosis, activity, and fibrosis "activity" (SAF-A) score 2-4 compared with lower SAF-A. The significant findings for miR-193a-5p were replicated in the additional NAFLD cohort. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n=80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Non-alcoholic fatty liver disease and diabetes.

Non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2DM) are common conditions that regularly co-exist and can act synergistically to drive adverse outcomes. The presence of both NAFLD and T2DM increases the likelihood of the development of complications of diabetes (including both macro- and micro- vascular complications) as well as augmenting the risk of more severe NAFLD, including cirrhosis, hepatocellular carcinoma and death. The mainstay of NAFLD management is currently to reduce modifiable metabolic risk. Achieving good glycaemic control and optimising weight loss are pivotal to restricting disease progression. Once cirrhosis has developed, it is necessary to screen for complications and minimise the risk of hepatic decompensation. Therapeutic disease modifying options for patients with NAFLD are currently limited. When diabetes and NAFLD co-exist, there are published data that can help inform the clinician as to the most appropriate oral hypoglycaemic agent or injectable therapy that may improve NAFLD, however most of these data are drawn from observations in retrospective series and there is a paucity of well-designed randomised double blind placebo controlled studies with gold-standard end-points. Furthermore, given the heterogeneity of inclusion criteria and primary outcomes, as well as duration of follow-up, it is difficult to draw robust conclusions that are applicable across the entire spectrum of NAFLD and diabetes. In this review, we have summarised and critically evaluated the available data, with the aim of helping to inform the reader as to the most pertinent issues when managing patients with co-existent NAFLD and T2DM