Emerging concepts in the detection of liver fibrosis in non-alcoholic fatty liver disease.

INTRODUCTION: The non-invasive identification of liver fibrosis related to Non-Alcoholic Fatty Liver Disease is crucial for risk-stratification of patients. Currently, the reference standard to stage hepatic fibrosis relies on liver biopsy, but multiple approaches are developed to allow for non-invasive diagnosis and risk stratification. Non-invasive tests, including blood-based scores and vibration-controlled transient elastography, have been widely validated and represent a good surrogate for risk stratification according to recent European and American guidelines. AREAS COVERED: Novel approaches are based on 'liquid' biomarkers of liver fibrogenesis, including collagen-derived markers (PRO-C3 or PRO-C6), or 'multi-omics' technologies (e.g. proteomic-based molecules or miRNA testing), bearing the advantage of tailoring the intrahepatic disease activity. Alternative approaches are based on 'dry' biomarkers, including magnetic resonance-based tools (including proton density fat fraction, magnetic resonance elastography, or corrected T1), which reach similar accuracy of liver histology and will potentially help identify the best candidates for pharmacological treatment of fibrosing non-alcoholic steatohepatitis. EXPERT OPINION: In the near future, the sequential use of non-invasive tests, as well as the complimentary use of liquid and dry biomarkers according to the clinical need (diagnosis, risk stratification, and prognosis, or treatment response) will guide and improve the management of this liver disease.

Evaluation of blood-based PRO-C3 testing as a diagnostic marker for staging liver fibrosis: A systematic review and meta-analysis.

BACKGROUND AND AIM: With the global increase in chronic liver disease and cirrhosis, there is an increasing need to identify non-invasive biomarkers to measure the severity of disease progression while reducing reliance on pathological biopsies. This study aimed to comprehensively evaluate the diagnostic value of PRO-C3 as a biomarker for staging liver fibrosis in patients with viral hepatitis or fatty liver disease. METHODS: Articles published until January 6, 2023, were searched in the PubMed, Embase, MEDLINE, Web of Science, and Cochrane Library databases. The Quality Assessment of Diagnostic Accuracy Studies-2 tool was used to evaluate the quality of the included studies. Pooled sensitivity, specificity, diagnostic odds ratio, and likelihood ratios were integrated using a random-effects model, and a summary receiver operating characteristic curve was constructed. Publication bias was also detected. Subgroup and meta-regression analyses, as well as sensitivity analysis, were also performed. RESULTS: Fourteen studies with 4315 patients were included. Summary area under the curve of PRO-C3 for the identification of significant fibrosis (≥ F2) and advanced fibrosis (≥ F3) was 0.80 (95% confidence interval: 0.76-0.83). Subgroup and meta-regression analyses suggested that disease type and sample size may be the primary factors of heterogeneity in PRO-C3 diagnosis of ≥ F2, while study design, study sample type, and enzyme-linked immunosorbent assay kit brand may be the primary sources of heterogeneity in PRO-C3 diagnosis of ≥ F3. CONCLUSIONS: PRO-C3 demonstrated clinically meaningful diagnostic accuracy when used alone as a non-invasive biomarker for diagnosing the liver fibrosis stage in individuals with viral hepatitis or fatty liver disease.

Effect of pegbelfermin on NASH and fibrosis-related biomarkers and correlation with histological response in the FALCON 1 trial.

Background & Aims: FALCON 1 was a phase IIb study of pegbelfermin in patients with non-alcoholic steatohepatitis (NASH) and stage 3 fibrosis. This FALCON 1 post hoc analysis aimed to further assess the effect of pegbelfermin on NASH-related biomarkers, correlations between histological assessments and non-invasive biomarkers, and concordance between the week 24 histologically assessed primary endpoint response and biomarkers. Methods: Blood-based composite fibrosis scores, blood-based biomarkers, and imaging biomarkers were evaluated for patients with available data from FALCON 1 at baseline through week 24. SomaSignal tests assessed protein signatures of NASH steatosis, inflammation, ballooning, and fibrosis in blood. Linear mixed-effect models were fit for each biomarker. Correlations and concordance were assessed between blood-based biomarkers, imaging, and histological metrics. Results: At week 24, pegbelfermin significantly improved blood-based composite fibrosis scores (ELF, FIB-4, APRI), fibrogenesis biomarkers (PRO-C3 and PC3X), adiponectin, CK-18, hepatic fat fraction measured by MRI-proton density fat fraction, and all four SomaSignal NASH component tests. Correlation analyses between histological and non-invasive measures identified four main categories: steatosis/metabolism, tissue injury, fibrosis, and biopsy-based metrics. Concordant and discordant effects of pegbelfermin on the primary endpoint vs. biomarker responses were observed; the most clear and concordant effects were on measures of liver steatosis and metabolism. A significant association between hepatic fat measured histologically and by imaging was observed in pegbelfermin arms. Conclusions: Pegbelfermin improved NASH-related biomarkers most consistently through improvement of liver steatosis, though biomarkers of tissue injury/inflammation and fibrosis were also improved. Concordance analysis shows that non-invasive assessments of NASH support and exceed the improvements detected by liver biopsy, suggesting that greater consideration should be given to the totality of available data when evaluating the efficacy of NASH therapeutics. Clinical trial number: Post hoc analysis of NCT03486899. Impact and implications: FALCON 1 was a study of pegbelfermin vs. placebo in patients with non-alcoholic steatohepatitis (NASH) without cirrhosis; in this study, patients who responded to pegbelfermin treatment were identified through examination of liver fibrosis in tissue samples collected through biopsy. In the current analysis, non-invasive blood- and imaging-based measures of fibrosis, liver fat, and liver injury were used to determine pegbelfermin treatment response to see how they compared with the biopsy-based results. We found that many of the non-invasive tests, particularly those that measured liver fat, identified patients who responded to pegbelfermin treatment, consistent with the liver biopsy findings. These results suggest that there may be additional value in using data from non-invasive tests, along with liver biopsy, to evaluate how well patients with NASH respond to treatment.

Serum and Urine Biomarkers Related to Kidney Fibrosis Predict Kidney Outcome in Czech Patients with IgA Nephropathy.

We evaluated biomarkers related to kidney fibrosis for the outcome of patients with IgA nephropathy (IgAN). Clinical parameters (estimated glomerular filtration rate, hypertension, proteinuria) and histological findings were assessed in 134 patients with IgAN at the time of diagnosis and followed up prospectively (mean follow-up time, 56.5 months). We measured biomarkers of collagen and laminin turnover in serum and urine collected at the time of kidney biopsy using a novel enzyme-linked immunosorbent assay. Linear discriminant analysis and logistic regression models were used to predict the patient's kidney outcome. Five serum and urine biomarkers of laminin and collagen turnover (sLG1M, sPRO-C3, sPRO-C6, uPRO-C6/Cr, uC3M/Cr) could significantly differentiae IgAN patients with a worse prognosis. Clinical parameters (glomerular filtration rate (GFR), proteinuria) distinguished patients at risk of IgAN progression with a specificity of 87.3% and a sensitivity of 45.2% (area under the curve-AUC 0.751). The addition of the biomarkers significantly increased the prognostic ability with a specificity of 85.1% and a sensitivity of 73.3% (AUC 0.905). We have identified three serum (sLG1M, sPRO-C3, sPRO-C6) and two urinary markers (uPRO-C6/Cr, u-C3M /Cr) that significantly improve the prognostic ability of markers of kidney function to identify an IgAN patient's risk of progressing to ESKD.

Cross-Sectional and Longitudinal Performance of Non-Invasive Tests of Liver Fibrosis in Patients with Non-Alcoholic Fatty Liver Disease.

Background and aims: Non-invasive tests (NITs) are needed in clinical practice to replace histology for the identification of liver fibrosis and prognostication in Non-Alcoholic Fatty Liver Disease (NAFLD). Novel collagen-derived fibrogenesis markers including N-terminal type III collagen pro-peptide (PRO-C3) are among the most promising tools in this field. The aim of this study was to assess the diagnostic accuracy of PRO-C3, the derivative ADAPT score, and other NITs for the identification of advanced fibrosis (stages 3-4) and changes over 12 months of follow-up. Methods: In this longitudinal study, 96 patients with biopsy-proven NAFLD were evaluated at baseline, of which 50 underwent a follow-up visit after 12 months. Clinical-biochemical parameters, liver stiffness (LS) by transient elastography, PRO-C3, and other NITs (ADAPT, FIB-4, NFS, APRI) were collected at baseline and follow-up. Results: LS showed the best accuracy for the identification of advanced fibrosis, with Area under the Receiving Operator Curve (AUROC) 0.82 (0.73-0.89) for a cut-off value of 9.4 kPa. Among the other NITs, the ADAPT score showed the best accuracy, with AUROC 0.80 (0.71-0.88) for a cut-off of 5.02 (Se 62%, Sp 89%, PPV 74%, NPV 83%). The comparison between the AUROC of LS with that of ADAPT was not statistically different (DeLong test p value 0.348). At follow-up, LS was slightly reduced, whilst PRO-C3 displayed a significant increase from baseline median 11.2 ng/mL to 13.9 ng/mL at follow-up (p = 0.017). Accordingly, ADAPT score increased from median 5.3 to 6.1 (p = 0.019). The other NITs did not significantly change over 12 months. Conclusions: The ADAPT score shows the best performance among non-invasive scores for the identification of advanced fibrosis, not different from LS. Collagen-derived biomarker PRO-C3 and the derivative score ADAPT display significant changes over time, and may be useful tools for monitoring the progression of liver disease or assessing responses to treatments.

A randomized, double-blind, placebo-controlled phase IIa trial of efruxifermin for patients with compensated NASH cirrhosis.

Background & Aims: Efruxifermin has shown clinical efficacy in patients with non-alcoholic steatohepatitis (NASH) and F1-F3 fibrosis. The primary objective of the BALANCED Cohort C was to assess the safety and tolerability of efruxifermin in patients with compensated NASH cirrhosis. Methods: Patients with NASH and stage 4 fibrosis (n = 30) were randomized 2:1 to receive efruxifermin 50 mg (n = 20) or placebo (n = 10) once-weekly for 16 weeks. The primary endpoint was safety and tolerability of efruxifermin. Secondary and exploratory endpoints included evaluation of non-invasive markers of liver injury and fibrosis, glucose and lipid metabolism, and changes in histology in a subset of patients who consented to end-of-study liver biopsy. Results: Efruxifermin was safe and well-tolerated; most adverse events (AEs) were grade 1 (n = 7, 23.3%) or grade 2 (n = 19, 63.3%). The most frequent AEs were gastrointestinal, including transient, mild to moderate diarrhea, and/or nausea. Significant improvements were noted in key markers of liver injury (alanine aminotransferase) and glucose and lipid metabolism. Sixteen-week treatment with efruxifermin was associated with significant reductions in non-invasive markers of fibrosis including Pro-C3 (least squares mean change from baseline [LSMCFB] -9 µg/L efruxifermin vs. -3.4 µg/L placebo; p = 0.0130) and ELF score (-0.4 efruxifermin vs. +0.4 placebo; p = 0.0036), with a trend towards reduced liver stiffness (LSMCFB -5.7 kPa efruxifermin vs. -1.1 kPa placebo; n.s.). Of 12 efruxifermin-treated patients with liver biopsy after 16 weeks, 4 (33%) achieved fibrosis improvement of at least one stage without worsening of NASH, while an additional 3 (25%) achieved resolution of NASH, compared to 0 of 5 placebo-treated patients. Conclusions: Efruxifermin appeared safe and well-tolerated with encouraging improvements in markers of liver injury, fibrosis, and glucose and lipid metabolism following 16 weeks of treatment, warranting confirmation in larger and longer term studies. Lay summary: Cirrhosis resulting from non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease, represents a major unmet medical need. Currently there are no approved drugs for the treatment of NASH. This proof-of-concept randomized, double-blind clinical trial demonstrated the potential therapeutic benefit of efruxifermin treatment compared to placebo in patients with cirrhosis due to NASH. Clinical Trial Number: NCT03976401.

Clinical and genetic determinants of the fatty liver-coagulation balance interplay in individuals with metabolic dysfunction.

Background & Aims: The aim of this study was to examine the determinants of the interplay between liver damage and the coagulation balance in individuals at risk of non-alcoholic fatty liver disease (NAFLD). Methods: We considered 581 healthy participants with ≥3 metabolic alterations undergoing clinical and genomic evaluation, measurement of liver stiffness (LSM) and controlled attenuation parameter (CAP) by Fibroscan, Pro-C3, coagulation balance (von Willebrand factor [vWF], factor VIII/protein C ratio [F8/PC] as the main outcome, D-dimer as marker of coagulation/fibrinolysis activation). Results: Liver fibrosis indices (both Fibrosis-4 [FIB-4] and liver stiffness measurement [LSM]), but not liver fat (CAP), were independently associated with higher F8/PC ratio (p <0.01), triggering D-dimer formation (p = 2E-21). In keeping with a causal role of liver damage in determining a procoagulant status, the main fatty liver inherited risk variant PNPLA3 p.I148M was independently associated with the F8/PC ratio (p = 0.048). Vice versa, the main determinant of the coagulation balance was ABO locus variation (p = 1E-16), through the impact on vWF (p = 8E-26). Both rs687289 ABO and factor V Leiden were independently associated with higher Pro-C3 (p <0.025), with the effect of ABO being mediated by the impact on vWF (p = 5E-10 for association with Pro-C3). Mendelian randomisation analysis was consistent with a causal association of procoagulant imbalance with heightened fibrogenesis (p = 0.001 at robust MR-Egger for Pro-C3), but not with fibrosis (for LSM; p = not significant). Conclusions: In individuals with metabolic dysfunction, liver damage severity and possibly the PNPLA3 p.I148M variant were associated with procoagulant status. Vice versa, evaluation of inherited variants in ABO and other genes influencing coagulation was consistent with a causal role of procoagulant imbalance in activation of early stages of fibrogenesis. Lay summary: In individuals with metabolic alterations at risk of metabolic fatty liver disease, there is a tendency toward heightened blood coagulation (clotting), but the cause and the impact on the progression of liver disease remain unclear. Here we show that liver damage severity and metabolic alterations, but not hepatic fat, are mainly responsible for heightened coagulation in patients with metabolic fatty liver disease. By using genetic approaches, we showed that hepatic inflammation due to lipotoxicity may favour heightened coagulation, which in turn can trigger liver fibrosis, igniting a vicious cycle that leads to progressive liver disease.

High Compliance to Mediterranean Diet Associates with Lower Platelet Activation and Liver Collagen Deposition in Patients with Nonalcoholic Fatty Liver Disease.

The Mediterranean diet (Med-Diet) is considered the most effective dietary patterns to obtain weight loss in NAFLD patients. Previous evidence suggested that Med-Diet adherence could reduce cardiovascular risk and have a beneficial effect on NAFLD severity. Aim of the study was to investigate the relationship between Med-Diet adherence, platelet activation (PA), and liver collagen deposition. The study was performed in 655 consecutive NAFLD outpatients from the PLINIO study, a prospective observational cohort study aimed to identify non-conventional predictors of liver fibrosis progression in NAFLD. PA was measured by the serum thromboxane B2 (TxB2), and liver collagen deposition by N-terminal propeptide of type III collagen (Pro-C3). Adherence to the Med-diet was investigated by a short nine-item validated dietary questionnaire. Patients with high Med-Diet adherence were older and had less metabolic syndrome and lower serum triglycerides, GGT, TxB2, and Pro-C3. At multivariate regression analyses, in the linear model, the Med-Diet score negatively correlated with both TxB2 (Beta = −0.106; p = 0.009) and Pro-C3 (Beta = −0.121; p = 0.002) and in the logistic model high adherence inversely correlated with higher TxB2 tertiles (II tertile: OR = 0.576, p = 0.044; III tertile: OR = 0.556, p = 0.026) and Pro-C3 tertile (III tertile: OR = 0.488, p = 0.013). Low consumption of red meat inversely correlated with higher TxB2 tertile (II tertile: OR = 0.448, p < 0.001, III tertile: OR = 0.567, p = 0.004). In conclusion, NAFLD patients with high adherence to the Med-Diet show lower PA and liver collagen deposition, suggesting a protective role of the Med-Diet against NAFLD progression and cardiovascular risk. In addition, the correlation between TxB2 and Pro-C3 suggests a link between NAFLD severity and cardiovascular risk.

Crosstalk between Irisin Levels, Liver Fibrogenesis and Liver Damage in Non-Obese, Non-Diabetic Individuals with Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Insulin resistance plays a relevant role in the onset of non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and fibrosis. Irisin is an exercise-induced myokine involved in the regulation of energy homeostasis and glucose metabolism. Additionally, pre-clinical models have shown a potential role of irisin in the pathogenesis of NAFLD. The aim of this study is to explore the association between irisin, histological features and biomarkers of liver fibrogenesis in non-diabetic, non-obese, biopsy-proven NAFLD individuals. METHODS: Forty-one patients with histological evidence of NAFLD were included. Circulating irisin and direct markers of fibrogenesis N-terminal type III collagen propeptide (PRO-C3) and type VI collagen cleavage product (PRO-C6) were measured by ELISA. RESULTS: Median age of the cohort was 45 years (41-51) and 80.4% were male. Significant fibrosis (stage ≥ 2) was present in 36.6% of cases. Circulating irisin, PRO-C3 and PRO-C6 levels were significantly higher in subjects with fibrosis stage ≥ 2 when compared to those with fibrosis stage < 2 (5.96 ng/mL (95% CI = 4.42-9.19) vs. 2.42 ng/mL (95% CI = 1.73-5.95), p = 0.033; 9.5 ng/mL (95% CI = 7.7-13.6) vs. 6.2 ng/mL (95% CI = 4.9-8.9), p = 0.016; 6.6 ng/mL (95% CI = 5.6-7.9) vs. 5.1 ng/mL (95% CI = 4.2-5.4), p = 0.013, respectively). Irisin levels were similarly distributed between the features of NASH. Circulating irisin positively correlated with both PRO-C3 and PRO-C6 levels (r = 0.47, p = 0.008 and r = 0.46, p = 0.002). CONCLUSIONS: Increased circulating irisin levels may identify a more aggressive phenotype of liver disease with increased fibrogenesis and more severe liver damage.

Pegbelfermin selectively reduces secondary bile acid concentrations in patients with non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Increased serum bile acids (BAs) have been observed in patients with non-alcoholic steatohepatitis (NASH). Pegbelfermin (PGBF), a polyethylene glycol-modified (PEGylated) analogue of human fibroblast growth factor 21 (FGF21), significantly decreased hepatic steatosis and improved fibrosis biomarkers and metabolic parameters in patients with NASH in a phase IIa trial. This exploratory analysis evaluated the effect of PGBF on serum BAs and explored potential underlying mechanisms. METHODS: Serum BAs and 7α-hydroxy-4-cholesten-3-one (C4) were measured by HPLC-mass spectrometry (MS) using serum collected in studies of patients with NASH (NCT02413372) and in overweight/obese adults (NCT03198182) who received PGBF. Stool samples were collected in NCT03198182 to evaluate faecal BAs by liquid chromatography (LC)-MS and the faecal microbiome by metagenetic and metatranscriptomic analyses. RESULTS: Significant reductions from baseline in serum concentrations of the secondary BA, deoxycholic acid (DCA), and conjugates, were observed with PGBF, but not placebo, in patients with NASH; primary BA concentrations did not significantly change in any arm. Similar effects of PGBF on BAs were observed in overweight/obese adults, allowing for an evaluation of the effects of PGBF on the faecal microbiome and BAs. Faecal transcriptomic analysis showed that the relative abundance of the gene encoding choloylglycine hydrolase, a critical enzyme for secondary BA synthesis, was reduced after PGBF, but not placebo, administration. Furthermore, a trend of reduction in faecal secondary BAs was observed. CONCLUSIONS: PGBF selectively reduced serum concentrations of DCA and conjugates in patients with NASH and in healthy overweight/obese adults. Reduced choloylglycine hydrolase gene expression and decreased faecal secondary BA levels suggest a potential role for PGBF in modulating secondary BA synthesis by gut microbiome. The clinical significance of DCA reduction post-PGBF treatment warrants further investigation. LAY SUMMARY: Pegbelfermin (PGBF) is a hormone that is currently being studied in clinical trials for the treatment of non-alcoholic fatty liver disease. In this study, we show that PGBF treatment can reduce bile acids that have previously been shown to have toxic effects on the liver. Additional studies to understand how PGBF regulates bile acids may provide additional information about its potential use as a treatment for fatty liver.

Systematic Review with Meta-Analysis: Diagnostic Accuracy of Pro-C3 for Hepatic Fibrosis in Patients with Non-Alcoholic Fatty Liver Disease.

The prevalence and severity of non-alcoholic fatty liver disease (NAFLD) is increasing, yet adequately validated tests for care paths are limited and non-invasive markers of disease progression are urgently needed. The aim of this work was to summarize the performance of Pro-C3, a biomarker of active fibrogenesis, in detecting significant fibrosis (F ≥ 2), advanced fibrosis (F ≥ 3), cirrhosis (F4) and non-alcoholic steatohepatitis (NASH) in patients with NAFLD. A sensitive search of five databases was performed in July 2021. Studies reporting Pro-C3 measurements and liver histology in adults with NAFLD without co-existing liver diseases were eligible. Meta-analysis was conducted by applying a bivariate random effects model to produce summary estimates of Pro-C3 accuracy. From 35 evaluated reports, eight studies met our inclusion criteria; 1568 patients were included in our meta-analysis of significant fibrosis and 2058 in that of advanced fibrosis. The area under the summary curve was 0.81 (95% CI 0.77-0.84) in detecting significant fibrosis and 0.79 (95% CI 0.73-0.82) for advanced fibrosis. Our results support Pro-C3 as an important candidate biomarker for non-invasive assessment of liver fibrosis in NAFLD. Further direct comparisons with currently recommended non-invasive tests will demonstrate whether Pro-C3 panels can outperform these tests, and improve care paths for patients with NAFLD.

Comparison of ADAPT, FIB-4 and APRI as non-invasive predictors of liver fibrosis and NASH within the CENTAUR screening population.

BACKGROUND & AIMS: The development of accurate non-invasive tests to detect and measure the extent of fibrosis and disease activity in patients with non-alcoholic steatohepatitis (NASH) - the progressive phenotype of non-alcoholic fatty liver disease (NAFLD) - is of great clinical importance. Herein, we aimed to validate the performance of PRO-C3 and ADAPT for the detection of moderate/severe fibrosis within the CENTAUR screening population. METHODS: PRO-C3 was assessed in plasma from the screening population of the phase IIb CENTAUR study (NCT02217475) in adults with NASH and liver fibrosis. The relation between PRO-C3 and histologic features of NASH was evaluated, as well as the demographics of patients with high and low levels of PRO-C3. The diagnostic ability of PRO-C3, as a standalone marker or incorporated into ADAPT, to identify patients with F≥2 and NASH was estimated using receiver-operating characteristic analysis and logistic regression models. RESULTS: A total of 517 individuals with matched biopsy and PRO-C3 measurements were included. Patients with PRO-C3 levels ≥20.2 ng/ml showed increased levels of insulin, HOMA-IR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and platelet count compared to patients with low PRO-C3 (p <0.05). PRO-C3 increased stepwise with increasing liver fibrosis, lobular inflammation, hepatocyte ballooning, steatosis, and NAFLD activity score (p <0.05), and could distinguish between NAFL and NASH (p <0.0001). PRO-C3 was independently associated with fibrosis and NASH when adjusted for clinical confounders. ADAPT outperformed Fibrosis-4, AST-to-platelet ratio index, and AST/ALT ratio as a predictor of advanced fibrosis and NASH (p <0.001). CONCLUSION: PRO-C3 was associated with NAFLD activity score and fibrosis. ADAPT outperformed other non-invasive scores for detecting NASH. These data support the use of PRO-C3 and ADAPT as diagnostic tools to identify patients with NASH eligible for inclusion in clinical trials. CLINICAL TRIAL NUMBER: NCT02217475 LAY SUMMARY: PRO-C3 is a serological biomarker associated with liver disease activity and fibrosis. Its performance for the detection of disease activity and fibrosis is improved when it is incorporated into the ADAPT score. Herein, we showed that ADAPT was better at selecting patients with non-alcoholic steatohepatitis for inclusion in clinical trials than other non-invasive scores.

TVB-2640 (FASN Inhibitor) for the Treatment of Nonalcoholic Steatohepatitis: FASCINATE-1, a Randomized, Placebo-Controlled Phase 2a Trial.

BACKGROUND & AIMS: Increased de novo lipogenesis creates excess intrahepatic fat and lipotoxins, propagating liver damage in nonalcoholic steatohepatitis. TVB-2640, a fatty acid synthase inhibitor, was designed to reduce excess liver fat and directly inhibit inflammatory and fibrogenic pathways. We assessed the safety and efficacy of TVB-2640 in patients with nonalcoholic steatohepatitis in the United States. METHODS: 3V2640-CLIN-005 (FASCINATE-1) was a randomized, placebo-controlled, single-blind study at 10 US sites. Adults with ≥8% liver fat, assessed by magnetic resonance imaging proton density fat fraction, and evidence of liver fibrosis by magnetic resonance elastography ≥2.5 kPa or liver biopsy were eligible. Ninety-nine patients were randomized to receive placebo or 25 mg or 50 mg of TVB-2640 (orally, once-daily for 12 weeks). The primary end points of this study were safety and relative change in liver fat after treatment. RESULTS: Liver fat increased in the placebo cohort by 4.5% relative to baseline; in contrast TVB-2640 reduced liver fat by 9.6% in the 25-mg cohort (n = 30; least squares mean: -15.5%; 95% confidence interval, -31.3 to -0.23; P = .053), and 28.1% in the 50-mg cohort (n = 28; least squares mean: -28.0%; 95% confidence interval, -44.5 to -11.6; P = .001). Eleven percent of patients in the placebo group achieved a ≥30% relative reduction of liver fat compared to 23% in the 25-mg group, and 61% in the 50-mg group (P < .001). Secondary analyses showed improvements of metabolic, pro-inflammatory and fibrotic markers. TVB-2640 was well tolerated; adverse events were mostly mild and balanced among the groups. CONCLUSIONS: TVB-2640 significantly reduced liver fat and improved biochemical, inflammatory, and fibrotic biomarkers after 12 weeks, in a dose-dependent manner in patients with nonalcoholic steatohepatitis. ClinicalTrials.gov, Number NCT03938246.

Potent suppression of hydrophobic bile acids by aldafermin, an FGF19 analogue, across metabolic and cholestatic liver diseases.

BACKGROUND & AIMS: Higher serum bile acid levels are associated with an increased risk of cirrhosis and liver-related morbidity and mortality. Herein, we report secondary analyses of aldafermin, an engineered analogue of the gut hormone fibroblast growth factor 19, on the circulating bile acid profile in prospective, phase II studies in patients with metabolic or cholestatic liver disease. METHODS: One hundred and seventy-six patients with biopsy-confirmed non-alcoholic steatohepatitis (NASH) and fibrosis and elevated liver fat content (≥8% by magnetic resonance imaging-proton density fat fraction) received 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), 6 mg (n = 28) aldafermin or placebo (n = 27) for 12 weeks. Sixty-two patients with primary sclerosing cholangitis (PSC) and elevated alkaline phosphatase (>1.5× upper limit of normal) received 1 mg (n = 21), 3 mg (n = 21) aldafermin or placebo (n = 20) for 12 weeks. Serum samples were collected on day 1 and week 12 for determination of bile acid profile and neoepitope-specific N-terminal pro-peptide of type III collagen (Pro-C3), a direct measure of fibrogenesis. RESULTS: Treatment with aldafermin resulted in significant dose-dependent reductions in serum bile acids. In particular, bile acids with higher hydrophobicity indices, such as deoxycholic acid, lithocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, and glycocholic acid, were markedly lowered by aldafermin in both NASH and PSC populations. Moreover, aldafermin predominantly suppressed the glycine-conjugated bile acids, rather than the taurine-conjugated bile acids. Changes in levels of bile acids correlated with changes in the novel fibrogenesis marker Pro-C3, which detects a neo-epitope of the type III collagen during its formation, in the pooled NASH and PSC populations. CONCLUSIONS: Aldafermin markedly reduced major hydrophobic bile acids that have greater detergent activity and cytotoxicity. Our data provide evidence that bile acids may contribute to sustaining a pro-fibrogenic microenvironment in the liver across metabolic and cholestatic liver diseases. LAY SUMMARY: Aldafermin is an analogue of a gut hormone, which is in development as a treatment for patients with chronic liver disease. Herein, we show that aldafermin can potently and robustly suppress the toxic, hydrophobic bile acids irrespective of disease aetiology. The therapeutic strategy utilising aldafermin may be broadly applicable to other chronic gastrointestinal and liver disorders. CLINICAL TRIALS REGISTRATION: The study is registered at Clinicaltrials.govNCT02443116 and NCT02704364.

Comprehensive assessment of ECM turnover using serum biomarkers establishes PBC as a high-turnover autoimmune liver disease.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH) are phenotypically distinct autoimmune liver diseases that progress to cirrhosis and liver failure; however, their histological fibrosis distribution differs. We investigated the extracellular matrix (ECM) profiles of patients with PSC, PBC, and AIH to establish whether the diseases display differential patterns of ECM turnover. METHODS: Serum samples were retrospectively collected from the UK (test cohort; PSC n = 78; PBC n = 74; AIH n = 58) and Norway (validation cohort; PSC n = 138; PBC n = 28; AIH n = 27). Patients with ulcerative colitis without liver disease (n = 194) served as controls. We assessed specific serological biomarkers of ECM turnover: type III and V collagen formation (PRO-C3, PRO-C5), degradation of type III and IV collagen (C3M, C4M), biglycan (BGM) and citrullinated vimentin (VICM). RESULTS: Most of the ECM markers showed elevated serum levels in PBC compared with PSC or AIH (p <0.01). PRO-C3 correlated well with liver stiffness and showed the most striking differences between advanced and non-advanced liver disease; several of the other ECM markers were also associated with stage. PRO-C3 and other ECM markers were inversely associated with ursodeoxycholic acid treatment response in PBC and remission in AIH. All ECM remodelling markers were significantly elevated (p <0.05) in patients with PSC, PBC, or AIH compared with ulcerative colitis. CONCLUSIONS: In this first study comparing ECM turnover in autoimmune liver diseases, we found increased ECM turnover in PBC compared with either PSC or AIH. The study indicates that ECM remodelling is different in PSC, PBC, and AIH, suggesting differing opportunities for therapeutic intervention. LAY SUMMARY: The level of scarring is linked to prognosis in autoimmune liver diseases such as primary sclerosing cholangitis, primary biliary cholangitis, and autoimmune hepatitis; hence, the scarring process is a possible target for novel therapy. Investigating the scarring process using highly specific technology, we show that the scarring process is different between the 3 autoimmune liver diseases, and this may have important implications for the development of medical treatment.

Clinical value of serum hyaluronan and propeptide of type III collagen in patients with pancreatic cancer.

Hyaluronan (HA) and collagen are highly expressed in pancreatic cancer (PC) stroma. HA and collagen accumulation increase tumor interstitial fluid pressure, compromising blood flow and drug penetration. The aim of this biomarker study was to determine the clinical utility of serum HA and the propeptide of type III collagen (PRO-C3) in patients with PC. A cohort from the Danish BIOPAC study (NCT03311776) including patients with histologically confirmed pancreatic ductal adenocarcinoma (n = 809), ampullary carcinoma (n = 44), distal biliary tract cancer (n = 31), chronic pancreatitis (n = 15), intraductal papillary mucinous neoplasm (n = 41), duodenal adenoma (n = 7) and no cancer (n = 25). Healthy controls were available for serum HA (n = 141) and PRO-C3 (n = 8). The main outcome was overall survival (OS) of patients with PC in relation to pretreatment serum HA and PRO-C3 levels. Patients with PC had higher baseline serum HA and PRO-C3 than healthy subjects and patients with benign conditions. Pretreatment serum baseline HA and PRO-C3 in patients with PC were associated with poorer survival and PRO-C3 remained prognostic also after adjusting for age, performance status, stage, the presence of liver and peritoneum metastasis, and CA19-9. Detection of HA and PRO-C3 may be useful in differentiating between malignant and benign pancreatic conditions. Serum HA and PRO-C3 were prognostic for OS in patients with PC.

Drug Development for Nonalcoholic Fatty Liver Disease: Landscape and Challenges.

Nonalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in industrialized economies. With no licensed treatment currently available, together with a growing prevalence that parallels global increases in obesity and type 2 diabetes, NAFLD will dominate the landscape of hepatology for the foreseeable future. A multifaceted etiopathogenesis, paucity of reproducible preclinical models that effectively recreate human NAFLD, and lack of robust surrogate trial endpoints have presented major hurdles in drug discovery and development. Smooth collaboration between bench scientists, biotechnology, pharmaceutical industries, and clinicians will be pivotal to target identification, development of effective therapies, biomarker discovery, and ultimately to bring pipeline drugs to market. This review examines the key challenges remaining in NAFLD drug development, outlines early and late phase clinical trials of candidate treatments, and discusses the journey toward biomarker discovery which may facilitate development of novel endpoints in NAFLD clinical trials, enabling meaningful response to be determined noninvasively.

Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.

There is an unmet need for non-invasive biomarkers in non-alcoholic fatty liver disease (NAFLD) that can diagnose advanced disease and identify patients suitable for clinical trials. The PRO-C3 collagen neo-epitope is a putative direct marker of fibrogenesis. We assessed the performance of PRO-C3 in a large, well-characterised international NAFLD cohort and report the development and validation of 2 novel panels for the diagnosis of advanced fibrosis (F≥3) in NAFLD, including a simplified clinical score which eliminates the need for online calculators. METHODS: Plasma PRO-C3 levels were determined in a prospectively recruited international cohort of 449 patients with biopsy diagnosed NAFLD across the full disease spectrum (F0: n = 90; F1: 100; F2: 92; F3: 101; F4: 66). The cohort was divided into a discovery group (n = 151) and a validation group (n = 298). Logistic regression was performed to establish complex (FIBC3) and simplified (ABC3D) diagnostic scores that accurately identify advanced fibrosis. Performance for each was compared to established non-invasive fibrosis scoring systems. RESULTS: Plasma PRO-C3 levels correlated with grade of histological steatohepatitis (rs = 0.367, p ≪0.0001) and stage of fibrosis (rs = 0.462, p ≪0.0001), exhibiting similar performance to current fibrosis scores such as FIB4 for the detection of F≥3 fibrosis. FIBC3 exhibited substantially improved accuracy (AUROC 0.89 and 0.83 in the discovery and validation sets, respectively) and outperformed FIB4 and other similar diagnostic panels. The simplified version, ABC3D, was concurrently developed and had comparable diagnostic accuracy (AUROC 0.88 and 0.81 in the discovery and validation sets, respectively). CONCLUSION: Plasma PRO-C3 levels correlate with severity of steatohepatitis and fibrosis stage. The FIBC3 panel is an accurate tool with a single threshold value that maintains both sensitivity and specificity for the identification of F≥3 fibrosis in NAFLD, eliminating indeterminate results and outperforming commonly used non-invasive tools. A greatly simplified version (ABC3D) that is readily amenable to use in the clinic has been validated and shown to perform with similar accuracy, and may prove a useful tool in routine clinical practice. LAY SUMMARY: We performed a comprehensive, independent evaluation of a collagen biomarker (PRO-C3) to detect and quantify liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). We report the development of 2 diagnostic panels using PRO-C3 to identify patients with advanced fibrosis, one optimal but more complex to calculate (FIBC3), the other easier to use (ABC3D) whilst still performing well.

Effect of NGM282, an FGF19 analogue, in primary sclerosing cholangitis: A multicenter, randomized, double-blind, placebo-controlled phase II trial.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an inflammatory, cholestatic and progressively fibrotic liver disease devoid of effective medical intervention. NGM282, an engineered, non-tumorigenic FGF19 analogue, potently regulates CYP7A1-mediated bile acid homeostasis. We assessed the activity and safety of NGM282 in patients with PSC. METHODS: In this double-blind, placebo-controlled phase II trial, 62 patients who had PSC confirmed by cholangiography or biopsy and an elevated alkaline phosphatase (ALP) >1.5 × the upper limit of normal were randomly assigned 1:1:1 to receive NGM282 1 mg, 3 mg or placebo once daily for 12 weeks. The primary outcome was the change in ALP from baseline to week 12. Secondary and exploratory outcomes included changes in serum biomarkers of bile acid metabolism and fibrosis. Efficacy analysis was by intention-to-treat. RESULTS: At 12 weeks, there were no significant differences in the mean change from baseline in ALP between the NGM282 and placebo groups, and therefore, the primary endpoint was not met. However, NGM282 significantly reduced levels of 7alpha-hydroxy-4-cholesten-3-one (a marker of hepatic CYP7A1 activity, LS mean differences -6.2 ng/ml (95% CI -10.7 to -1.7; p = 0.008) and -9.4 ng/ml (-14.0 to -4.9; p <0.001) in the NGM282 1 mg and 3 mg groups, respectively, compared with placebo) and bile acids. Importantly, fibrosis biomarkers that predict transplant-free survival, including Enhanced Liver Fibrosis score and Pro-C3, were significantly improved following NGM282 treatment. Most adverse events were mild to moderate in severity, with gastrointestinal symptoms more frequent in the NGM282 treatment groups. CONCLUSIONS: In patients with PSC, NGM282 potently inhibited bile acid synthesis and decreased fibrosis markers, without significantly affecting ALP levels. LAY SUMMARY: We present for the first time, the clinical and laboratory effects of a first-in-class, engineered analogue of the endocrine hormone FGF19 in patients with primary sclerosing cholangitis (PSC). By incorporating non-invasive markers of fibrosis, beyond standard liver injury markers, we show that NGM282 impacted on fibrosis turnover and hepatic inflammation without changing alkaline phosphatase. Our findings demonstrate the complexities of using highly potent rational agents in PSC, and furthermore challenge the dogma about what the appropriate endpoints should be for trials in PSC.