Neo-epitope detection identifies extracellular matrix turnover in systemic inflammation and sepsis: an exploratory study.

BACKGROUND: Sepsis is associated with high morbidity and mortality, primarily due to systemic inflammation-induced tissue damage, resulting organ failure, and impaired recovery. Regulated extracellular matrix (ECM) turnover is crucial for maintaining tissue homeostasis in health and in response to disease-related changes in the tissue microenvironment. Conversely, uncontrolled turnover can contribute to tissue damage. Systemic Inflammation is implicated to play a role in the regulation of ECM turnover, but the relationship between the two is largely unclear. METHODS: We performed an exploratory study in 10 healthy male volunteers who were intravenously challenged with 2 ng/kg lipopolysaccharide (LPS, derived from Escherichia coli) to induce systemic inflammation. Plasma samples were collected before (T0) and after (T 1 h, 3 h, 6 h and 24 h) the LPS challenge. Furthermore, plasma was collected from 43 patients with septic shock on day 1 of ICU admission. Circulating neo-epitopes of extracellular matrix turnover, including ECM degradation neo-epitopes of collagen type I (C1M), type III (C3M), type IV (C4Ma3), and type VI (C6M), elastin (ELP-3) and fibrin (X-FIB), as well as the ECM synthesis neo-epitopes of collagen type III (PRO-C3), collagen type IV (PRO-C4) and collagen type VI (PRO-C6) were measured by ELISA. Patient outcome data were obtained from electronic patient records. RESULTS: Twenty-four hours after LPS administration, all measured ECM turnover neo-epitopes, except ELP-3, were increased compared to baseline levels. In septic shock patients, concentrations of all measured ECM neo-epitopes were higher compared to healthy controls. In addition, concentrations of C6M, ELP-3 and X-FIB were higher in patients with septic shock who ultimately did not survive (N = 7) compared to those who recovered (N = 36). CONCLUSION: ECM turnover is induced in a model of systemic inflammation in healthy volunteers and was observed in patients with septic shock. Understanding interactions between systemic inflammation and ECM turnover may provide further insight into mechanisms underlying acute and persistent organ failure in sepsis.

Serum PRO-C3 is useful for risk prediction and fibrosis assessment in MAFLD with chronic kidney disease in an Asian cohort.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is an emerging risk factor for chronic kidney disease (CKD). N-terminal propeptide of collagen type 3 (PRO-C3) is a biomarker of advanced fibrosis in MAFLD and PRO-C3 may be involved in renal fibrosis. We aimed to use PRO-C3 measurements to generate a new algorithmic score to test the prediction of MAFLD with chronic kidney disease (MAFLD-CKD). METHODS: A derivation and independent validation cohort of 750 and 129 Asian patients with biopsy-confirmed MAFLD were included. Serum PRO-C3 concentration was measured and regression analyses were performed to examine associations with MAFLD-CKD. A derivative algorithm for MAFLD-CKD risk prediction was evaluated with receiver operator characteristic (ROC) curve analysis. RESULTS: The study included two Asian cohorts (n = 180 with MAFLD-CKD; mean-eGFR: 94.93 mL/min/1.73 m2; median-urinary albumin-to-creatinine ratio: 6.58 mg/mmol). PRO-C3 was associated with the severity of MAFLD-CKD and independently associated with MAFLD-CKD (adjusted odds ratio = 1.16, 95% confidence interval [CI]: 1.08-1.23, p < .001). A new non-invasive score (termed PERIOD) including PRO-C3 efficiently predicted MAFLD-CKD (AUROC = .842, 95% CI: .805-.875). Accuracy, specificity and negative predictive values were 80.2%, 85.1% and 88.4%, respectively. In the validation cohort, the PERIOD score had good diagnostic performance (AUROC = .807, 95% CI: .691-.893) with similar results in all patient subgroups. In the MAFLD-CKD subgroup, the accuracy for identifying advanced fibrosis was further improved by combining the PRO-C3-based ADAPT with the Agile 3+ scores (AUROC = .90, 95% CI: .836-.964). CONCLUSIONS: The PERIOD score is helpful for accurately predicting the risk of MAFLD-CKD. PRO-C3 can also be used to assess liver fibrosis in people with MAFLD-CKD.

Review: Opportunities and barriers for omics-based biomarker discovery in steatotic liver diseases.

The rising prevalence of liver diseases related to obesity and excessive use of alcohol is fuelling an increasing demand for accurate biomarkers aimed at community screening, diagnosis of steatohepatitis and significant fibrosis, monitoring, prognosis and prediction of treatment efficacy. Breakthroughs in omics methodologies and the power of bioinformatics have created an excellent opportunity to combine clinical needs with technological advancements. Omics technologies allow for advanced investigations into biological processes from the genes to transcription and regulation, to circulating protein, metabolite and lipid levels, as well as the microbiome including bacteria, viruses and fungi. We consequently find ourselves in a period of rapid progress in technology and bioinformatics that may allow for development of precision biomarkers for personalised medicine. However, there are important barriers to consider in omics biomarker discovery and validation, including the use of semi-quantitative measurements from untargeted platforms, which may exhibit high analytical, inter- and intra-individual variance. Standardising methods and the need to validate across diverse populations, presents a challenge, partly due to disease complexity and the dynamic nature of biomarker expression in different disease stages. Lack of validity causes lost opportunities when studies fail to provide the knowledge needed for regulatory approvals, all of which contributes to a delayed translation of these discoveries into clinical practice. While no omics-based biomarkers have matured to clinical implementation, the extent of data generated through omics-technologies holds the power of hypothesis-free discovery of a plethora of candidate biomarkers to be further validated. To explore the many opportunities of omics technologies, hepatologists need detailed knowledge of commonalities and differences between the various omics layers, and both the barriers to and advantages of these approaches.

A Serological Neoepitope Biomarker of Neutrophil Elastase-Degraded Calprotectin, Associated with Neutrophil Activity, Identifies Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease More Effectively Than Total Calprotectin.

Neutrophil activation can release neutrophil extracellular traps (NETs) in acute inflammation. NETs result in the release of human neutrophil elastase (HNE) and calprotectin, where the former can degrade the latter and generate protein fragments associated with neutrophil activity. We investigated this in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) using the novel neoepitope biomarker CPa9-HNE, quantifying a specific HNE-mediated fragment of calprotectin in serum. CPa9-HNE was compared to total calprotectin. Initially, CPa9-HNE was measured in healthy (n = 39), COPD (n = 67), and IPF (n = 16) serum using a neoepitope-specific competitive enzyme-linked immunosorbent assay. Then, a head-to-head comparison of CPa9-HNE and total calprotectin, a non-neoepitope, was conducted in healthy (n = 19), COPD (n = 25), and IPF (n = 19) participants. CPa9-HNE levels were significantly increased in COPD (p < 0.0001) and IPF subjects (p = 0.0001) when compared to healthy participants. Additionally, CPa9-HNE distinguished IPF (p < 0.0001) and COPD (p < 0.0001) from healthy participants more effectively than total calprotectin for IPF (p = 0.0051) and COPD (p = 0.0069). Here, CPa9-HNE also distinguished IPF from COPD (p = 0.045) participants, which was not observed for total calprotectin (p = 0.98). Neutrophil activity was significantly higher, as assessed via serum CPa9-HNE, for COPD and IPF compared to healthy participants. Additionally, CPa9-HNE exceeded the ability of non-neoepitope calprotectin serum measurements to separate healthy from lung disease and even COPD from IPF participants, indicating that neutrophil activity is essential for both COPD and IPF.

Citrullinated and MMP-degraded vimentin is associated with chronic pulmonary diseases and genetic variants in PADI3/PADI4 and CFH in postmenopausal women.

Citrullinated vimentin has been linked to several chronic and autoimmune diseases, but how citrullinated vimentin is associated with disease prevalence and genetic variants in a clinical setting remains unknown. The aim of this study was to obtain a better understanding of the genetic variants and pathologies associated with citrullinated and MMP-degraded vimentin. Patient Registry data, serum samples and genotypes were collected for a total of 4369 Danish post-menopausal women enrolled in the Prospective Epidemiologic and Risk Factor study (PERF). Circulating citrullinated and MMP-degraded vimentin (VICM) was measured. Genome-wide association studies (GWAS) and phenome wide association studies (PheWAS) with levels of VICM were performed. High levels of VICM were significantly associated with the prevalence of chronic pulmonary diseases and death from respiratory and cardiovascular diseases (CVD). GWAS identified 33 single nucleotide polymorphisms (SNPs) with a significant association with VICM. These variants were in the peptidylarginine deiminase 3/4 (PADI3/PADI4) and Complement Factor H (CFH)/KCNT2 gene loci on chromosome 1. Serum levels of VICM, a marker of citrullinated and MMP-degraded vimentin, were associated with chronic pulmonary diseases and genetic variance in PADI3/PADI4 and CFH/ KCNT2. This points to the potential for VICM to be used as an activity marker of both citrullination and inflammation, identifying responders to targeted treatment and patients likely to experience disease progression.

Serum levels of fibrogenesis biomarkers reveal distinct endotypes predictive of response to weight loss in advanced nonalcoholic fatty liver disease.

BACKGROUND: NAFLD is associated with activation of fibroblasts and hepatic fibrosis. Substantial patient heterogeneity exists, so it remains challenging to risk-stratify patients. We hypothesized that the amount of fibroblast activity, as assessed by circulating biomarkers of collagen formation, can define a "high-risk, high-fibrogenesis" patient endotype that exhibits greater fibroblast activity and potentially more progressive disease, and this endotype may be more amendable to dietary intervention. METHODS: Patients with clinically confirmed advanced NAFLD were prescribed a very low-calorie diet (VLCD) intervention (∼800 kcal/d) to induce weight loss, achieved using total diet replacement. Serum markers of type III (PRO-C3) and IV collagen (PRO-C4) fibrogenesis were assessed at baseline every second week until the end of the VLCD, and 4 weeks post-VLCD and at 9 months follow-up. RESULTS: Twenty-six subjects had a mean weight loss of 9.7% with VLCD. This was associated with significant improvements in liver biochemistry. When stratified by baseline PRO-C3 and PRO-C4 into distinct fibrosis endotypes, these predicted substantial differences in collagen fibrogenesis marker dynamics in response to VLCD. Patients in the high activity group (PRO-C3 >11.4 ng/mL and/or PRO-C4 >236.5 ng/mL) exhibited a marked reduction of collagen fibrogenesis, ranging from a 40%-55% decrease in PRO-C3 and PRO-C4, while fibrogenesis remained unchanged in the low activity group. The biochemical response to weight loss was substantially greater in patients a priori exhibiting a high fibroblast activity endotype in contrast to patients with low activity. CONCLUSIONS: Thus, the likelihood of treatment response may be predicted at baseline by quantification of fibrogenesis biomarkers.

Biomarkers of Type IV Collagen Turnover Reflect Disease Activity in Patients with Early-Stage Non-Alcoholic Fatty Liver (NAFL).

BACKGROUND: Identification of progressive liver disease necessitates the finding of novel non-invasive methods to identify and monitor patients in need of early intervention. Investigating patients with early-liver injury may help identify unique biomarkers. Early-liver injury is characterized by remodeling of the hepatocyte basement membrane (BM) of the extracellular matrix. Thus, we quantified biomarkers targeting two distinct neo-epitopes of the major BM collagen, type IV collagen (PRO-C4 and C4M), in patients spanning the non-alcoholic fatty liver disease (NAFLD) spectrum. METHODS: We evaluated PRO-C4 and C4M in a cross-sectional study with 97 patients with NAFLD confirmed on histology. Serological levels of PRO-C4 and C4M were quantified using validated competitive enzyme-linked immunosorbent assays (ELISA). Using the fatty liver inhibition of progression (FLIP) algorithm, we stratified patients into two groups: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Biomarker levels were investigated in the two groups in patients stratified by the NAFLD activity score (NAS). In both groups, biomarker measurements were analyzed in relation to histological scorings of steatosis, inflammation, ballooning, and fibrosis. RESULTS: Patients had a body mass index (BMI) of 30.9 ± 5.6 kg/m2, age of 53 ± 13 years and a NAS range of 1-8. Upon stratification by FLIP, the NASH patients had higher platelets, ALT, and AST levels than the NAFL group. Both PRO-C4 (p = 0.0125) and C4M (p = 0.003) increased with increasing NAS solely within the NAFL group; however, a large variability was present in the NASH group. Furthermore, both markers were significantly associated with lobular inflammation (p = 0.020 and p = 0.048) and steatosis (p = 0.004 and p = 0.015) in patients with NAFL. CONCLUSIONS: This study found that type IV collagen turnover increased with the increase in NAS in patients with NAFL; however, this was not the case in patients with NASH. These findings support the assessments of the BM turnover using biomarkers in patients with early-disease development. These biomarkers may be used to track specific processes involved in the early pathobiology of NAFL.

Von Willebrand factor processing in patients with advanced chronic liver disease and its relation to portal hypertension and clinical outcome.

BACKGROUND AND AIMS: Endothelial dysfunction and portal hypertension (PH) are reflected by increased von Willebrand factor antigen (VWF-Ag) levels in advanced chronic liver disease (ACLD). This study investigated VWF release and cleavage and their association with PH and clinical outcomes. METHODS: Levels of VWF-Ag, VWF-N (VWF-propeptide), and VWF-A (VWF processed by the main VWF-cleaving protease ADAMTS13) were assessed in 229 patients with clinically stable ACLD (hepatic venous pressure gradient [HVPG] ≥ 6 mmHg; absence of bacterial infections or acute decompensation) undergoing HVPG-measurement. Liver-healthy individuals served as controls (n = 24). RESULTS: VWF-Ag and VWF-N were similarly accurate for the identification of clinically significant PH (CSPH; HVPG ≥ 10 mmHg) in compensated ACLD (AUROC: VWF-Ag 0.748; VWF-N 0.728). ADAMTS13 activity was similar between patients with ACLD and controls and did not correlate with PH and disease severity, whereas VWF cleavage decreased in patients with CSPH (i.e., VWF-Ag/-A-ratio increased). In vitro VWF activity strongly reflected VWF-Ag levels (Spearman's r = 0.874, p < 0.001), but decreased (vs. controls) in patients with CSPH when normalized to VWF-Ag levels (VWF-activity/-Ag-ratio). VWF-Act/-Ag ratio correlated negatively with ADAMTS13 activity (r =- 0.256, p < 0.001). ADAMTS13 activity was independently predictive for (i) portal vein thrombosis (PVT) and (ii) hepatic decompensation or liver-related death. CONCLUSIONS: VWF-Ag levels and its propeptide are similarly suitable surrogates of PH in patients with compensated ACLD. ADAMTS13-Act was not linked to disease and PH severity, however, when normalized to VWF-Ag, both VWF cleavage and VWF activity were decreased in patients with CSPH, as compared to liver-healthy individuals. Low ADAMTS13-Act was associated with presumably more procoagulant VWF and adverse outcomes. CLINICAL TRIAL NUMBER: NCT03267615.

PRO-C3 is a predictor of clinical outcomes in distinct cohorts of patients with advanced liver disease.

Background & Aims: Fibroblast activity is a key feature of fibrosis progression and organ function loss, leading to liver-related complications and mortality. The fibrogenesis marker, PRO-C3, has been shown to have prognostic significance in relation to fibrosis progression and as a treatment efficacy marker. We investigated whether PRO-C3 was prognostic for clinical outcome and mortality in two distinct cohorts of compensated cirrhosis. Methods: Cohort 1 was a rapid fibrosis progression cohort including 104 patients with HCV and biopsy-proven Ishak fibrosis stage ≥3 without prior clinical events. Cohort 2 was a prospective cohort including 172 patients with compensated cirrhosis of mixed aetiology. Patients were assessed for clinical outcomes. PRO-C3 was assessed in serum at baseline in cohorts 1 and 2, and compared with model for end-stage liver disease and albumin-bilirubin (ALBI) scores. Results: In cohort 1, a 2-fold increase in PRO-C3 was associated with 2.7-fold increased hazard of liver-related events (95% CI 1.6-4.6), whereas a one unit increase in ALBI score was associated with a 6.5-fold increased hazard (95% CI 2.9-14.6). In cohort 2, a 2-fold increase in PRO-C3 was associated with a 2.7-fold increased hazard (95% CI 1.8-3.9), whereas a one unit increase in ALBI score was associated with a 6.3-fold increased hazard (95% CI 3.0-13.2). A multivariable Cox regression analysis identified PRO-C3 and ALBI as being independently associated with the hazard of liver-related outcomes. Conclusions: PRO-C3 and ALBI were independent prognostic factors for predicting liver-related clinical outcomes. Understanding the dynamic range of PRO-C3 might enhance its use for both drug development and clinical practice. Impact and Implications: We tested novel proteins of liver scarring (PRO-C3) in two groups of liver patients with advanced disease to see if they could predict clinical events. We found that this marker and an established test called ALBI were both independently associated with future liver-related clinical outcomes.

A serologically assessed neo-epitope biomarker of cellular fibronectin degradation is related to pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive extracellular matrix (ECM) remodeling, herein ECM degradation. Fibronectin (FN) is an important component of the ECM that is produced by multiple cell types, including fibroblasts. Extra domain B (EDB) is specific for a cellular FN isoform which is found in the ECM. We sought to develop a non-invasive test to investigate whether matrix metalloproteinase 8 (MMP-8) degradation of EDB in cellular FN results in a specific protein fragment that can be assessed serologically and if levels relate to pulmonary fibrosis. METHOD: Cellular FN was cleaved in vitro by MMP-8 and a protein fragment was identified by mass spectrometry. A monoclonal antibody (mAb) was generated, targeting a neo-epitope originating from EDB in cellular FN. Utilizing this mAb, a neo-epitope specific enzyme-linked immunosorbent assay (FN-EDB) was developed and technically validated. Serum FN-EDB was assessed in an IPF cohort (n = 98), registered at clinicaltrials.gov (NCT02818712), and in healthy controls (n = 35). RESULTS: The FN-EDB assay had high specificity for the MMP-8 degraded neo-epitope and was technically robust. FN-EDB serum levels were not influenced by age, sex, ethnicity, or BMI. Moreover, FN-EDB serum levels were significantly higher in IPF patients (median 31.38 [IQR 25.79-46.84] ng/mL) as compared to healthy controls (median 28.05 [IQR 21.58-33.88] ng/mL, p = 0.023). CONCLUSION: We developed the neo-epitope specific FN-EDB assay, a competitive ELISA, as a tool for serological assessment of MMP-8 mediated degradation of EDB in cellular FN. This study indicates that degradation of EDB in cellular FN is elevated in IPF and warrants further investigation.

Validation of scores of PRO-C3 to predict liver-related events in alcohol-related liver disease.

BACKGROUND AND AIMS: Risk prediction in alcohol-related liver disease (ArLD) is an unmet need. We aimed to assess PRO-C3 models to predict liver-related events (LRE) in patients with a history of excessive alcohol use without an established diagnosis of chronic liver disease. METHODS: A prospective cohort study of 462 patients with ArLD, split into a derivation cohort of 221 secondary care patients and a validation cohort of 241 primary care patients. Baseline variables, including fibrogenesis marker PRO-C3, were used to develop a prediction model. Prognostic accuracy was compared to enhanced liver fibrosis (ELF), fibrosis-4-index (FIB-4), transient elastography (TE) and ADAPT. RESULTS: In the derivation and validation cohorts, 67 (30%) and 19 (8%) experienced an LRE during a median follow-up of 5.2 years (IQR: 3.2-6.8) and 4.0 years (IQR: 2.7-5.6). On top of PRO-C3 and ADAPT score, we generated a model (ALPACA) of independent predictors of LREs (PRO-C3, AST/ALT, platelets). ALPACA had high prognostic accuracy with a C-statistic of 0.85 in the derivation cohort, comparable to ELF (0.83) and TE (0.84) and significantly higher than FIB-4 (0.78), PRO-C3 (0.80) and ADAPT (0.81). In the validation cohort, all tests had comparable C-statistics. Compared to low-risk patients (ALPACA ≤11), high-risk patients (>11) had a subhazard ratio for LREs of 12.6 (95% CI 5.9-26.8, p < .001) and higher cumulative incidence (57% vs. 7%, p < .001; derivation cohort). We observed similar subhazard ratio in the validation cohort. CONCLUSIONS: PRO-C3-based scores are reliable tools to predict LREs in ArLD patients and are suitable for risk stratification in primary and secondary care.

Quantification of extracellular matrix remodeling for the non-invasive identification of graft fibrosis after liver transplantation.

Detecting patients with early post-transplant fibrosis after liver transplantation (LT) is very important. Non-invasive tests are needed to avoid liver biopsies. We aimed to detect fibrosis in liver transplant recipients (LTR) using extracellular matrix (ECM) remodeling biomarkers. ECM biomarkers for type III (PRO-C3), IV (PRO-C4), VI (PRO-C6) and XVIII (PRO-C18L) collagen formation and type IV collagen degradation (C4M) were measured by ELISA in prospectively collected, cryopreserved plasma samples (n = 100) of LTR with paired liver biopsies from a protocol biopsy program. Fibrosis ≥ F2 was present in 29% of patients (median 44 months post-LT). APRI and FIB-4 neither identified significant fibrosis nor were correlated with histopathological fibrosis scores, while ECM biomarkers (AUCs 0.67-0.74) did. The median levels of PRO-C3 (15.7 vs. 11.6 ng/ml; p = 0.002) and C4M (22.9 vs. 11.6 ng/ml; p = 0.006) levels were elevated in T-cell-mediated rejection compared to normal graft function. The median levels of PRO-C4 (178.9 vs. 151.8 ng/ml; p = 0.009) and C4M (18.9 vs. 16.8 ng/ml; p = 0.004) levels were increased if donor-specific antibodies were present. PRO-C6 had the highest sensitivity (100%), NPV (100%) and negative likelihood-ratio (0) for graft fibrosis. To conclude, ECM biomarkers are helpful in identifying patients at risk of relevant graft fibrosis.

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study.

BACKGROUND AND AIMS: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. APPROACH AND RESULTS: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). CONCLUSIONS: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis.

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.

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.

Circulating levels of endotrophin and cross-linked type III collagen reflect liver fibrosis in people with HIV.

BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.

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 prospective pilot study of a gluten-free diet for primary sclerosing cholangitis and associated colitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a progressive bile duct disease associated with inflammatory bowel disease (PSC-IBD). AIM: To investigate whether patients with PSC-IBD benefit from a gluten-free and amylase trypsin inhibitor (ATI)-free diet (GFD). METHODS: We performed a prospective clinical pilot study administering an eight-week GFD. The primary outcomes were colonic inflammation assessed by proctosigmoidoscopy, and liver stiffness (surrogate for fibrosis, inflammation and cholestasis) measured by transient elastography before and after GFD. Amongst the secondary (exploratory) outcomes were colonic mucosal and serum cytokine/chemokine changes, the intestinal microbiome and transcriptome dynamics, and shifts in serum markers of hepatic fibrogenesis. RESULTS: Fifteen patients with PSC-IBD completed the study. The study did not meet its primary outcome: the endoscopic score and liver stiffness remained unchanged. However, the expression of pro-inflammatory mucosal cytokines and chemokines such as IL6, IL8, CCL2, and TNFα was significantly down-regulated. Two critical markers of liver fibrosis and matrix remodelling, thrombospondin-2 and -4, decreased significantly. The microbiota composition changed slightly, including a decrease in the pathogen Romboutsia ilealis. The intestinal transcriptome indicated a gut barrier improvement. Pruritus, fatigue, overall well-being, faecal calprotectin levels, and serum alkaline phosphatase did not change significantly. CONCLUSIONS: This study did not demonstrate a clinical improvement with short-term GFD in patients with PSC-IBD. However, a gluten/ATI-free diet may improve biomarkers of intestinal inflammation and barrier function in these patients with associated changes in the enteric microbiota. Further investigation of the therapeutic potential of the GFD in PSC-IBD is warranted.

N-terminal propeptide of type 3 collagen-based sequential algorithm can identify high-risk steatohepatitis and fibrosis in MAFLD.

BACKGROUND AND AIMS: With metabolic dysfunction-associated fatty liver disease (MAFLD) incidence and prevalence sharply increasing globally, there is an urgent need for non-invasive diagnostic tests to accurately screen high-risk MAFLD patients for liver inflammation and fibrosis. We aimed to develop a novel sequential algorithm based on N-terminal propeptide of type 3 collagen (PRO-C3) for disease risk stratification in patients with MAFLD. METHODS: A derivation and independent validation cohort of 327 and 142 patients with biopsy-confirmed MAFLD were studied. We compared the diagnostic performances of various non-invasive scores in different disease states, and a novel sequential algorithm was constructed by combining the best performing non-invasive scores. RESULTS: For patients with high-risk progressive steatohepatitis (i.e., steatohepatitis + NAFLD activity score ≥ 4 + F ≥ 2), the AUROC of FAST score was 0.801 (95% confidence interval (CI): 0.739-0.863), and the negative predictive value (NPV) was 0.951. For advanced fibrosis (≥ F3) and cirrhosis (F4), the AUROCs of ADAPT and Agile 4 were 0.879 (95%CI 0.825-0.933) and 0.943 (95%CI 0.892-0.994), and the NPV were 0.972 and 0.992. Sequential algorithm of ADAPT + Agile 4 combination was better than other combinations for risk stratification of patients with severe fibrosis (AUROC = 0.88), with similar results in the validation cohort. Meanwhile, in all subgroup analyses (stratifying by sex, age, diabetes, NAS, BMI and ALT), ADAPT + Agile 4 had a good diagnostic performance. CONCLUSIONS: The new sequential algorithm reliably identifies liver inflammation and fibrosis in MAFLD, making it easier to exclude low-risk patients and recommending high-risk MAFLD patients for clinical trials and emerging pharmacotherapies.

Biomarkers reflecting pericellular fibrosis improve together with liver histology after bariatric surgery in early non-alcoholic fatty liver disease.

AIM: Non-invasive tests for non-alcoholic fatty liver disease (NAFLD) are needed for assessing disease stage, prognosis and treatment efficacy. Extracellular matrix biomarkers, such as PRO-C3, are useful as biomarkers of advanced liver fibrosis. However, non-invasive biomarkers of early-stage NAFLD, characterized by pericellular fibrosis, are lacking. Here, we measured serological biomarkers of type IV and VIII collagens reflecting the remodeling of the pericellular basement membrane to explore the effect of bariatric surgery on pericellular fibrosis in patients with early NAFLD. METHODS: Seventy patients with severe obesity underwent bariatric surgery. The cohort consisted of 61 % females who had a mean age of 44. Patients had a median NAFLD activity score of 3 and mild-to-moderate fibrosis F0 (3 %), F1 (86 %), and F2 (11 %). Blood samples were taken at baseline, three, six and 12 months after surgery. At 12 months, 40 patients had a follow-up liver biopsy. The biomarkers PRO-C3, PRO-C4, C4M, and PRO-C8 were measured using indirect competitive ELISAs. RESULTS: Twelve months after surgery patients had significantly lower levels of ALT, GGT, HbA1c, fasting glucose, and CRP. The pericellular fibrosis biomarkers, C4M, PRO-C4, and PRO-C8 decreased by 24 %, 18 % and 44 %, respectively (p < 0.0001), while the interstitial matrix fibrosis marker PRO-C3 remained unchanged. Furthermore, baseline C4M was associated with histologically assessed hepatocyte ballooning and lobular inflammation in patients with (p = 0.032) and without (p = 0.032) steatosis, respectively. CONCLUSION: Biomarkers of pericellular fibrosis decrease in early-stage NAFLD after patients undergo bariatric surgery and potentially reflect an improvement in liver histology.