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.

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.

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.

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.

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.

Nintedanib modulates type III collagen turnover in viable precision-cut lung slices from bleomycin-treated rats and patients with pulmonary fibrosis.

BACKGROUND: Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone. METHODS: Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants. RESULTS: In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied. CONCLUSIONS: This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.

Systemic inflammation is linked to liver fibrogenesis in patients with advanced chronic liver disease.

BACKGROUND & AIMS: Experimental evidence indicates that systemic inflammation (SI) promotes liver fibrogenesis. This study investigated the potential link between SI and fibrogenesis in patients with advanced chronic liver disease (ACLD). METHODS: Serum biomarkers of SI (CRP, IL-6, procalcitonin [PCT]) and extracellular matrix (ECM) turnover (i.e., fibrogenesis/fibrolysis) were analysed in 215 prospectively recruited patients with ACLD (hepatic venous pressure gradient [HVPG] ≥6 mm Hg) undergoing hepatic vein catheterization. Patients with non-elective hospitalization or bacterial infection were excluded. Histological alpha-smooth muscle actin (α-SMA) area was quantified on full biopsy scans by automated morphometric quantification in a subset of 34 patients who underwent concomitant transjugular liver biopsy. RESULTS: Histological α-SMA proportionate area correlated with enhanced liver fibrosis (ELF) score (Spearman's ρ = 0.660, p < .001), markers of collagen formation (PRO-C3, ρ = 0.717, p < .001; PRO-C6, ρ = 0.526, p = .002) and tissue inhibitor of metalloproteinases-1 (TIMP1; ρ = 0.547, p < .001), indicating that these blood biomarkers are capable of reflecting the dynamic process of ECM turnover. CRP, IL-6 and PCT levels correlated with ELF, biomarkers of collagen synthesis/degradation and TIMP1, both in compensated and decompensated patients. Multivariate linear regression models (adjusted for HVPG) confirmed that CRP, IL-6 and PCT were independently linked to markers of liver fibrogenesis and ECM turnover. CONCLUSION: Systemic inflammation is linked to both liver fibrogenesis and ECM turnover in ACLD and this association is not confounded by the severity of liver disease, as evaluated by HVPG. Our study confirms experimental data on the detrimental impact of SI on ECM deposition and fibrosis progression in a thoroughly characterized cohort of patients with ACLD.

LPA1 antagonist BMS-986020 changes collagen dynamics and exerts antifibrotic effects in vitro and in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease with limited treatment options. A phase 2 trial (NCT01766817) showed that twice-daily treatment with BMS-986020, a lysophosphatidic acid receptor 1 (LPA1) antagonist, significantly decreased the slope of forced vital capacity (FVC) decline over 26 weeks compared with placebo in patients with IPF. This analysis aimed to better understand the impact of LPA1 antagonism on extracellular matrix (ECM)-neoepitope biomarkers and lung function through a post hoc analysis of the phase 2 study, along with an in vitro fibrogenesis model. METHODS: Serum levels of nine ECM-neoepitope biomarkers were measured in patients with IPF. The association of biomarkers with baseline and change from baseline FVC and quantitative lung fibrosis as measured with high-resolution computed tomography, and differences between treatment arms using linear mixed models, were assessed. The Scar-in-a-Jar in vitro fibrogenesis model was used to further elucidate the antifibrotic mechanism of BMS-986020. RESULTS: In 140 patients with IPF, baseline ECM-neoepitope biomarker levels did not predict FVC progression but was significantly correlated with baseline FVC and lung fibrosis measurements. Most serum ECM-neoepitope biomarker levels were significantly reduced following BMS-986020 treatment compared with placebo, and several of the reductions correlated with FVC and/or lung fibrosis improvement. In the Scar-in-a-Jar in vitro model, BMS-986020 potently inhibited LPA1-induced fibrogenesis. CONCLUSIONS: BMS-986020 reduced serum ECM-neoepitope biomarkers, which were previously associated with IPF prognosis. In vitro, LPA promoted fibrogenesis, which was LPA1 dependent and inhibited by BMS-986020. Together these data elucidate a novel antifibrotic mechanism of action for pharmacological LPA1 blockade. Trial registration ClinicalTrials.gov identifier: NCT01766817; First posted: January 11, 2013; https://clinicaltrials.gov/ct2/show/NCT01766817 .

Stage-dependent expression of fibrogenic markers in alcohol-related liver disease.

Alcohol-related liver disease (ALD) is the primary cause of liver-related mortality and morbidity. Liver fibrosis remains the best validated surrogate marker for patient prognosis and is usually assessed in liver biopsies using histochemical stains detecting collagen as the major extracellular matrix (ECM) component in ALD. Distinction of collagen subtypes is of clinical interest, as they, including their cleavage products, have different functions. Changes in production, distribution, and composition of specific collagen subtypes and other extracellular matrix (ECM) components as well as markers for their main cellular source (activated hepatic stellate cells (aHSCs) and myofibroblasts (MFBs)) have not been fully elucidated in ALD. Our aims were to investigate the stage-dependent expression of collagen subtypes and markers for aHSCs and MFBs in ALD. We included liver biopsies from 49 ALD patients with fibrosis stages F0-F4. Biopsies were classified according to the semi-quantitative non-alcoholic fatty liver disease (NAFLD) activity score (NAS-CRN). Slides were stained with H&E, Sirius Red, and immunohistochemically with antibodies against collagen types I, III, IV and VI, and aHSC/MFB markers α-SMA, osteonectin, and CD271. Expression was examined using automated digital imaging analysis (DIA), by calculating the positive area relative to the total liver biopsy area (proportionate area). Likewise, collagen-proportionate area (CPA) was assessed using DIA of Sirius Red stained slides. CPA correlated highly with fibrosis stage (rs = 0.88, P = 5.9E-17) and moderately with activity score (rs = 0.58, P = 0.00001). Collagen type I proportionate area increased from 0.3% (± 0.1) at F1 to 10.2% (± 1.6) at F4 (P < 0.001). Collagen type III proportionate area increased from 0.6% (± 0.2) at F0 to 11.9% (± 1.7) at F4 (P < 0.001). Collagen type IV proportionate area increased from 17.0% (± 2.5) at F0 to 27.0% (± 3.9) at F4 (P = 0.079). Collagen type VI proportionate area increased from 14.8% (± 1.4) at F0 to 31.4% (± 2.4) at F4 (P < 0.001). Proportionate areas for α-SMA and CD271 increased from 10.7% (± 1.6) and 6.5% (± 1.0) at F0 to 29.5% (± 3.4) and 22.1% (± 2.2) at F4 (P < 0.001). Proportionate area for osteonectin increased from 1.4-1.8% at F0-F2 to 3.1% (± 0.5) at F3 and 3.2% (± 0.6) at F4 (P < 0.05). In conclusion, our data indicate that collagen types I, III and VI and the aHSC/MFB markers α-SMA and CD271 show a stage-dependent increase in ALD. In early ALD, collagen types IV and VI are important components of the perisinusoidal and pericellular fibrosis. Immunohistochemistry is a valuable additional tool to characterize the ECM changes in ALD. Further research is needed to explore the functional role of CD271 and the stage-dependent expression of other collagen subtypes in ALD.

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.

Among simple non-invasive scores, Pro-C3 and ADAPT best exclude advanced fibrosis in Asian patients with MAFLD.

BACKGROUND: With metabolic dysfunction-associated fatty liver disease (MAFLD) incidence and prevalence increasing, it is necessary to identify patients with advanced fibrosis (F3-F4 stages). We evaluated the performance of new biomarkers and algorithms for diagnosing advanced fibrosis in an Asian population. METHODS: Data from two Asian cohorts (including 851 biopsy-proven MAFLD [578 from Wenzhou, 273 from Hong Kong]) were studied. The association between N-terminal propeptide of type 3 collagen (PRO-C3) and the histologic stage of liver fibrosis was analyzed by multivariable linear regression. The area under the receiver operating characteristic curve (AUROC) was used to test the diagnostic performance of serum PRO-C3 and the ADAPT score for advanced fibrosis and compared them to other established non-invasive tests. RESULTS: Serum PRO-C3 levels increased progressively across liver fibrosis stages and correlated with advanced fibrosis (P < 0.001). The ADAPT score had an AUROC of 0.865 (95% confidence interval 0.829-0.901) for advanced fibrosis; the accuracy, sensitivity and negative predictive values were 81.4%, 82.2% and 96.1%, respectively. This result was better compared to that of PRO-C3 alone or other non-invasive fibrosis biomarkers (aspartate aminotransferase-to-platelet ratio index, Fibrosis-4, BARD, and NAFLD fibrosis score). In subgroup analyses (including sex, age, diabetes, NAFLD activity score, body mass index or serum alanine aminotransferase levels), the ADAPT score had good diagnostic performance. CONCLUSION: PRO-C3 and the ADAPT score reliably exclude advanced fibrosis in MAFLD patients and reduce the need for liver biopsy.

Serologically assessed heat shock protein 47 is related to fibrosis stage in early compensated alcohol-related liver disease.

BACKGROUND AND AIMS: Heat shock protein (HSP)47 is a collagen-specific chaperone, essential for the correct formation of fibrillar procollagens. Collagen accumulation in the extracellular matrix (ECM) is a hallmark of fibrogenesis. The expression of HSP47 is proportional to the rate of collagen formation. Thus, HSP47 is a potential drug target for fibrotic diseases. We hypothesized that a C-terminal fragment of HSP47 (HSP47-C) could be quantified serologically and related to liver fibrosis stage. For this, a novel competitive enzyme-linked immunosorbent assay (ELISA) was developed. METHOD: An ELISA employing a monoclonal antibody targeting HSP47-C was developed and technically validated. The assay was evaluated in serum from a cross-sectional biopsy-controlled study of 281 patients with alcohol-related liver disease (ALD) and 50 gender, age and BMI matched healthy controls (HC). All liver biopsies from ALD patients were scored by one pathologist according to fibrosis stage (F0-4). RESULTS: The HSP47-C assay was technically robust and specific for the target sequence. HSP47-C was 39% higher in ALD patients (median 17.7 ng/mL, IQR 12.4-24.0 ng/mL) compared to HC (median 12.7 ng/mL, IQR 9.4-15.7 ng/mL, p < 0.0001). In addition, HSP47-C was elevated in patients with severe fibrosis (F3-4, median 22.8 ng/mL, IQR 17.5-33.3 ng/mL) compared to none-to-moderate fibrosis (F0-2, median 16.5 ng/mL, IQR 11.8-22.5 ng/mL) with an AUROC of 0.72 (p < 0.0001). HSP47-C also correlated with other liver disease parameters, albumin, bilirubin and aspartate transaminase. CONCLUSION: We developed a competitive ELISA for serological detection of HSP47-C. The study supports HSP47 as a potential marker of liver fibrosis in ALD.

Determining a healthy reference range and factors potentially influencing PRO-C3 - A biomarker of liver fibrosis.

BACKGROUND & AIMS: Progressive fibrosis has been identified as the major predictor of mortality in patients with non-alcoholic fatty liver disease (NAFLD). Several biomarkers are currently being evaluated for their ability to substitute the liver biopsy as the reference standard. Recent clinical studies in NAFLD/NASH patients support the utility of PRO-C3, a marker of type III collagen formation, as a marker for the degree of fibrosis, disease activity, and effect of treatment. Here we establish the healthy reference range, optimal sample handling conditions for both short- and long-term serum storage, and robustness for the PRO-C3 assay. METHODS: PRO-C3 was measured in 269 healthy volunteers and in 222 NAFLD patients. Robustness of the PRO-C3 assay was measured according to Clinical and Laboratory Standards Institute standards and included validation of interference, precision, and reagent stability, whilst sample stability was defined for storage at different temperatures and for 3 freeze-thaw cycles. Fibrosis scoring was based on histological assessments and used as a reference for the diagnostic ability of PRO-C3 to discriminate between patients with different levels of fibrosis. RESULTS: Robustness of the PRO-C3 analysis validated by interference, precision, and reagent stability was found to be within the predefined acceptance criteria. The healthy reference range was determined to be 6.1-14.7 ng/ml. Levels of PRO-C3 were not affected by sex, age, BMI, or ethnicity. Levels of PRO-C3 were able to identify patients with clinically significant fibrosis and advanced fibrosis (AUC = 0.83 (95% CI [0.77-0.88], p <0.0001), and AUC = 0.79 (95% CI [0.73-0.85], p <0.0001), respectively). CONCLUSIONS: The assay proved to be robust and sample stability was found to comply with hospital sample handling requirements. PRO-C3 measured in samples from patients with NAFLD/NASH was diagnostic for significant and advanced liver fibrosis. LAY SUMMARY: We showed that PRO-C3 levels were stable under conditions conforming with hospital sample-handling requirements. We determined a healthy reference range and showed that PRO-C3 levels were not associated with sex, age, BMI, or ethnicity. Finally, we provide further evidence of an association of PRO-C3 with increasing liver fibrosis.

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.

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

BACKGROUND: Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test. RESULTS: Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5). CONCLUSION: C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

A Sequential Algorithm Combining ADAPT and Liver Stiffness Can Stage Metabolic-Associated Fatty Liver Disease in Hospital-Based and Primary Care Patients.

INTRODUCTION: Metabolic-associated fatty liver disease is common, with fibrosis the major determinant of adverse outcomes. Population-based screening tools with high diagnostic accuracy for the staging of fibrosis are lacking. METHODS: Three independent cohorts, 2 with both liver biopsy and liver stiffness measurements (LSMs, n = 254 and 65) and a population sample (n = 713), were studied. The performance of a recently developed noninvasive algorithm (ADAPT [age, diabetes, PRO-C3 and platelets panel]) as well as aspartate aminotransferase-to-platelet ratio index, fibrosis-4, nonalcoholic fatty liver disease fibrosis score, and LSM was used to stage patients for significant (≥F2) and advanced (≥F3) fibrosis. RESULTS: In the hospital-based cohorts, the N-terminal propeptide of type 3 collagen (Pro-C3) increased with fibrosis stage (P < 0.0001) and independently associated with advanced fibrosis (odds ratio = 1.091, 95% confidence interval [CI]: 1.053-1.113, P = 0.0001). ADAPT showed areas under the receiver operating characteristics curve of 0.831 (95% CI: 0.779-0.875) in the derivation and 0.879 (95% CI: 0.774-0.946) in the validation cohort for advanced fibrosis. This was superior to the existing fibrosis scores, aspartate aminotransferase-to-platelet ratio index, fibrosis-4, BARD (BMI, aspartate aminotransferase to alanine aminotransferase ratio [AAR], diabetes), and nonalcoholic fatty liver disease fibrosis score in most comparisons and comparable with LSM. Serial use of ADAPT and LSM had diagnostic accuracy of 92.5%, with 98% and 100% negative predictive value in the derivation and validation cohorts, respectively. In the population cohort, PRO-C3 associated with advanced fibrosis (P = 0.04), while ADAPT had a negative predictive value of 98% for excluding advanced fibrosis. DISCUSSION: PRO-C3 and ADAPT reliably exclude advanced fibrosis in low-risk populations. The serial combination of ADAPT with LSM has high diagnostic accuracy with a low requirement for liver biopsy. The proposed algorithm would help stratify those who need biopsies and narrow down those patients who would need to be referred to specialty clinics.

Cross-Linked Multimeric Pro-Peptides of Type III Collagen (PC3X) in Hepatocellular Carcinoma - A Biomarker That Provides Additional Prognostic Value in AFP Positive Patients.

PURPOSE: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis is present in 80-90% of HCC patients. Cirrhosis is characterized by deposition and cross-linking of collagens that have crucial roles in HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC. PATIENTS AND METHODS: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS). RESULTS: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (p=0.0002, p<0.0001). In patients with normal AFP (<20 IU/mL), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.72 and 0.68, respectively. High PC3X and AFP predicted for poor PFS (HRPC3X=1.80, p=0.032; HRAFP=1.70, p=0.031) and OS (HRPC3X=2.12, p=0.024; HRAFP=2.55; p=0.003), whereas PRO-C3 did not (PFS: HR=1.19, p=0.059 and OS: HR=1.12, p=0.324). PC3X was independent of AFP (PFS: HR=1.74, p=0.045 and OS: HR=2.21, p=0.018) and combining the two improved prognostic value (PFS: HR=2.66, p=0.004 and OS: HR=5.86, p<0.0001). CONCLUSION: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC.

A Fragment of Collagen Type VI alpha-3 chain is Elevated in Serum from Patients with Gastrointestinal Disorders.

Extracellular matrix (ECM) remodeling is a hallmark of the pathology of gastrointestinal disorders. Collagen type VI (COL6) is produced by fibroblasts, and the COL6 α3-chain has shown to be elevated in patients with ulcerative colitis (UC), Crohn's disease (CD) and colorectal cancer (CRC). Measuring COL6α3 in serum may therefore have potential as a biomarker for gastrointestinal disorders. The aims of this study were to develop and validate a competitive ELISA targeting a specific neo-epitope of COL6α3 and evaluate its associations with the gastrointestinal disorders UC, CD and CRC, in comparison to healthy controls. A monoclonal antibody was raised against a matrix metalloproteinase-2 and -9 specific cleavage site of COL6α3 (C6Mα3) and employed in a competitive enzyme-linked immunosorbent assay (ELISA). The assay was developed and technically validated. Levels of C6Mα3 were measured in serum from patients with UC (n = 58), CD (n = 44) and CRC (n = 39) and compared to healthy controls (n = 32). The levels of C6Mα3 were elevated in patients with UC, CD and CRC patients compared to healthy controls (all p < 0.0001). The area under the receiver operating characteristics (AUROC) curve for separation of patients with UC from healthy controls was 0.972 (95% CI: 0.925-1.020, p < 0.0001), with CD from healthy controls was 0.947 (95% CI: 0.885-1.009, p < 0.0001) and with CRC from healthy controls was 0.890 (95% CI: 0.809-0.972, p < 0.0001). We developed a technically robust assay targeting a fragment of COL6, which was elevated in serum from patients with UC, CD and CRC.