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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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A. H. Hansen, D. G. K. Rasmussen, F. Genovese, L. L. Langholm, T. Manon-Jensen, D. J. Leeming, M. A. Karsdal, and J. M. B. Sand, are employed at Nordic Bioscience which is a company involved in discovery and development of biochemical markers. Moreover, D. G. K. Rasmussen, T. Manon-Jensen, M. A. Karsdal, D. J. Leeming, L. L. Langholm and J. M. B. Sand are shareholders in Nordic Bioscience Holding A/S. H.W. Breisnes is a PhD student at University of Copenhagen, conducting a collaboration project with Nordic Bioscience., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests QMA is the coordinator of the Innovative Medicines Initiative 2 (IMI2) LITMUS consortium funded by the IMI2 Program of the EU (777377) and is a Newcastle National Institute for Health and Care Research Biomedical Research Centre investigator. The multistakeholder LITMUS consortium includes industry partners and received funding from the European Federation of Pharmaceutical Industries and Associations. QMA also declares research grant funding from AbbVie, AstraZeneca, Boehringer Ingelheim, Glympse Bio, Intercept, Novartis, and Pfizer; is a consultant on behalf of Newcastle University to Alimentiv, Akero, AstraZeneca, Axcella, 89Bio, Boehringer Ingelheim, Bristol-Myers Squibb, Galmed, Genfit, Genentech, Gilead, GlaxoSmithKline, Hanmi, HistoIndex, Intercept, Inventiva, Ionis, IQVIA, Janssen, Madrigal, Medpace, Merck, NGMBio, Novartis, Novo Nordisk, PathAI, Pfizer, Poxel, Resolution Therapeutics, Roche, Ridgeline Therapeutics, RTI International, Shionogi, and Terns; has received speaker fees from Fishawack, Integritas Communications, Kenes, Novo Nordisk, Madrigal, Medscape, and Springer Healthcare; is on the North Sea Therapeutics Data Safety and Monitoring Committee at Medpace on behalf of Newcastle University; and has received royalties from Elsevier. JB declares research grant funding from Echosens, Intercept, Inventiva, and Siemens; consulting fees from Echosens, Intercept, and Siemens; speaker fees from Gilead, Intercept, Lilly, and Siemens; and is a member of advisory boards of Bristol-Myers Squibb, Echosens, Intercept, Novo Nordisk, and Merck Sharp & Dohme. DT declares grants from and contracts with Histoindex; consulting fees from Alimentiv, Clinnovate Health UK, Allergan, Cirius Therapeutics, Intercept Pharmaceuticals, ICON Clinical Research, and Ionis Pharmaceuticals; and was the past President of the European Society of Pathology (unpaid). PB is the owner and Director of Liverpat. AG declares support from the IMI2 LITMUS project and research grants from Falk and Intercept; consulting fees from AbbVie, Alexion, Bayer, Bristol-Meyers Squibb, Eisai, Gilead, Intercept, Ipsen, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Sanofi-Aventis, and Sequana; research fees from AbbVie, Alexion, Bristol-Meyers Squibb, Burger Stein, CSL Behring, Falk, Gilead, Intercept, Mert, Merck Sharp & Dohme, Novartis, Roche, and Sequana; travel and meeting support from Intercept, Gilead, AbbVie, and Falk; and is a member of an advisory board of Falk. SF has a Senior Clinical Investigator fellowship from the Research Foundation Flanders (1802154N); declares grants to his institution (University of Antwerp, Belgium) from Genfit, Gilead Sciences, Roche, and Bristol-Meyers Squibb; has been a consultant to Roche, Intercept, Gilead Sciences, Echosens, Allergan, Genentech, AbbVie, Novo Nordisk, Bayer, Novartis, Bristol-Meyers Squibb, AstraZeneca, Boehringer Ingelheim, Galmed, Merck Sharp & Dohme, Promethera, Janssen Pharmaceutica, Coherus, Actelion Madrigal, Astellas, Julius Clinical, Genfit, NGM Bio, and Inventiva; and has been lecturer for AbbVie, Allergan, Bayer, Genfit, and Gilead Sciences. MA declares research grants from GlaxoSmithKline, Takeda, and AstraZeneca and consulting fees from Intercept and AstraZeneca. GP declares support from AbbVie, Gilead Sciences, Janssen, and Takeda; consulting fees from AbbVie, Amgen, Astellas, Astra Zeneca, Gilead Sciences, GlaxoSmithKline, Janssen, Ipsen, Merck Sharp & Dohme, Novartis, Novo Nordisk, and Roche; speaker fees from AbbVie, Gilead Sciences, GlaxoSmithKline, Ipsen, Merck Sharp & Dohme, Novartis, Novo Nordisk, Roche, and Takeda; and travel support from AbbVie, Gilead Sciences, Ipsen, Janssen, and Novo Nordisk. HC-P declares support from the IMI2 LITMUS project; consulting fees from Intercept, Novo Nordisk, and Orphalan; and speaker fees from Eisai and Roche Portugal. J-FD is a member of the advisory committees of Alentis, AstraZeneca, Bayer, Bristol-Myers Squibb, Enyo, Esai, Falk, Genfit, Gilead Sciences, Intercept, Inventiva, Ipsen, Lilly, Madrigal, Merck, Novartis, Novo Nordisk, and Roche; declares travel and meeting support from Gilead and speaking and teaching fees from Bayer, Bristol-Myers Squibb, Intercept, Gilead Sciences, Novartis, and Roche; has received fees from the journal GUT as an Associate Editor and Uptodate; and is President of the Swiss NASH Foundation and the Swiss Foundation against Liver Cancer. DJL declares support from the IMI2 LITMUS project and Nordic Bioscience and has stock in Nordic Bioscience. SAH declares grants from Akero Therapeutics, Axcella Health, Cirius Therapeutics, CiVi Biopharma, Cymabay, Enyo Pharma, Galectin, Galmed Research and Development, Genfit, Gilead Sciences, Hepion Pharmaceuticals, Hightide Therapeutics, Intercept Pharmaceuticals, Madrigal Pharmaceuticals, Metacrine, NGM Bio, Northsea Therapeutics, Novartis Pharmaceuticals, Novo Nordisk, Poxel, Sagimet Biosciences, and Viking Therapeutics; consulting and speaker fees from AgomAB, Akero Therapeutics, Alentis Therapeutics, Alimentiv, Altimmune, Axcella Health, Boston Pharmaceuticals, Riley FBR, BVF Partners, Cohbar, Canfite, Corcept Therapeutics, Cymabay, Echosens North America, Enyo Pharma, Fibronostics, Foresite Labs, Fortress Biotech, Galectin Therapeutics, Genfit, Genius Group, Hepion Pharmaceuticals, Hightide Therapeutics, HistoIndex, Inipharm, Intercept Pharmaceuticals, Ionis, Kowa Research Institute, Madrigal Pharmaceuticals, Medpace, Metacrine, Microba, NGM Biopharmaceuticals, Northsea Therapeutics, Novo Nordisk, Nutrasource, Perspectum Diagnostics, Piper, Sandler, Poxel, Prometic Pharma, Ridgeline, Sagimet Biosciences, Sonic Incytes, Medical, Terns, and Viking Therapeutics; is a member of advisory boards of 89 Bio, Akero Therapeutics, Altimmune, Arrowhead, Axcella Health, Chronwell, CiVi, Cymabay, Echosens North America, Foresite Labs, Galectin Therapeutics, Galmed Research and Development, Genfit, Gilead Sciences, Hepion Pharmaceuticals, Hightide Therapeutics, HistoIndex, Indalo, Intercept Pharmaceuticals, Madrigal Pharmaceuticals, Medpace, Metacrine, NGM Bio, Northsea Therapeutics, Novartis Pharmaceuticals, Novo Nordisk, PathAI, Poxel, Prometic Pharma, Ridgeline, Sagimet Biosciences, Sonic Incytes Medical, Terns, and Theratechnologies; has stock in Akero Therapeutics, Chronwell, Cirius Therapeutics, Galectin Therapeutics, Genfit, Hepion Pharmaceuticals, HistoIndex, Metacrine, NGM Bio, Northsea Therapeutics, Sonic Incytes, and Medical Corp; and declares consultancy services to Akero Therapeutics, Axcella Health, Cirius Therapeutics, CiVi Biopharma, Cymabay, Enyo Pharma, Galectin Therapeutics, Galmed Research and Development, Genfit, Gilead Sciences, Hepion Pharmaceuticals, Hightide Therapeutics, Intercept Pharmaceuticals, Madrigal Pharmaceuticals, Metacrine, NGM Bio, Northsea Therapeutics, Novartis Pharmaceuticals, Novo Nordisk, Poxel, Sagimet Biosciences, and Viking Therapeutics. YC is an employee of Eli Lilly and the company and hold stocks and shares in Eli Lilly. JFC declares support from Oxford National Institute for Health and Care Research Biomedical Research Centre; consulting fees from Intercept and Alnylam; speaker fees from AstraZeneca; and is a member of an advisory board of Intercept. MP declares support from the IMI2 LITMUS project and grants from Cancer Research UK (C30358/A29725); has a patent for medical imaging (WO2015155521A1); and is a shareholder in Perspectum. HY-J declares support from the IMI2 LITMUS project; grants from Academy of Finland, Sigrid Juselius Foundation, and EVO foundation; consulting fees from Boehringer Ingelheim; speaker fees from Novo Nordisk and GlaxoSmithKline; and is a member of advisory boards of Merck Sharp & Dohme, Eli Lilly, and Hamni. JC declares grants and research support from Gilead Sciences, AbbVie, and Intercept Pharmaceuticals; consultant fees from Gilead Sciences, AbbVie, Merck Sharp & Dohme, Shionogi, Intercept Pharmaceuticals, Janssen Pharmaceuticals, Celgene, and Alexion; speaker support from Gilead Sciences, AbbVie, Shionogi, Janssen Pharmaceuticals, and Celgene; meeting and travel support from Gilead Sciences, AbbVie, and Janssen Pharmaceuticals; and is President of the Spanish Society of Digestive Pathology. MK declares support from the IMI2 LITMUS project and Nordic Bioscience. He is a Nordic Bioscience stock share holder. RO is an employee of SomaLogic; declares support from the IMI2 LITMUS project (SomaLogic funded the SomaScan proteomic assay results as a contribution to LITMUS); is on the patent application of SomaLogic for NASH biomarkers; and has stock in SomaLogic. RT is an employee of and holds stock in AstraZeneca, Sweden. KD is an employee of Eli Lilly and the company and hold stocks and shares in Eli Lilly. CY is an employee and stock and share holder of Pfizer. CB is a former employee of Novartis Pharma and declares financial support from Novartis Pharma; has pending patents for treatment of NASH (Novartis); and has stock in Novartis Pharma and Merck. ME is a member of the advisory board of AMRA Medical. GPA declares support from the IMI2 LITMUS project and consulting fees from Clinicpace, Servier Pharmaceuticals, GlaxoSmithKline, NuCana, AstraZeneca, and Benevolent AI. JMS declares grants from Gilead Sciences, Boehringer Ingelheim, Nordic Bioscience, and Siemens Healthcare; consulting fees from Apollo Endosurgery, Albireo Pharma, Bayer, Bristol-Meyers Squibb, Boehringer Ingelheim, Echosens, Genfit, Gilead Sciences, GlaxoSmithKline, Heel, Intercept Pharmaceuticals, Ipsen, Inventiva Pharma, Julius Clinical, Madrigal, Merck Sharp & Dohme, Nordic Bioscience, Novartis, Novo Nordisk, Pfizer, Roche, Sanofi, Shinogi, Siemens Healthcare, and Summit Clinical Research; speaker fees from Apollo Endosurgery, MedPublico, and Boehringer Ingelheim; and is a member of advisory boards of Novo Nordisk and Boehringer Ingelheim. EB declares consulting fees from Gilead, Novo Nordisk, Boehringer Ingelheim, and Intercept; speaker fees from Novo Nordisk, Intercept, and Merck Sharp & Dohme; and is a member of advisory boards of Novo Nordisk and Intercept. MR-G declares grants from Siemens, Gilead, and Intercept; consulting fees from AbbVie, Alpha-sigma, Allergen, AstraZeneca, Inventia, Kaleido, Novo Nordisk, Pfizer, Axcella, Bristol-Meyers Squibb, Boehringer Ingelheim, and Gilead; speaker fees from Inventia, Sobi, Novo Nordisk, Rubio, and Shionogi; meeting and travel support from Abbvie and Gilead; and is a member of an advisory board of Galmed. VR declares grants from Gilead Sciences and Intercept Pharmaceuticals and consulting fees from GlaxoSmithKline, Galmed, Novo Nordisk, Prosciento, Terns, NorthSea Therapeutics, Enyo, Sagimet, NGM Pharmaceuticals, and Madrigal. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

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., (© 2023. The Author(s).)

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., (© 2023 The Authors.)

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., (© 2022. Asian Pacific Association for the Study of the Liver.)

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.

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., (© 2022. The Author(s).)

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., (Copyright © 2021. Published by Elsevier Inc.)

Background: Asthma is a heterogeneous disease; therefore, biomarkers that can assist in the identification of subtypes and direct therapy are highly desirable. Asthma is a chronic inflammatory disease that leads to changes in the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) degradation causing fragments of type I collagen that is released into circulation., Objective: Here, we asked if MMP-generated type I collagen (C1M) was associated with subtypes of asthma., Methods: C1M was serologically assessed at baseline in the adult participants of the All Age Asthma study (ALLIANCE) (n = 233), and in The Prospective Epidemiological Risk Factor study (PERF) (n = 283). In addition, C1M was assessed in mice sensitized to ovalbumin (OVA) and challenged with OVA aerosol. C1M was evaluated in mice with and without acute neutrophilic inflammation provoked by poly(cytidylic-inosinic) acid and mice treated with CP17, a peptide inhibiting neutrophil accumulation., Results: Serum C1M was significantly increased in asthmatics compared to healthy controls (p = 0.0005). We found the increased C1M levels in asthmatics were related to blood neutrophil and body mass index (BMI) in the ALLIANCE cohort, which was validated in the PERF cohort. When patients were stratified into obese (BMI > 30) asthmatics with high neutrophil levels and uncontrolled asthma, this group had a significant increase in C1M compared to normal-weight (BMI < 25) asthmatics with low neutrophil levels and controlled asthma (p = 0.0277). C1M was significantly elevated in OVA mice with acute neutrophilic inflammation compared to controls (P = 0.0002) and decreased in mice treated with an inhibitor of neutrophil infiltration (p = 0.047)., Conclusion & Clinical Relevance: C1M holds the potential to identify a subtype of asthma that relates to severity, obesity, and high neutrophils. These data suggest that C1M is linked to a subtype of overall inflammation, not only derived from the lung. The link between C1M and neutrophils were further validated in in vivo model., Trial Registration: (ALLIANCE, NCT02419274 )., (© 2022. The Author(s).)

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 (LPA 1 ) 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 LPA 1 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 LPA 1 -induced fibrogenesis., Conclusions: BMS-986020 reduced serum ECM-neoepitope biomarkers, which were previously associated with IPF prognosis. In vitro, LPA promoted fibrogenesis, which was LPA 1  dependent and inhibited by BMS-986020. Together these data elucidate a novel antifibrotic mechanism of action for pharmacological LPA 1 blockade. Trial registration ClinicalTrials.gov identifier: NCT01766817; First posted: January 11, 2013; https://clinicaltrials.gov/ct2/show/NCT01766817 ., (© 2022. The Author(s).)

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., Competing Interests: Declaration of competing interest Although the PRO-C3 ELISA was carried out at Nordic Biosciences under a research collaboration, we confirm that cohort generation, research conceptualization, analysis, and manuscript drafting were carried out independent of the Nordic Biosciences team. ADAPT is not developed as a proprietary test. The PRO-C3 ELISA is not currently commercially available, but can be obtained as a Nordic Bioscience research test for research use only. Diana Julie Leeming is employed by, and owns stock in Nordic Bioscience. Pei Jiang and Li-Mei Gao are employed by Fosun Diagnostics (Shanghai). Grace Lai-Hung Wong and Vincent Wai-Sun Wong have served as speakers and/or consultants for Echosens. Other authors have no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: JS has acted as consultant to Boehringer Ingelheim, BMS, Echosens, Genfit, Gilead Sciences, Intercept Pharmaceuticals, Madrigal, Novartis, Novo Nordisk, Nordic Bioscience, Pfizer, Roche, Sanofi, Siemens Healthcare GmbH, Zydus, has received research funding from Gilead Sciences, and has acted as speaker for Falk Foundation MSD Sharp & Dohme GmbH. All outside the submitted work. EE, DGKR, PF, DJL, and MAK, are employees of Nordic Bioscience, a company engaged in the development of biochemical markers. MAK and DJL are stockholders of Nordic Bioscience. LLG has acted as consultant for Novo Nordisk and received funding for research from Novo Nordisk, Gilead, Alexion, and Vingmed, all outside the submitted work. DS is an employee of Bristol Myers Squibb. GA has associations with Shire, Agios, and GlaxoSmithKline, and advises the Medicines and Healthcare products Regulatory Agency (MHRA), all outside the submitted work., (© 2021 The Author(s).)

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., Competing Interests: MJN, DJL, TM-J, and MAK are full-time employees at Nordic Bioscience. MAK and DJL hold stocks in Nordic Bioscience. MJN, DJL and MAK are among the original inventors and patent holders for PRO-C3, PRO-C5, C3M, and C4M. MV has received fees as an advisory board member for Intercept. DT has received fees for an advisory board with Intercept and speakers fees from Intercept and Falk. JRH has served on advisory boards for Orkla Health and Novartis, and received research support from Biogen, all unrelated to the present study. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2020 The Authors.)

Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)-BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ-BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.

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.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal epithelial repair process that may result in intra-airway accumulation of fibrin. Given that plasma fibrinogen is the only FDA approved biomarker that predicts mortality and COPD exacerbations, we hypothesized that changes in the processing of fibrinogen may provide additional characterization of disease phenotype and COPD progression., Methods: A subpopulation of subjects with COPD, (n = 983) smoker (n = 205) and non-smoker controls (n = 98) were included from The Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort. Two biomarkers that specifically target the thrombin-mediated conversion of fibrinogen into fibrin (PRO-FIB), and plasmin-mediated degradation of cross-linked fibrin (X-FIB) were measured and compared with fibrinogen measurements., Results: X-FIB had a predictive value for two-year mortality, with an adjusted hazard ratio of 1.48 per SD (n = 980; 95% Cl 1.18-1.84; p < 0.0001), and comparable to the fibrinogen hazard ratio of 1.59 per SD (n = 983; 95% Cl 1.29-1.96; p = 0.0003). X-FIB (p < 0.001), fibrinogen (p < 0.0001) and PRO-FIB (p < 0.05) were significantly elevated in symptomatic COPD (mMRC ≥ 2) as compared to asymptomatic COPD. X-FIB was the only biomarker that was associated with emphysema (p < 0.001), and only plasma fibrinogen (p < 0.05) was associated with exacerbations., Conclusion: There is a need for biomarkers to characterize the heterogeneity of COPD, to continuously improve clinical trial design and to identify disease progressors for efficient health care utilization. Each of three fibrinogen biomarkers studied provide information representing distinct aspects of COPD which may be used to characterize disease endotypes and to assess mortality risk in COPD., (Copyright © 2019. Published by Elsevier Ltd.)

Chronic obstructive pulmonary disease (COPD) is characterized by a slow heterogeneous progression. Therefore, improved biomarkers that can accurately identify patients with the highest likelihood of progression and therefore the ability to benefit from a given treatment, are needed. Elastin is an essential structural protein of the lungs. In this study, we investigated whether elastin degradation products generated by the enzymes proteinase 3, cathepsin G, neutrophil elastase, MMP7 or MMP9/12 were prognostic biomarkers for COPD-related outcomes. The elastin degradome was assessed in a subpopulation (n = 1307) of the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort with 3 years of clinical follow-up. Elastin degraded by proteinase 3 could distinguish between COPD participants and non-smoking controls (p = 0.0006). A total of 30 participants (3%) died over the 3 years of observation. After adjusting for confounders, plasma levels of elastin degraded by proteinase 3 and cathepsin G were independently associated with mortality outcome with a hazard ratio per 1 SD of 1.49 (95%CI 1.24-1.80, p < 0.0001) and 1.31 (95%CI 1.10-1.57, p = 0.0029), respectively. Assessing the elastin degradome demonstrated that specific elastin degradation fragments have potential utility as biomarkers identifying subtypes of COPD patients at risk of poor prognosis and supports further exploration in confirmatory studies.

Protein cross-links are formed in regulated biochemical processes in many biological systems, but they are also generated inadvertently via the reactions of exogenous or endogenous oxidants. Site-specific identification and characterization of such cross-links is challenging, and the goal was, therefore, to develop mass-spectrometry-based approaches tailored for proteins subjected to oxidative challenges that also are applicable for the analysis of complex samples. Using trypsin-mediated 18 O isotopic labeling, different types of data acquisition workflows, and designated database software tools, we successfully identified tyrosine-tyrosine, tyrosine-tryptophan, tyrosine-lysine, and histidine-lysine cross-links in proteins subjected to sensitizer-mediated photo-oxidation with rose bengal or chemical oxidation with peroxyl radicals generated from the water-soluble compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Subsequently, AAPH was also applied to a protein extract from the Gram-positive bacterium Lactococcus lactis, demonstrating the feasibility to identify tyrosine-tyrosine, tyrosine-tryptophan, and tryptophan-tryptophan cross-linked peptides in a complex system. Different fragmentation techniques were evaluated, and it was observed that higher-energy collisional dissociation (HCD) resulted in a higher number of identified cross-link peptides, while electron-transfer dissociation supplemented with HCD (EThcD) generally provides higher fragment ion coverage of the cross-linked peptides.

Objectives: Type VIII collagen is involved in angiogenesis and remodeling of arteries. We hypothesized that type VIII collagen was upregulated in diseases associated with vascular remodeling, e.g. pulmonary fibrosis and cancer. In this paper we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) targeting type VIII collagen in serum and plasma., Design and Methods: A monoclonal antibody was raised against the C-terminal of type VIII collagen (C8-C) and a competitive ELISA was developed. The assay was evaluated in relation to epitope specificity, technical performance, and in relevant disease cohorts. The developed ELISA was applied for the assessment of type VIII collagen in serum from patients diagnosed with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and various cancers., Results: The C8-C ELISA was technically stable and applicable for measurements in serum and plasma samples. Concentrations of C8-C was increased in serum from patients diagnosed with COPD (n=68) (p<0.0001), SCC lung- (n=10) (p<0.0001), breast- (n=13) (p<0.0001), colon- (n=7) (p=0.002), melanoma- (n=7) (p=0.001), non-small cell lung- (n=12) (p<0.0001), ovary- (n=10) (p=0.0001), pancreas- (n=5) (p=0.017), prostate- (n=14) (p=0.001) and small cell lung cancer (n=8) (p=0.0002) when compared to controls (n=43). Concentrations of C8-C were not significantly increased in serum from patients diagnosed with IPF., Conclusions: The C8-C assay was technically robust and specific for type VIII collagen. Concentrations of C8-C were significantly elevated in serum from patients diagnosed with COPD and within 9 different cancer types, but not IPF. Further research is required to test C8-C as an efficacy marker for angiostatic treatments., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)