Multiplex Serum Protein Analysis Identifies Novel Biomarkers of Advanced Fibrosis in Patients with Chronic Liver Disease with the Potential to Improve Diagnostic Accuracy of Established Biomarkers

RESEARCH ARTICLE Multiplex Serum Protein Analysis Identifies Novel Biomarkers of Advanced Fibrosis in Patients with Chronic Liver Disease with the Potential to Improve Diagnostic Accuracy of Established Biomarkers a11111 Katharine M. Irvine1*, Leesa F. Wockner2, Isabell Hoffmann2, Leigh U. Horsfall1,3, Kevin J. Fagan1, Veonice Bijin4, Bernett Lee4, Andrew D. Clouston1, Guy Lampe5, John E. Connolly4, Elizabeth E. Powell1,3 1 Centre for Liver Disease Research, School of Medicine, The University of Queensland, Brisbane, Australia, 2 Statistics Unit, QIMR Berghofer Medical Research Institute, Brisbane, Australia, 3 Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, Australia, 4 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673; Singapore Immunology Network, Singapore 138648; and Institute of Biomedical Studies, Baylor University, Waco, Texas, 76798, United States of America, 5 Pathology Queensland, Princess Alexandra Hospital, Brisbane, Australia OPEN ACCESS Citation: Irvine KM, Wockner LF, Hoffmann I, Horsfall LU, Fagan KJ, Bijin V, et al. (2016) Multiplex Serum Protein Analysis Identifies Novel Biomarkers of Advanced Fibrosis in Patients with Chronic Liver Disease with the Potential to Improve Diagnostic Accuracy of Established Biomarkers. PLoS ONE 11(11): e0167001. doi:10.1371/journal. pone.0167001 Editor: Han-Chieh Lin, Taipei Veterans General Hospital, TAIWAN * Abstract Background and Aims Non-invasive markers of liver fibrosis are urgently required, especially for use in non-specialist settings. The aim of this study was to identify novel serum biomarkers of advanced fibrosis. Received: August 4, 2016 Accepted: November 7, 2016 Published: November 18, 2016 Copyright: © 2016 Irvine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data are available in supporting tables 1 and 2. Funding: This study was funded by Pathology Queensland (https://www.health.qld.gov.au/ pathology-queensland), an Australian Liver Foundation Pauline Hall Fellowship to KMI (https:// www.liver.org.au) and a National Medical Research Council Practitioner Fellowship to EEP (APP1004242). There was no additional external funding received for this study. The funders had no Methods We performed an unbiased screen of 120 serum analytes including cytokines, chemokines and proteases in 70 patients (35 without fibrosis, 35 with cirrhosis on biopsy), and selected a panel of 44 candidate biomarkers, which were subsequently measured in a mixed-etiology cohort of 432 patients with known serum HA, PIIINP and TIMP1 (which comprise the validated Enhanced Liver Fibrosis (ELF) test). Multivariate logistic regression modelling was used to generate models for the prediction of advanced or significant fibrosis (METAVIR F3 and F2, respectively); in addition to identifying biomarkers of disease activity and steatohepatitis. Results Seventeen analytes were significantly differentially expressed between patients with no advanced fibrosis and patients with advanced fibrosis, the most significant being hyaluronic acid (HA) and matrix metalloproteinase (MMP) 7 (p = 2.9E-41 and p = 1.0E-26, respectively). The optimal model for the prediction of advanced fibrosis comprised HA, MMP7, MMP1, alphafetoprotein (AFP) and the AST to platelet ratio index (APRI). We demonstrate PLOS ONE | DOI:10.1371/journal.pone.0167001 November 18, 2016 1 / 13 Serum Biomarkers of Advanced Liver Fibrosis role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. enhanced diagnostic accuracy (AUROC = 0.938) compared to a model comprising HA, PIIINP and TIMP1 alone (ELF) (AUROC = 0.898, p<0.0001, De Long’s test). Competing Interests: The authors have declared that no competing interests exist. Conclusions We have identified novel serum biomarkers of advanced liver fibrosis, which have the potential to enhance the diagnostic accuracy of established biomarkers. Our data suggest MMP7 is a valuable indicator of advanced fibrosis and may play a role in liver fibrogenesis. Introduction Liver fibrosis is the main cause of chronic liver disease (CLD)-related morbidity and mortality. The severity of fibrosis, the precursor to cirrhosis, predicts the emergence of complications of portal hypertension and liver-related morbidity and mortality, and therefore influences clinical management. Liver biopsy is the gold standard method for staging hepatic fibrosis; as well as grading inflammatory activity, distinguishing non-alcoholic steatohepatitis (NASH) from fatty liver, and for identifying non-alcoholic fatty liver disease (NAFLD) in patients with other chronic liver diseases. Despite the diagnostic advantages of liver biopsy, the procedure is invasive, costly, requires specialised expertise; and is also limited by its semi-quantitative nature, sampling error and intra-observer variability[1]. With the growing prevalence of CLD, especially NAFLD, and the introduction of highly efficacious direct acting antiviral (DAA) agents for HCV, there is an increasing need for non-invasive biomarkers to stratify risk and assess disease progression/regression; to facilitate larger scale screening and provide efficacy endpoints for clinical trials. A number of non-invasive methods of fibrosis assessment have been identified and widely validated, including imaging techniques and serum biomarkers. The use of non-invasive biomarkers for excluding advanced fibrosis to reduce the number of liver biopsies has been incorporated into clinical practice guidelines, however they are still considered insufficiently accurate for assessing intermediate stages of fibrosis, disease progression or the effect of therapy[1]. Transient elastography, which can reliably identify advanced fibrosis, is one of the most frequently used non-invasive methods; however its use is largely limited to specialist centres due to the need for specialised instrumentation and expertise. Serum biomarkers are more suitable for use in general clinical practice, and several serum tests have been developed using combinations of direct (associated with liver fibrogenesis) and/or indirect (reflecting liver function) biomarkers. Serum panels offer several advantages over liver biopsy, as they are quantitative and have the potential to reflect the dynamic nature of fibrogenesis, providing a more sensitive assessment of the dynamic changes associated with fibrosis progression/regression compared to static fibrosis stage. Complex panels incorporating direct markers, such as the Enhanced Liver Fibrosis (ELF) test[2], FibroTest[3] and Hepascore[4], are generally thought to be superior to simple panels such as the aspartate aminotransferase (AST) to plat (...truncated)