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

Elisabeth Erhardtsen; Daniel G K Rasmussen; Peder Frederiksen; Diana Julie Leeming; Diane Shevell; Lise Lotte Gluud; Morten Asser Karsdal; Guruprasad P Aithal; Jörn M Schattenberg

doi:10.1016/j.jhepr.2021.100317

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

JHEP Rep. 2021 Jul 10;3(4):100317. doi: 10.1016/j.jhepr.2021.100317. eCollection 2021 Aug.

Authors

Affiliations

¹ Nordic Bioscience Biomarkers and Research, Herlev, Denmark.
² Innovative Medicine, Bristol Myers-Squibb, Princeton, NJ, USA.
³ Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁴ Gastro Unit, Medical Division, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark.
⁵ Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, UK.
⁶ NIHR Nottingham Biomedical Research Centre at the Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, UK.
⁷ Medical Research Council (MRC), Nottingham Molecular Pathology Node, University of Nottingham, Nottingham, UK.
⁸ Department of Internal Medicine I, University Medical Centre of the Johannes Gutenberg-University, Mainz, Germany.

Abstract

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.

Keywords: ADAM, A Disintegrin and Metalloproteases; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristics curve; Biomarkers; Biopsy; Body mass index; CLSI, Clinical and Laboratory Standards Institute; Collagen type III; ELF™ test, Enhanced Liver Fibrosis test; Ethnic groups; FIB-4, fibrosis-4; Fibrosis; Healthy volunteers; Hospitals; Humans; LITMUS, Liver Investigation: Testing Marker Utility in Steatohepatitis (consortium); NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD Activity Score; NASH, non-alcoholic steatohepatitis; NASH-CRN, NASH Clinical Research Network; NIMBLE, Non-Invasive Biomarkers of Metabolic Liver Disease (consortium); NPV, negative predictive value; Non-alcoholic fatty liver disease; PPV, positive predictive value; Reference standards; Reference values; T2DM, type 2 diabetes mellitus.