title: Multiplex protein analysis to determine fibrosis stage and progression in patients with chronic hepatitis C.
abstract: OBJECTIVE
  Noninvasive tests cannot differentiate between adjacent stages of fibrosis, which limits assessment of disease progression and regression during therapy. We investigated whether levels of cytokines and extracellular matrix proteins in serum and biopsy samples can be used to determine actual stage of liver fibrosis in patients with chronic hepatitis C (CHC) and in prognosis.
  
  
  METHODS
  We collected data from 383 treatment-naive patients with CHC from the Duke Hepatology Clinical Research Database and Biorepository, from 2006 through 2009, for use in the training set. Serum samples were obtained from 100 individuals without CHC (controls). We selected 37 serum biomarkers for customized array analysis by using the SearchLight multiplex sandwich enzyme-linked immunosorbent assay. Data from 434 treatment-naive patients with CHC, which were obtained from the Trent HCV cohort, were used in the validation analysis. Multivariable modeling, marker selection, and validation included randomForest and Obuchowski measures, with independent comparison with FibroSURE.
  
  
  RESULTS
  Four serum markers (levels of hyaluronic acid, vascular cell adhesion molecule 1, alpha-2 macroglobulin, and retinol-binding protein 4) and age associated with fibrosis stage (F0-1, F2-3, or F4); these had Obuchowski measures of 0.85-0.89, with misclassification rates of 38% and 29% in training and validation sets, compared with 50% for the FibroSURE test. In the training set, area under the curve values for the multiplex markers were similar to those from the FibroSURE test: stages F0 vs F1 (0.51 vs 0.53), F1 vs F2 (0.60 vs 0.59), F2 vs F3 (0.69 vs 0.72), and F3 vs F4 (0.51 vs 0.52). Area under the curve values were similar in the validation cohort. In longitudinal analyses of 133 paired biopsies, 9 markers (level of alanine aminotransferase, γ-glutamyltransferase, hyaluronic acid, intracellular adhesion molecule 1, interleukin 4, CXCL10, CXCL9, and vascular cell adhesion molecule 1) were associated with change in the histologic activity index (P values ranging from .000 to .049), and 4 (granulocyte-macrophage colony-stimulating factor, interleukin 12, interleukin 2, and matrix metalloproteinase 13) were associated with a change in fibrosis stage (P values ranging from .001 to .042).
  
  
  CONCLUSIONS
  We identified serum biomarkers that can be measured by multiplex enzyme-linked immunosorbent assay to determine levels of fibrosis in patients with CHC, although misclassification is frequent and results are comparable with those from the FibroSURE test. Changes in protein levels in biopsy samples were associated with progression of fibrosis in patients.
authors.affiliation: Duke Clinical Research Institute, Durham, North Carolina; Liver Cell Biology, Centenary Institute, University of Sydney, Sydney, NSW, Australia. Electronic address: keyur.patel@duke.edu.
authors.forename: Keyur
authors.initials: K
authors.lastname: Patel
authors.affiliation: GlaxoSmithKline, Research Triangle Park, North Carolina.
authors.forename: Katja S
authors.initials: KS
authors.lastname: Remlinger
authors.affiliation: OpGen, Gaithersburg, Maryland.
authors.forename: Terence G
authors.initials: TG
authors.lastname: Walker
authors.affiliation: BioAgilytix Labs, Research Triangle Park, North Carolina.
authors.forename: Peter
authors.initials: P
authors.lastname: Leitner
authors.affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Lucas
authors.affiliation: GlaxoSmithKline, Research Triangle Park, North Carolina.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Gardner
authors.affiliation: Duke Clinical Research Institute, Durham, North Carolina; Gilead Sciences, Foster City, California.
authors.forename: John G
authors.initials: JG
authors.lastname: McHutchison
authors.affiliation: National Institute for Health Research Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, United Kingdom.
authors.forename: Will
authors.initials: W
authors.lastname: Irving
authors.affiliation: National Institute for Health Research Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust and the University of Nottingham, Nottingham, United Kingdom.
authors.forename: Indra Neil
authors.initials: IN
authors.lastname: Guha
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood*; D001706:Biopsy; D001774:Blood Chemical Analysis; D015331:Cohort Studies; D016207:Cytokines / Q000032:analysis / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005260:Female; D005355:Fibrosis / Q000175:diagnosis* / Q000473:pathology*; D019698:Hepatitis C, Chronic / Q000175:diagnosis* / Q000473:pathology*; D006651:Histocytochemistry; D006801:Humans; D008099:Liver / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D011379:Prognosis
chemical_list: D015415:Biomarkers; D016207:Cytokines; D016326:Extracellular Matrix Proteins
keywords: ALT; Alpha-2 Macroglobulin; Fibrogenesis; Retinol-binding Protein 4; Vascular Cell Adhesion Molecule