title: Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications--a study from the Lunenburg Lymphoma Biomarker Consortium.
abstract: OBJECTIVE
  The results of immunohistochemical class prediction and prognostic stratification of diffuse large B-cell lymphoma (DLBCL) have been remarkably various thus far. Apart from biologic variations, this may be caused by differences in laboratory techniques, scoring definitions, and inter- and intraobserver variations. In this study, an international collaboration of clinical lymphoma research groups from Europe, United States, and Canada concentrated on validation and standardization of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL.
  
  
  METHODS
  Sections of a tissue microarray from 36 patients with DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM1, and MIB-1 according to local methods. The study was performed in two rounds firstly focused on the evaluation of laboratory staining variation and secondly on the scoring variation.
  
  
  RESULTS
  Different laboratory staining techniques resulted in unexpectedly highly variable results and very poor reproducibility in scoring for almost all markers. No single laboratory stood out as uniformly poor or excellent. With elimination of variation due to staining, high agreement was found for CD20, HLA-DR, and CD10. Poor agreement was found for bcl-6 and Ki-67. Optimization of techniques and uniformly agreed on scoring criteria improved reproducibility.
  
  
  CONCLUSIONS
  This study shows that semiquantitative immunohistochemistry for subclassification of DLBCL is feasible and reproducible, but exhibits varying rates of concordance for different markers. These findings may explain the wide variation of biomarker prognostic impact reported in the literature. Harmonization of techniques and centralized consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.
authors.affiliation: Netherlands Cancer Institute, Amsterdam, The Netherlands. d.d.jong@nki.nl
authors.forename: Daphne
authors.initials: D
authors.lastname: de Jong
authors.forename: Andreas
authors.initials: A
authors.lastname: Rosenwald
authors.forename: Mukesh
authors.initials: M
authors.lastname: Chhanabhai
authors.forename: Philippe
authors.initials: P
authors.lastname: Gaulard
authors.forename: Wolfram
authors.initials: W
authors.lastname: Klapper
authors.forename: Abigail
authors.initials: A
authors.lastname: Lee
authors.forename: Birgitta
authors.initials: B
authors.lastname: Sander
authors.forename: Christoph
authors.initials: C
authors.lastname: Thorns
authors.forename: Elias
authors.initials: E
authors.lastname: Campo
authors.forename: Thierry
authors.initials: T
authors.lastname: Molina
authors.forename: Andrew
authors.initials: A
authors.lastname: Norton
authors.forename: Anton
authors.initials: A
authors.lastname: Hagenbeek
authors.forename: Sandra
authors.initials: S
authors.lastname: Horning
authors.forename: Andrew
authors.initials: A
authors.lastname: Lister
authors.forename: John
authors.initials: J
authors.lastname: Raemaekers
authors.forename: Randy D
authors.initials: RD
authors.lastname: Gascoyne
authors.forename: Gilles
authors.initials: G
authors.lastname: Salles
authors.forename: Edie
authors.initials: E
authors.lastname: Weller
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D018956:CD5 Antigens / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry; D016393:Lymphoma, B-Cell / Q000737:chemistry* / Q000145:classification / Q000276:immunology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000737:chemistry* / Q000145:classification / Q000276:immunology; D015260:Neprilysin / Q000032:analysis; D011379:Prognosis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051560:Proto-Oncogene Proteins c-bcl-6; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: C084103:BCL6 protein, human; D014408:Biomarkers, Tumor; D018956:CD5 Antigens; D004268:DNA-Binding Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051560:Proto-Oncogene Proteins c-bcl-6; D015260:Neprilysin