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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

Daphne de Jong, Andreas Rosenwald, Mukesh Chhanabhai, Philippe Gaulard, Wolfram Klapper, Abigail Lee, Birgitta Sander, Christoph Thorns, Elias Campo, Thierry Molina, Andrew Norton, Anton Hagenbeek, Sandra Horning, Andrew Lister, John Raemaekers, Randy D. Gascoyne, Gilles Salles, Edie Weller

From the Netherlands Cancer Institute; Academic Medical Center, Amsterdam; University Medical Center Nijmegen, the Netherlands; Institute of Pathology, University of Würzburg, Würzburg; Department of Pathology, Hematopathology Section, University Hospital Schleswig-Holstein, Campus Kiel; University Clinic Schleswig-Holstein, Campus Luebeck, Germany; Department of Pathology & Medical Oncology, British Columbia Cancer Agency, University of British Columbia, Vancouver, Canada; Department of Pathology, Inserm U617, Hôpital Henri Mondor, Créteil; Université Paris-Descartes; AP-HP, Hôtel-Dieu, Paris; Hospices Civils de Lyon & Université Claude Bernard Lyon-1, Lyon, France; CR-UK Medical Oncology Unit, St Bartholomew's Hospital, London, United Kingdom; Karolinska Institutet, Stockholm, Sweden; Hospital Clinic, University of Barcelona, Barcelona, Spain; Stanford University Medical Center, Palo Alto, CA; and Dana-Farber Cancer Institute, Boston, MA

Address reprint requests to Daphne de Jong, MD, PhD, Department of Pathology, the Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands; e-mail: d.d.jong{at}nki.nl

Purpose: 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.

Patients and 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.

Conclusion: 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.

Supported by the van Vlissingen Lymphoma Foundation. In addition, unrestricted grants were received from Genentech, Millennium Pharmaceuticals Inc, Roche International, and Schering AG.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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