Retracted: 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)

Background and Aims: The results of class prediction and the determination of prognostic markers in diffuse large B-cell lymphoma (DLBCL) have been variably reported. Apart from biological variations, this may be caused by differences in laboratory techniques, scoring definitions and inter- and intra-observer variation. In this study, an international collaboration of clinical lymphoma research groups has concentrated on validation and standardisation of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. Methods: Sections of a tissue microarray with 36 cases of DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM-1 and Ki-67 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 techniques resulted in highly variable results and poor reproducibility for almost all markers. Reproducibility of the nuclear markers was highly sensitive to technical variations, including immunological enhancement techniques (agreements 34%). With elimination of variation due to staining and uniformly agreed on scoring criteria, significant improvement was seen; however less so for bcl-6 and Ki-67 (agreement 53–58%). Absence of internal controls that preclude scoring, significantly influenced the results for CD10 and bcl-6. Conclusion: Semi-quantitative immunohistochemistry for subclassification of DLBCL is feasible, but with varying rates of concordance for different markers and only using optimised techniques and strict scoring criteria. These findings may explain the wide variation in prognostic impact reported in the literature. Harmonisation of techniques and centralised consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

[1]  J Diebold,et al.  Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin's lymphoma. Groupe d'Etude des Lymphomes de l'Adulte (GELA). , 1996, Blood.

[2]  M. Kenward,et al.  An Introduction to the Bootstrap , 2007 .

[3]  M. Kersten,et al.  Beyond the International Prognostic Index: new prognostic factors in follicular lymphoma and diffuse large-cell lymphoma A meeting report of the Second International Lunenburg Lymphoma Workshop. , 2004, The hematology journal : the official journal of the European Haematology Association.

[4]  T. Golub,et al.  Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. , 2004, Blood.

[5]  S. Barrans,et al.  Germinal center phenotype and bcl-2 expression combined with the International Prognostic Index improves patient risk stratification in diffuse large B-cell lymphoma. , 2002, Blood.

[6]  R. Rosenquist,et al.  Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis , 2005, Modern Pathology.

[7]  Emili Montserrat,et al.  Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma. , 2003, Blood.

[8]  T. Golub,et al.  NFkappaB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes. , 2005, Blood.

[9]  R. Gascoyne,et al.  Prognostic significance of Bcl-6 protein expression in DLBCL treated with CHOP or R-CHOP: a prospective correlative study. , 2006, Blood.

[10]  Peter A Kaufman,et al.  HER2 testing by local, central, and reference laboratories in specimens from the North Central Cancer Treatment Group N9831 intergroup adjuvant trial. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[11]  Mark Levine,et al.  Radiation therapy and tamoxifen: concurrent or sequential? That is the question. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[12]  P. de Paepe,et al.  Large cleaved and immunoblastic lymphoma may represent two distinct clinicopathologic entities within the group of diffuse large B-cell lymphomas. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  J Hermans,et al.  Clinical significance of bcl2 and p53 protein expression in diffuse large B-cell lymphoma: a population-based study. , 1996, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[14]  J C Reed,et al.  Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin's lymphoma. , 1997, Blood.

[15]  W. Klapper,et al.  Diffuse large B-cell lymphoma in pediatric patients belongs predominantly to the germinal-center type B-cell lymphomas: a clinicopathologic analysis of cases included in the German BFM (Berlin-Frankfurt-Munster) Multicenter Trial. , 2006, Blood.

[16]  P. de Paepe,et al.  Forkhead box protein P1 expression in mucosa-associated lymphoid tissue lymphomas predicts poor prognosis and transformation to diffuse large B-cell lymphoma. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  L. Staudt,et al.  The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. , 2002, The New England journal of medicine.

[18]  S. Barrans,et al.  Strong expression of FOXP1 identifies a distinct subset of diffuse large B-cell lymphoma (DLBCL) patients with poor outcome. , 2004, Blood.

[19]  E. Schuuring,et al.  Prognostic impact of germinal center-associated proteins and chromosomal breakpoints in poor-risk diffuse large B-cell lymphoma. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[20]  P. Gaulard,et al.  Rituximab plus CHOP (R-CHOP) overcomes bcl-2--associated resistance to chemotherapy in elderly patients with diffuse large B-cell lymphoma (DLBCL). , 2003, Blood.

[21]  R. Gascoyne,et al.  Expression of the FOXP1 transcription factor is strongly associated with inferior survival in patients with diffuse large B-cell lymphoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[22]  Chung-Che Chang,et al.  Immunohistochemical Expression Patterns of Germinal Center and Activation B-cell Markers Correlate With Prognosis in Diffuse Large B-cell Lymphoma , 2004, The American journal of surgical pathology.

[23]  R. Tibshirani,et al.  An introduction to the bootstrap , 1993 .

[24]  J Diebold,et al.  Prognostic significance of survivin expression in diffuse large B-cell lymphomas. , 2000, Blood.

[25]  D C Linch,et al.  Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma Investigation Study. , 1996, Blood.

[26]  R Tibshirani,et al.  Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma. , 2001, Blood.

[27]  A. Olshen,et al.  Cell of origin, germinal center versus nongerminal center, determined by immunohistochemistry on tissue microarray, does not correlate with outcome in patients with relapsed and refractory DLBCL. , 2005, Blood.

[28]  L. Staudt,et al.  BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large B-cell lymphoma. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[29]  Kajia Cao,et al.  BCL2 translocation defines a unique tumor subset within the germinal center B-cell-like diffuse large B-cell lymphoma. , 2004, The American journal of pathology.

[30]  S. Pileri,et al.  Identification of outcome predictors in diffuse large B-cell lymphoma. Immunohistochemical profiling of homogeneously treated de novo tumors with nodal presentation on tissue micro-arrays. , 2005, Haematologica.

[31]  Youli Zu,et al.  Validation of tissue microarray immunohistochemistry staining and interpretation in diffuse large B-cell lymphoma , 2005, Leukemia & lymphoma.

[32]  L. Staudt,et al.  Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. , 2004, Blood.

[33]  Manuela Herman,et al.  Statistical Methods for the Analysis of Biomedical Data , 2003 .

[34]  Ash A. Alizadeh,et al.  Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling , 2000, Nature.