Morphologic evaluation and survival analysis.

(A) Example of marrow biopsy sample with diffuse involvement pattern (left) and absence of MUM1/IRF4 expression (right), defined as less than 20% of CD20⁺ B cells stained positively for MUM1/IRF4. Double staining for MUM1/IRF4 (brown; nuclear staining) and CD20 (red; membrane staining) shows that most CD20⁺ B cells do not express MUM1/IRF4. Cells that are positive for MUM1/IRF4 but negative for CD20 represent activated T cells or plasma cells (arrows). Double staining was performed, after heat retrieval, by incubating the marrow biopsy sections with monoclonal antibody against MUM1/IRF4 (1:200 dilution) for 30 minutes, detected by DAB chromogen (DAKO), followed by the second incubation with monoclonal antibody against CD20 (1:800 dilution) for 30 minutes, detected by Vector-Nova Red (Vector, Burlingame, CA). All slides were stained using the DAKO automated immunostainer. In some patients, the monoclonal antibody (MUM1p, a kind gift from Dr B. Falini, University of Perugia, Italy) was used for double staining. Results were comparable between the 2 antibodies. (B) Patients without MUM1/IRF4 expression had significantly worse OS than those with MUM1/IRF4 expression (Kaplan-Meier survival analysis;P < .03, log-rank test). (C) Among patients with interstitial/nodular marrow involvement, patients with MUM1/IRF4 expression had significantly better OS than those without (Kaplan-Meier survival analysis; P < .02, log-rank test).