CD79b expression in B cell chronic lymphocytic leukemia: its implication for minimal residual disease detection

Abstract

The surface expression of CD79b, using the monoclonal antibody (Mab) CB3–1, on B lymphocytes from normal individuals and patients with B cell chronic lymphocytic leukemia (CLL) has been analyzed using triple-staining cells for flow cytometry. In addition, the clinical significance of CD79b expression in CLL patients and its possible value for the evaluation of minimal residual disease (MRD) was explored. A total of 15 peripheral blood (PB) samples from healthy blood donors, five bone marrow (BM) samples from normal donors and 40 PB samples from CLL untreated patients were included in the study. In addition we studied the expression of CD79b in B lymphocytes from five CLL patients after fludarabine treatment in order to support our method. The expression of CD79b in B lymphocytes from PB was analyzed by flow cytometry, using simultaneous staining with the Mabs CD22, CD79b, CD19 and CD5, CD79b and CD19. Since normal immature bone marrow B cells are CD79b^−/dim+ on their surface, in BM samples we used the combination CD45, CD79b and CD19 selecting mature B lymphocytes according to their bright CD45 intensity. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD19⁺ cells. For data analysis, the PAINT-A-GATE PRO software (Becton Dickinson) was used. Dilution experiments of CD79b⁻ CLL cells and CD79b^dim+ CLL cells with normal PB and BM cells were performed in order to assess the sensitivity level of the technique for detection of CD79b^−/dim+residual CLL cells. All B lymphocytes from normal samples showed reactivity for the CD79b antigen. In contrast, CD79b was absent in 18/40 CLL patients (42.5%) and 20/40 CLL cases (50%) exhibited a low CD79b expression. Therefore, CD79b⁻ B lymphocytes would be restricted to the CLL population and thus could be considered a ‘tumor phenotype’ for monitoring MRD in CLL patients. Dilution experiments indicate that the detection limit with this marker almost reaches the levels obtained by molecular biology methods as the PCR technique. All cases studied after fludarabine presented leukemic cells in their PB or BM samples detected by flow cytometry. Upon comparing the clinical and morphological characteristics of CD79b⁻ and CD79b⁺ cases, all atypical CLL cases included in the present study were CD79b⁺ and advanced clinical stage (B and C Binet stage) was most frequently observed in CD79b⁺ cases than in CD79b⁻ cases.