Leukemia (1999) 13, 1501–1505 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu CD79b expression in B cell chronic lymphocytic leukemia: its implication for minimal residual disease detection JA Garcia Vela, I Delgado, L Benito, MC Monteserin, L Garcia Alonso, N Somolinos, MA Andreu and F On˜a Department of Hematology, Hospital Universitario de Getafe, Madrid, Spain The surface expression of CD79b, using the monoclonal anti- antigen expression and antigen overexpression) in order to body (Mab) CB3–1, on B lymphocytes from normal individuals establish the applicability of immunophenotypic aberrances and patients with B cell chronic lymphocytic leukemia (CLL) for monitoring MRD as in acute leukemias with a sensitivity has been analyzed using triple-staining cells for flow cytome- −4 try. In addition, the clinical significance of CD79b expression level of 10 (one aberrant CLL cell among 10000 normal in CLL patients and its possible value for the evaluation of mini- cells). We have previously published that CD5 is overex- mal residual disease (MRD) was explored. A total of 15 periph- pressed in most CLL cases.2 This aberrantly CD5high/CD19+ eral blood (PB) samples from healthy blood donors, five bone expression was present in 90% of our CLL. Dilutional experi- marrow (BM) samples from normal donors and 40 PB samples ments showed that CD5high/CD19+ were identified at fre- from CLL untreated patients were included in the study. In −4 addition we studied the expression of CD79b in B lymphocytes quencies as low as 10 . from five CLL patients after fludarabine treatment in order to Recently, different groups have communicated that CD79b support our method. The expression of CD79b in B lympho- (B cell receptor (BCR), B29 protein) is undetectable in the sur- cytes from PB was analyzed by flow cytometry, using simul- face of most CLL cells.5,6 This aberrant phenotype (all CD19+ taneous staining with the Mabs CD22, CD79b, CD19 and CD5, mature lymphocytes express CD79b) has not been studied for CD79b and CD19. Since normal immature bone marrow B cells monitoring MRD in CLL. The aim of our group was to study are CD79b−/dim+ on their surface, in BM samples we used the combination CD45, CD79b and CD19 selecting mature B lym- the expression of CD79b in CLL, to estimate quantitatively the phocytes according to their bright CD45 intensity. Cell acqui- number of molecules per cell, and finally to explore if this sition was performed in two consecutive steps using a live gate antigen could be used in the detection of MRD by flow cyto- drawn on SSC/CD19+ cells. For data analysis, the PAINT-A- metry after treatment. GATE PRO software (Becton Dickinson) was used. Dilution experiments of CD79b− CLL cells and CD79bdim+ 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+ Materials and methods 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 Patients and samples (50%) exhibited a low CD79b expression. Therefore, CD79b− B lymphocytes would be restricted to the CLL population and A total of 20 healthy donors: 15 peripheral blood (PB) samples thus could be considered a ‘tumor phenotype’ for monitoring MRD in CLL patients. Dilution experiments indicate that the and five bone marrow (BM) samples and 40 PB samples from detection limit with this marker almost reaches the levels untreated CLL patients were included in the present study. PB obtained by molecular biology methods as the PCR technique. and BM aspirates were available for review in all patients, All cases studied after fludarabine presented leukemic cells in bone marrow trephines in 33 patients and lymph node aspir- their PB or BM samples detected by flow cytometry. Upon com- ates or biopsies in 13 cases. All slides were reviewed and paring the clinical and morphological characteristics of CD79b− + classified according to both the REAL classification and the and CD79b cases, all atypical CLL cases included in the 7,8 present study were CD79b+ and advanced clinical stage (B and FAB cooperative group criteria. The diagnosis of CLL was C Binet stage) was most frequently observed in CD79b+ cases confirmed by immunophenotype using a large panel of mono- than in CD79b− cases. clonal antibodies (Mabs) with a direct immunofluorescence Keywords: CD79b; CLL; minimal residual disease; immunopheno- technique: FMC7, CD5, CD10, CD11c, CD19, CD20, CD22, type CD23, CD25, CD43, CD103 and polyclonal anti-␬ and anti- ␭ surface light chains (slg) to determinate B cell clonal excess. A typical CLL immunophenotype was defined as CD5+/ Introduction CD23+/CD19+/CD22−/dim+/CD20dim+/CD10−/CD103−/FMC7−/dim+ and slgdim. CLL was further subdivided into typical and atypi- It has been shown that immunophenotypic analysis is a useful cal CLL on morphology without considering the immuno- approach for the investigation of minimal residual disease phenotype.9 Atypical CLL included cases with more than 10% (MRD) in acute leukemia patients.1 At present this approach circulating prolymphocytes (CLL/PL) and others with more has not been used in B cell chronic lymphocytic leukemia than 15% lymphocytes with lymphoplasmacytic features (CLL) for MRD detection, with the exception of the and/or cleaved nuclei. According to the REAL classification kappa/lambda ratio but the sensitivity of this parameter is low and FAB criteria, all cases included were classified as CLL. (10−2).1 We and others2–4 have explored the incidence of There were no cases with mantle zone lymphoma, follicle phenotypic aberrances in CLL (maturational asynchronous center cell lymphoma, lymphoplasmacytoid lymphoma and splenic marginal zone B cell lymphoma. Six out of 40 CLL cases displayed atypical lymphocyte morphology in the blood film. Correspondence: JA Garcia Vela, Department of Hematology, Hospi- tal Universitario de Getafe, Carretera de Toledo KM 12,500, Getafe The mean age of normal blood donors (eight males, seven 28905, Madrid, Spain; Fax: 34 91 6833541 females) was 49.5 ± 12.9 years and 68.2 ± 11.3 for CLL Received 7 July 1998; accepted 25 May 1999 patients. According to the Binet clinical staging classi- CD79b expression in CLL JA Garcia Vela et al 1502 fication,10 CLL patients were distributed as follows: A 67.5%, San Juan, PR). Each marker was considered to be positive B 17.5% and C 15%. when the mean MESF obtained was at least double that Five patients were studied prior to treatment and at the time observed in the isotype-negative control tube. of maximal response after fludarabine treatment which was defined as: CR, nodular PR (nPR) or PR. Criteria for CR were normal physical examination, absence of constitutional symp- Dilution experiments toms, PB lymphocyte count Ͻ4 × 109/l, platelets Ͼ100 × 109/l, Hb Ͼ11 g/dl and Ͻ30% lymphoid cells in the BM aspirate Dilution experiments were performed in order to assess the with normal histology. The presence of residual nodular or sensitivity level of this technique for the detection of interstitial lymphoid aggregates in the BM was considered as CD79b−/dim+/CD19+ leukemic cells. For this purpose, progress- nPR. The presence of more than 30% lymphocytes in the BM ive dilutions of a CLL sample with normal PB and BM were aspirate after normalization of all other parameters was carried out at ratios of up to one CLL cell in 106 normal cells defined as PR. prior to staining with Mabs as previously described. Five ml of EDTA-anticoagulated PB or BM samples were obtained in all cases after informed consent according to the Hospital Ethical Committee. Statistical analysis The Mann–Whitney test was used to compare the MESF value Immunophenotypical studies of CD79b among the two groups, CLL and normal donors, and to demonstrate the correlation between the expression of In all cases unseparated PB samples (1–2 × 106/tube) were CD79b and the other markers analyzed in the present study. stained with a simultaneous triple labeling using the Mabs CD5 and CD22 conjugated with fluorescein isothiocyanate (FITC), CD79b conjugated with phycoerythrin (PE) and CD19 Results conjugated with the PE/Cyanin5 fluorochrome tandem (Tricolor). Since normal immature BM B cells are CD79b−/dim+ The mean frequency of B lymphocytes CD19+ in the normal as CLL cells, in all BM samples we used the combination PB samples analyzed was 8.4 ± 4.0% (range: 4.3–14%) of CD19-FITC/CD79b-PE/CD45-PerCP. Immature B cells lymphocytes, whereas in CLL PB samples it was (CD45−/dim+) were separate from mature B lymphocytes 74.09 ± 16.68% (range: 34–98%). The overall proportion of (CD45bright) according to the different CD45 expression.11 The CD19+ cells in the normal BM samples was 3.14 ± 1.68% CD5, CD19, CD22 and CD45 Mabs were purchased from (range: 0.7–6.4%) and 70.8 ± 14.3% of them showed a bright Becton Dickinson (San Jose´, CA, USA), CD79b (CB3-1) from expression of CD45. In normal PB, B lymphocytes expressing Immunotech (Marseille, France) and CD19-PE/Cy5 from Cal- CD5 represented 2.5 ± 1.4% of lymphocytes (MESF tag Laboratories (San Francisco, CA, USA). Samples were 6.6 ± 0.4 × 103). In the CLL group, all cases were CD5+ and it incubated for 15 min at room temperature in the dark with was expressed in practically all CD19+ cells (MESF saturating amounts of the fluorochrome-conjugated Mab 78.4 ± 43.1 × 103); 24 out of 40 cases (60%) showed a weak reagents. Afterwards, 2 ml of FACS lysing solution (Becton expression of CD22 (MESF 7.5 ± 2.8 × 103).