Leukemia (2004) 18, 491–498 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation P Menendez1,2, A Vargas3, C Bueno1,2, S Barrena1,2, J Almeida1,2 M de Santiago1,2,ALo´pez1,2, S Roa2, JF San Miguel2,4 and A Orfao1,2 1Servicio General de Citometrı´a, Universidad de Salamanca, Salamanca, Spain; 2Departamento de Medicina and Centro de Investigacio´n del Ca´ncer, Universidad de Salamanca, Salamanca, Spain; 3Servicio de Inmunodiagno´stico, Departamento de Patologı´a Clı´nica, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru´, ; and 4Servicio de Hematologı´a, Hospital Universitario, Salamanca, Spain Lack of apoptosis has been linked to prolonged survival of results in the overexpression of the bcl-2 protein and it malignant B cells expressing bcl-2. The aim of the present represented the first clear example of a common step in study was to analyze the amount of bcl-2 protein expressed 9,10 along normal human B-cell maturation and to establish the oncogenesis mediated by decreased cell death. Currently, frequency of aberrant bcl-2 expression in B-cell malignancies. the exact antiapoptotic pathways through which bcl-2 exerts its In normal bone marrow (n ¼ 11), bcl-2 expression obtained by role are only partially understood, involving decreased mito- quantitative multiparametric flow cytometry was highly vari- chondrial release of cytochrome c, which in turn is required for þ À able: very low in both CD34 and CD34 B-cell precursors, high the activation of procaspase-9 and the subsequent initiation of in mature B-lymphocytes and very high in plasma cells. Bcl-2 the apoptotic cascade.11 expression of mature B-lymphocytes from peripheral blood Despite being a characteristic feature of FL bearing t(14;18),7 (n ¼ 10), spleen (n ¼ 8) and lymph node (n ¼ 5) was significantly higher (P 0.02) in CD23À as compared to CD23 þ B cells, overexpression of the bcl-2 protein is not specific of this subtype o 12 independent of the type of tissue analyzed. Upon comparison of non-Hodgkin’s lymphoma (NHL). High amounts of bcl-2 with normal human B-cell maturation, bcl-2 expression in protein have been reported in other mature peripheral B-cell neoplastic B cells from 144 patients was found to be aberrant neoplasms such as B-cell chronic lymphocytic leukemia (B- in 66% of the cases, usually corresponding to bcl-2 over- CLL),13,14 diffuse large B-cell lymphoma (DLCL),15,16 multiple expression (63%). Follicular lymphoma (FL) carrying t(14;18) myeloma (MM),17–19 monoclonal gammopathy of undetermined and MALT lymphoma were the only diagnostic groups con- 19 stantly showing overexpression of bcl-2. Bcl-2 overexpression significance (MGUS) and in cases of B-cell precursor acute 20 was also frequently found in precursor B-acute lymphoblastic lymphoblastic leukemia (BCP-ALL). Such apparent lack of leukemia (84%), typical (77%) and atypical (75%) B-cell chronic specificity of bcl-2 overexpression has contributed to the general lymphocytic leukemia, prolymphocytic leukemia (two of three belief that its assessment is of limited utility for the diagnostic À cases), mantle cell lymphoma (55%), but not in t(14;18) FL, subclassification of B-cell malignancies. However, a careful splenic marginal zone lymphoma, Burkitt lymphoma and multi- ple myeloma. analysis of the literature shows that information on the levels of Leukemia (2004) 18, 491–498. doi:10.1038/sj.leu.2403231 bcl-2 expressed during normal B-cell differentiation is scanty; Published online 15 January 2004 even more, to the best of our knowledge, no study has been Keywords: bcl-2; flow cytometry; human B-cell differentiation; reported in which quantitative evaluation of bcl-2 expression B-cell leukemic/B-cell lymphoma during normal B-cell maturation has been taken into account prior to establishing whether the amount of this protein in neoplastic cells from different B-cell disorders was abnormally increased or not, as compared to the normal B cells. Introduction The aim of the present study is to analyze quantitatively the amount of bcl-2 protein expressed along the different stages of Control of cell survival is essential in tissues with high cell normal human B-cell maturation, in order to establish the turnover, such as the lympho-hematopoietic system, in order to incidence of aberrant bcl-2 expression in a large series of 144 maintain the normal tissue homeostasis; disruption of the patients suffering from different B-lineage hematopoietic malig- balance between cell production and death is a critical step in nancies. tumorigenesis.1,2 Under physiological conditions, cell death occurs in a programmed way through apoptosis.1–5 The bcl-2 proto-oncogene and its homologues play a key role Material and methods in regulating physiological cell death.1,6 Bcl-2 was initially identified by its involvement in the translocation Controls and patients t(14;18)(q32;q21) – a hallmark of follicular lymphoma (FL)7,8 – 9,10 where it acts as a key negative regulator of apoptosis. In For the evaluation of bcl-2 expression along the normal B-cell t(14;18), the bcl-2 gene from chromosome 18 is translocated to maturation, a total of 34 normal samples were studied. These the long arm of chromosome 14, into the vicinity of the corresponded to 10 peripheral blood (PB) and 11 bone marrow immunoglobulin heavy-chain (IgH) gene, where its expression 7,8 (BM) samples obtained from an identical number of adult comes under the control of the IgH gene promoter. This healthy volunteers and to eight reactive spleen plus five reactive lymph node samples. The mean age of normal donors was Correspondence: Professor A Orfao, Servicio General de Citometrı´a, 46726 years (range: 23–61 years). Hospital Universitario de Salamanca, Paseo San Vicente 58-182, A total of 47 BM and 97 PB samples from 144 newly Salamanca 37007, Spain; Fax: þ 34 923 294624; E-mail: [email protected] diagnosed untreated patients with B-cell malignancies were Received 7 April 2003; accepted 13 October 2003; Published online studied. Diagnosis of the different B-cell malignances was based 15 January 2004 on clinical, morphologic, immunophenotypic and molecular Bcl-2 expression in normal and neoplastic human B cells P Menendez et al 492 criteria, according to the WHO classification.21,22 According to reactive spleen and reactive lymph node samples (Figure 1). In diagnosis, patients were distributed as follows: B-cell precursor samples from patients with malignant B-cell disorders, bcl-2 ALL, 12 cases (mean age: 24720 years); B-CLL, 64 cases – 56 expression was specifically evaluated for the neoplastic B cells. typical (typ) and eight atypical (atyp) CLL (67714 and 7578 Bcl-2 expression was evaluated as the mean fluorescence years, respectively); prolymphocytic leukemia (PLL), three cases intensity (MFI) obtained after subtracting from the MFI values of (61730 years); hairy cell leukemia (HCL), two cases (55721 bcl-2-stained samples the MFI values obtained for the corre- years); mantle cell lymphoma (MCL), 13 cases (67716 years); sponding isotypic-negative controls for each specific normal and splenic marginal zone lymphoma (SMZL), five cases (68724 neoplastic B-cell subset analyzed. In order to normalize the years); MALT lymphoma, three cases (73718 years); FL, 12 measurements of bcl-2-associated fluorescence, a ratio between cases (6479 years); DLCL, six cases (65721 years); Burkitt the bcl-2 MFI value obtained for each B-cell population and the lymphoma, three cases (44730 years); and MM, 21 cases bcl-2 MFI found for the normal resting T-lymphocytes present in (67710 years). In all cases, informed consent according to the the same sample was established in each sample (bcl-2 B/T Ethics Committee of the University Hospital of Salamanca ratio). For this purpose, resting T cells were identified as those (Spain) was given prior to entering the study. events displaying an FSClow/SSClow pattern typical of mature lymphocytes and that were bcl-2hi, CD38À, CD19À and CD23À.26 Flow cytometry immunophenotypic studies Both normal and neoplastic PB and BM samples were collected in tubes containing K3 EDTA as an anticoagulant. BM samples were immediately diluted 1/1 (vol/vol) in phosphate-buffered Statistical analyses saline (PBS). Single-cell suspensions were obtained from spleen and lymph node tissues, by mechanical separation according to The mean values and their standard deviation as well as median, well-established procedures.23 25th and 75th percentiles and range were calculated for each Bcl-2 expression was explored in both the normal and variable. In order to determine the statistical significance of the malignant B-cell subpopulations, by combined immunofluores- differences observed between groups, either the Mann–Whitney cence stainings directed against cell surface and intracytoplas- U and Kruskal–Wallis tests or the Wilcoxon and Friedman tests mic proteins,24 using the following four-color combinations of were used for unrelated and related variables, respectively. monoclonal antibodies (MoAbs) directly conjugated with Statistical analyses were performed with the SPSS 9.0 software fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-cya- program (SPSS Inc, Chicago, IL, USA). P-values p0.05 were nine 5 (PECy5) and allophycocyanine (APC): bcl-2-FITC (clone considered to be statistically significant. 124, DAKO cytomation, Glostrup, Denmark), either CD34-PE (clone HPCA2, Becton Dickinson Biosciences (BDB), San Jose´, CA, USA) – in BM samples – or CD23-PE (clone