Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing bcl-2. The aim of the present study was to analyze the amount of bcl-2 protein expressed along normal human B-cell maturation and to establish the frequency of aberrant bcl-2 expression in B-cell malignancies. In normal bone marrow (n=11), bcl-2 expression obtained by quantitative multiparametric flow cytometry was highly variable: very low in both CD34+ and CD34− B-cell precursors, high in mature B-lymphocytes and very high in plasma cells. Bcl-2 expression of mature B-lymphocytes from peripheral blood (n=10), spleen (n=8) and lymph node (n=5) was significantly higher (P<0.02) in CD23− as compared to CD23+ B cells, independent of the type of tissue analyzed. Upon comparison with normal human B-cell maturation, bcl-2 expression in neoplastic B cells from 144 patients was found to be aberrant in 66% of the cases, usually corresponding to bcl-2 overexpression (63%). Follicular lymphoma (FL) carrying t(14;18) and MALT lymphoma were the only diagnostic groups constantly showing overexpression of bcl-2. Bcl-2 overexpression was also frequently found in precursor B-acute lymphoblastic leukemia (84%), typical (77%) and atypical (75%) B-cell chronic lymphocytic leukemia, prolymphocytic leukemia (two of three cases), mantle cell lymphoma (55%), but not in t(14;18)− FL, splenic marginal zone lymphoma, Burkitt lymphoma and multiple myeloma.
Control of cell survival is essential in tissues with high cell turnover, such as the lympho-hematopoietic system, in order to maintain the normal tissue homeostasis; disruption of the balance between cell production and death is a critical step in tumorigenesis.1,2 Under physiological conditions, cell death occurs in a programmed way through apoptosis.1,2,3,4,5
The bcl-2 proto-oncogene and its homologues play a key role in regulating physiological cell death.1,6 Bcl-2 was initially identified by its involvement in the translocation t(14;18)(q32;q21) – a hallmark of follicular lymphoma (FL)7,8 – where it acts as a key negative regulator of apoptosis.9,10 In t(14;18), the bcl-2 gene from chromosome 18 is translocated to the long arm of chromosome 14, into the vicinity of the immunoglobulin heavy-chain (IgH) gene, where its expression comes under the control of the IgH gene promoter.7,8 This results in the overexpression of the bcl-2 protein and it represented the first clear example of a common step in oncogenesis mediated by decreased cell death.9,10 Currently, the exact antiapoptotic pathways through which bcl-2 exerts its role are only partially understood, involving decreased mitochondrial release of cytochrome c, which in turn is required for the activation of procaspase-9 and the subsequent initiation of the apoptotic cascade.11
Despite being a characteristic feature of FL bearing t(14;18),7 overexpression of the bcl-2 protein is not specific of this subtype of non-Hodgkin's lymphoma (NHL).12 High amounts of bcl-2 protein have been reported in other mature peripheral B-cell neoplasms such as B-cell chronic lymphocytic leukemia (B-CLL),13,14 diffuse large B-cell lymphoma (DLCL),15,16 multiple myeloma (MM),17,18,19 monoclonal gammopathy of undetermined significance (MGUS)19 and in cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL).20 Such apparent lack of specificity of bcl-2 overexpression has contributed to the general belief that its assessment is of limited utility for the diagnostic subclassification of B-cell malignancies. However, a careful analysis of the literature shows that information on the levels of bcl-2 expressed during normal B-cell differentiation is scanty; even more, to the best of our knowledge, no study has been reported in which quantitative evaluation of bcl-2 expression 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.
The aim of the present study is to analyze quantitatively the amount of bcl-2 protein expressed along the different stages of normal human B-cell maturation, in order to establish the incidence of aberrant bcl-2 expression in a large series of 144 patients suffering from different B-lineage hematopoietic malignancies.
Material and methods
Controls and patients
For the evaluation of bcl-2 expression along the normal B-cell maturation, a total of 34 normal samples were studied. These corresponded to 10 peripheral blood (PB) and 11 bone marrow (BM) samples obtained from an identical number of adult healthy volunteers and to eight reactive spleen plus five reactive lymph node samples. The mean age of normal donors was 46±26 years (range: 23–61 years).
A total of 47 BM and 97 PB samples from 144 newly diagnosed untreated patients with B-cell malignancies were studied. Diagnosis of the different B-cell malignances was based on clinical, morphologic, immunophenotypic and molecular criteria, according to the WHO classification.21,22 According to diagnosis, patients were distributed as follows: B-cell precursor ALL, 12 cases (mean age: 24±20 years); B-CLL, 64 cases – 56 typical (typ) and eight atypical (atyp) CLL (67±14 and 75±8 years, respectively); prolymphocytic leukemia (PLL), three cases (61±30 years); hairy cell leukemia (HCL), two cases (55±21 years); mantle cell lymphoma (MCL), 13 cases (67±16 years); splenic marginal zone lymphoma (SMZL), five cases (68±24 years); MALT lymphoma, three cases (73±18 years); FL, 12 cases (64±9 years); DLCL, six cases (65±21 years); Burkitt lymphoma, three cases (44±30 years); and MM, 21 cases (67±10 years). In all cases, informed consent according to the Ethics Committee of the University Hospital of Salamanca (Spain) was given prior to entering the study.
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 saline (PBS). Single-cell suspensions were obtained from spleen and lymph node tissues, by mechanical separation according to well-established procedures.23
Bcl-2 expression was explored in both the normal and malignant B-cell subpopulations, by combined immunofluorescence stainings directed against cell surface and intracytoplasmic proteins,24 using the following four-color combinations of monoclonal antibodies (MoAbs) directly conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-cyanine 5 (PECy5) and allophycocyanine (APC): bcl-2-FITC (clone 124, DAKO cytomation, Glostrup, Denmark), either CD34-PE (clone HPCA2, Becton Dickinson Biosciences (BDB), San José, CA, USA) – in BM samples – or CD23-PE (clone EBVCS-5, BDB) – in PB, spleen and lymph node samples, CD19-PECy5 (clone SJ25C1, Caltag Laboratories, San Francisco, CA, USA) and CD38-APC (clone HB7, BDB). Cell fixation and permeabilization prior to bcl-2 protein staining was performed using the Fix&Perm reagent (Caltag Laboratories) according to the methods that have been previously described in detail.25 For each sample analyzed, a second tube containing cells stained with the same combination of MoAb directed against the cell surface antigens plus an FITC-conjugated, isotype-matched nonspecific mouse immunoglobulin was analyzed in parallel as negative control.
In all cases, either a two- or three-step data acquisition was performed in a FACSCalibur flow cytometer (BDB) using the CellQuest software program (BDB). In the first step, information was stored in a minimum of 3 × 104 total nucleated cells present in the sample; this information was used to assess the relative distribution of the different B-cell subpopulations present in each normal and neoplastic sample. In the second step, acquisition through an electronic ‘live gate’, drawn on the CD19+ region in which B cells are located, was carried out in all samples analyzed; in BM samples, a further third acquisition step through a ‘live gate’ drawn on the CD38+++ region was carried out to analyze plasma cells specifically. In these two latter steps, information on a minimum of 104 CD19+ B cells or CD38+++ plasma cells was acquired, respectively. For data analysis, the Paint-A-Gate PRO software (BDB) was used. The expression of cytoplasmic bcl-2 was assessed in the following subpopulations of CD19+ B cells and/or CD38+++ plasma cells: CD34+/CD38++/CD19+, CD34−/CD38++/CD19+, CD34−/CD38−/+/CD19+ and CD34−/CD38+++/CD19−/+ in BM or CD23−/CD19+ and CD23+/CD19+ in normal PB, reactive spleen and reactive lymph node samples (Figure 1). In samples from patients with malignant B-cell disorders, bcl-2 expression was specifically evaluated for the neoplastic B cells.
Bcl-2 expression was evaluated as the mean fluorescence intensity (MFI) obtained after subtracting from the MFI values of bcl-2-stained samples the MFI values obtained for the corresponding isotypic-negative controls for each specific normal and neoplastic B-cell subset analyzed. In order to normalize the measurements of bcl-2-associated fluorescence, a ratio between the bcl-2 MFI value obtained for each B-cell population and the bcl-2 MFI found for the normal resting T-lymphocytes present in the same sample was established in each sample (bcl-2 B/T ratio). For this purpose, resting T cells were identified as those events displaying an FSClow/SSClow pattern typical of mature lymphocytes and that were bcl-2hi, CD38−, CD19− and CD23−.26
The mean values and their standard deviation as well as median, 25th and 75th percentiles and range were calculated for each variable. In order to determine the statistical significance of the differences observed between groups, either the Mann–Whitney U and Kruskal–Wallis tests or the Wilcoxon and Friedman tests were used for unrelated and related variables, respectively. Statistical analyses were performed with the SPSS 9.0 software program (SPSS Inc, Chicago, IL, USA). P-values ⩽0.05 were considered to be statistically significant.
Bcl-2 expression during normal B-cell maturation
Intracellular expression of the bcl-2 protein along the normal BM B-cell maturation as well as in the major subsets of CD23− and CD23+ B cells from PB, spleen and lymph node is illustrated in Figure 1 and summarized in Table 1. As may be seen in this table, the most immature CD34+ BM B-cell progenitors showed dim bcl-2 expression (bcl-2 MFI of 11±6; bcl-2 B/T ratio of 0.45±0.3). The maturation of CD34+/CD38++/CD19+ progenitors into CD34−/CD38++/CD19+ B-cell precursors was associated with a slight (P>0.05) decreased expression of bcl-2 (bcl-2 MFI 6±4 and bcl-2 B/T ratio of 0.27±0.3). As these cells differentiated into CD34−/CD38+/−/CD19+ B cells, a significant (P<0.005) increase in bcl-2 expression was observed (bcl-2 MFI of 44±18, and bcl-2 B/T ratio of 1.68±0.6). Differentiated BM plasma cells (CD38+++/CD19−/+/CD34−) showed the highest bcl-2 levels (bcl-2 MFI of 161±48 and bcl-2 B/T ratio of 5.94±3).
In normal PB, CD23+ B-lymphocytes showed a significantly (P=0.008) lower expression of cytoplasmic bcl-2 than CD23− B-cells (bcl-2 MFI of 40±12 vs 57±18 and bcl-2 B/T ratio of 0.96±0.27 vs 1.22±0.35). Likewise, in both reactive spleen and lymph node samples, CD23− B-lymphocytes displayed a higher reactivity for bcl-2 than CD23+ B cells (P=0.01 and 0.02, respectively) (Table 1). Interestingly, no statistically significant differences were found among CD23+ or among CD23− B-lymphocytes from PB, spleen and lymph nodes (Table 1).
Bcl-2 expression in neoplastic cells from B-cell malignancies
According to their similarities to normal B-cell subsets, neoplastic B-cell disorders were classified into four major groups: (1) those originating in CD34+ BM B-cell precursors (BCP-ALL); (2) mature peripheral B-cell neoplasms of either CD23+ (B-CLL) or CD23− (PLL) mature PB B cells; (3) mature B-cell neoplasms deriving from mature B cells from lymphoid tissues such as the spleen (HCL and SMZL) or the lymph node (FL, MCL, MALT lymphoma, DLCL and Burkitt lymphoma), which could be either CD23+ or CD23−; and (4) MM patients showing BM infiltration by neoplastic plasma cells.
Neoplastic cells from all cases analyzed, including those from Burkitt lymphoma, showed the expression of the bcl-2 protein above the negative control values. As may be seen in Figure 2a in BCP-ALL cases (n=12), low amounts of intracellular bcl-2 were detected (bcl-2 MFI of 38±14). After normalization of bcl-2 measurements, CD34+ BCP-ALL cases displayed a significantly higher bcl-2 B/T ratio than normal CD34+ B-cell precursors (0.96±0.23 vs 0.45±0.3, P<0.001) (Figure2b and 2c and Table 1).
The analysis of bcl-2 expression in mature B-cell neoplasms, including tissue lymphomas, showed the presence of variable but constantly higher levels of bcl-2 on the neoplastic B-cells, as compared to normal B cells when the bcl-2 MFI (P<0.04) was considered; Burkitt lymphoma cases were the only exception, since they showed the lowest bcl-2 MFI values (Figure 2a). Once bcl-2 expression by neoplastic B cells was normalized for the bcl-2 levels found in resting T cells from the same sample (bcl-2 B/T ratio) only in six out of the 11 groups in which the mature B-cell neoplasms were distributed, the statistical significance (P<0.05) was retained, these including both typical and atypical CLL, PLL, MCL, MALT lymphomas and FL with t(14;18)+, but not t(14;18)− FL, HCL, SMZL, DLCL and Burkitt lymphomas. However, once this bcl-2 B/T ratio was compared for each diagnostic subgroup with the equivalent ratio found in the normal B cells, two- to five-fold higher mean levels of bcl-2 expression were found in all the subgroups analyzed, except HCL and Burkitt lymphoma (Figure 2c). The highest values obtained for the ratio between bcl-2 expression in neoplastic B cells and in normal B cells were found among patients with t(14;18)+ FL and MALT lymphomas (mean values of 5.2 and 4.1, respectively) as well as among the six MCL cases who had t(11;14)+, but not within t(11;14 )− MCL (2.8 and 1.5, respectively).
In MM, high levels of bcl-2 MFI were found in clonal plasma cells (Figure 2a). However, once these levels were normalized according to bcl-2 expression obtained for the normal resting T cells present in the same sample, no significant differences (P>0.05) were detected in myelomatous as compared to normal BM plasma cells (Figure 2b and Table 1). Actually, the ratio between bcl-2 expression in myelomatous and normal plasma cells was slightly lower than one (Figure 2c), indicating that bcl-2 overexpression is not a common feature of MM plasma cells.
Frequency of aberrant bcl-2 expression in B-cell malignancies according to diagnosis
Overall, 66% of all cases studied showed bcl-2 expression at either abnormally high (63%) or low (3%) levels, as compared to the amount of bcl-2 found in the corresponding normal B cells. As shown in Figure 3, bcl-2 overexpression was found in variable proportions in the different diagnostic subgroups, the highest frequencies corresponding to t(14;18)+ FL (eight of eight cases) and to MALT lymphoma (three of three cases), whereas the lowest frequencies were observed in MM (19%), SMZL (one of five cases) and t(14;18)− FL (one of four cases). Among the other diagnostic groups, intermediate frequencies of bcl-2 overexpression were found: BCP-ALL, 84%; typ B-CLL, 77%; atyp B-CLL, 75%; PLL, two of three cases; HCL, one of two cases; MCL, five of six cases with t(11;14)+ and one of five without t(11;14)−; and DLCL, four of six patients. Conversely, the underexpression of bcl-2 was only found among MM patients (19%), Burkitt lymphoma (two of three cases) and in one of the two HCL patients studied, but in none of the other diagnostic subgroups.
The bcl-2 protein plays a key role in preventing apoptosis both in normal and malignant hematopoietic cells.1,27,28 To date, high amounts of the bcl-2 protein have been reported in neoplastic cells from patients with B-cell NHL carrying t(14;18)+ as well as in t(14;18)− B-CLL, DLCL, MM, MGUS and BCP-ALL.12,13,14,15,16,17,18,19,20 Such observations have limited the use of bcl-2 as a diagnostic marker. However, a careful analysis of the literature shows that no study has been reported so far, in which a quantitative evaluation of bcl-2 expression was performed in neoplastic B cells from BCP-ALL, MM and other mature B-cell malignancies as compared to normal B-cell precursors, BM plasma cells and mature B-lymphocytes.
The first goal of our study was to analyze quantitatively the expression of the bcl-2 protein along the different stages of normal human B-cell maturation. Our results show that in normal BM, low expression of bcl-2 occurs among the earliest CD34+ CD19+ B-cell progenitors; interestingly, as these CD34+ B cells differentiate into CD34−/CD38++ B-cell precursors and undergo immunoglobulin gene rearrangements,1,11 bcl-2 expression decreases to barely detectable amounts. In contrast, a high bcl-2 expression is found among the more mature B-lymphocytes, especially in plasma cells that are long-living cells that need regulatory mechanisms to prevent apoptosis.29,30 High bcl-2 protein levels were also found among the normal mature B-lymphocytes present in PB, spleen and lymph nodes. Interestingly, no significant differences were observed in any of these tissues with regard to bcl-2 expression in CD5+ vs CD5− B cells (data not shown). In contrast, bcl-2 expression was significantly higher in the CD23− B cells as compared to the CD23+ compartment of mature B-lymphocytes, independent of whether PB, spleen or lymph node samples were considered. It should be noted that CD23 is expressed in mature B cells upon antigen recognition and subsequent cell activation;31 accordingly, the lower amounts of the bcl-2 protein found in CD23+ B cells may reflect an increased susceptibility to death of B-lymphocytes, following antigen-driven cell activation. Overall, these results confirm and extend previous observations showing that the expression of bcl-2 largely varies in normal individuals along the B-cell differentiation, the amount of this protein per cell correlating with the expected cellular lifespan.27,32,33 Especially interesting is the observation that during the rearrangement of the immunoglobulin genes in BM B-cell precursors, bcl-2 expression is extremely low probably reflecting that at this stage, B-cell precursors are particularly prone to apoptosis that would facilitate the prevention of survival of nonfunctional B cells.
Once the levels of bcl-2 expression in the different normal BM, PB, lymph node and spleen B-cell compartments were established, our major interest focused on establishing the frequency of abnormal bcl-2 expression in B-cell malignancies. Overall, neoplastic B cells from each of the cases from all diagnostic groups studied showed expression of bcl-2, including patients with Burkitt lymphoma; these findings suggest that flow cytometry may be more sensitive than conventional immunohistochemistry for the detection of low levels of bcl-2.34 Typically, once B-cell neoplasms were grouped according to their maturation stage (B-cell precursors, mature B-lymphocytes and plasma cells), bcl-2 expression in neoplastic B cells varied in a similar way to what occurs in the normal B-cell maturation, with the exception of Burkitt lymphoma that had unexpectedly low bcl-2 levels.
In order to standardize the quantitative measurements of bcl-2 by flow cytometry, a ratio between the MFI value of the target cell population and that of resting T cells present in the same sample was established both for normal and neoplastic B cells. This allowed to carry out not only a direct comparison between the bcl-2 B/T ratio found in neoplastic cells and that found in normal cells, but also to establish the frequency at which abnormally high or low bcl-2 expression occurs in the different diagnostic subgroups.
As expected, patients with FL carrying t(14;18) (q32;q21) showed the highest bcl-2 B/T ratio, all cases studied overexpressing bcl-2. However, it should be noted that neoplastic B cells from most BCP-ALL, B-CLL, PLL, MALT lymphoma, MCL and DLCL, despite lacking t(14;18), also displayed overexpression of bcl-2; moreover, no cases showing an abnormally low expression of bcl-2 were found in any of these diagnostic subgroups. This is in clear agreement with previous studies reporting that despite the close association between bcl-2 expression and the presence of t(14;18) (q32;q21), high bcl-2 levels may frequently be found in the absence of this translocation.8,35,36,37 Nevertheless, it should be noted that bcl-2 levels were clearly higher among FL patients carrying t(14;18) and that similarly increased bcl-2 levels were found only in the three MALT lymphomas and a few MCL cases, from all patients studied. Interestingly, among MCL cases, bcl-2 expression was two-fold higher among those cases carrying t(11;14)(q13;q32) as compared to those who did not, suggesting that overexpression of cyclin D1 might be associated with an upregulation of bcl-2 as previously pointed out.38 Further studies in a larger series of patients are necessary to elucidate the exact relationship between cyclin D1 and bcl-2 overexpression in MCL. Altogether, these results indicate that extremely high bcl-2 expression in the context of a phenotype compatible with FL could be used to screen for t(14;18); in contrast, normal bcl-2 expression in neoplastic B cells bearing an FL phenotype would rule out the presence of this cytogenetic abnormality.
Despite the fact that the mutational status of the IgH gene was not analyzed in most cases included in this study, no statistically significant differences were found with regard to clinical, biological and survival features of B-CLL cases showing overexpression of bcl-2 and those who did not, suggesting that bcl-2 overexpression in B-CLL does not confer a different clinical behavior of the disease.
In MM, plasma cells typically displayed high levels of bcl-2. Despite this, bcl-2 expression in MM plasma cells was similar to that of normal BM plasma cells, abnormally high or low bcl-2 levels being found in only a minor proportion of cases. To the best of our knowledge, this is the first report in which bcl-2 expression is compared in normal and myelomatous plasma cells. Previous studies have suggested that abnormally increased bcl-2 expression could occur in plasma cells from most MM patients.17,18,19 Here, we clearly show in a relatively large series of newly diagnosed untreated MM patients that this would only occur in a minor proportion of cases, indicating that bcl-2 expression is not enhanced in most myelomatous plasma cells. These results could contribute to explain the lack of correlation reported in MM between bcl-2 expression and both response to treatment and survival.19,39 In addition, no correlation was found in the present study between bcl-2 expression and neither the proportion of cycling plasma cells nor the presence of monosomy 13/13q− (data not shown), further supporting the lack of prognostic impact for bcl-2 expression in MM.40,41
In summary, our results show that the quantitative expression of bcl-2 largely varies during normal human B-cell differentiation; once such variations are taken into account, bcl-2 overexpression appears to be a constant finding only in FL with t(14;18)+ and MALT lymphomas. Despite this, bcl-2 overexpression is also frequently observed in BCP-ALL, B-CLL, PLL and MCL, but not in t(14;18)− FL, SMZL, Burkitt lymphoma and MM.
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P Menéndez was supported by a grant from Fondo de Investigaciones Sanitarias, Madrid, Spain (FIS, BEFI 98/9669). This work was supported in part by a grant from the Red Temática “Mieloma mùltiple y otras gammapatias monoclonales”. Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Madrid, Spain.
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Menendez, P., Vargas, A., Bueno, C. et al. Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation. Leukemia 18, 491–498 (2004) doi:10.1038/sj.leu.2403231
- flow cytometry
- human B-cell differentiation
- B-cell leukemic/B-cell lymphoma
Journal of Clinical Medicine (2019)
Retrospective Review of the Use of High-Dose Cyclophosphamide, Bortezomib, Doxorubicin, and Dexamethasone for the Treatment of Multiple Myeloma and Plasma Cell Leukemia
Clinical Lymphoma Myeloma and Leukemia (2019)
Antioxidants & Redox Signaling (2018)
Journal of Cancer Therapy (2017)
Development of a flow cytometric method for quantification of BCL-2 family members in chronic lymphocytic leukemia and correlation with sensitivity to BCL-2 family inhibitors
Cytometry Part B: Clinical Cytometry (2017)