|Year : 2012 | Volume
| Issue : 4 | Page : 207-212
Expression of myelomonocytic antigens in Egyptian B-cell chronic lymphocytic leukemia ( impact on prognosis)
Deena M.M. Habashy1, Amira F. Barakat2
1 Clinical Pathology Department, Hematology Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||20-Mar-2012|
|Date of Acceptance||22-Apr-2012|
|Date of Web Publication||21-Jun-2014|
Amira F. Barakat
Department of Internal Medicine, Faculty of Medicine, Mansoura University, P.O. Box 35516, Mansoura
Source of Support: None, Conflict of Interest: None
B-cell chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays immense clinical heterogeneity. Therefore, there has been considerable interest in identifying prognostic markers that can be used to distinguish those patients who may have an aggressive form of CLL. Clinical staging systems lack the ability to predict disease progression. Numerous cellular and molecular markers with potential prognostic and therapeutic significance have been identified. Among these markers are the myelomonocyte-associated markers whose expression on neoplastic lymphocytes in CLL has been reported to correlate with an unfavorable prognosis.
Aim of the study
We aimed to study the expression of the myelomonocytic markers CD13 and CD11b in Egyptian CLL patients in comparison with a healthy control group and assess their relation to other prognostic factors and patient outcomes. This study also aimed to reveal the association of CD38 and FMC7 with prognosis and disease progression.
Participants and methods
Expressions of CD13 and CD11b were detected by flow cytometry in 30 newly diagnosed CLL cases and 10 healthy individuals. Percentages of CD38-positive and FMC7-positive cells were also measured.
CD13 was expressed in three cases (10%), whereas CD11b was expressed in four cases (13.3%) of CLL. CD38 was expressed in 13 cases (43.3%) and FMC7 in 11 cases (36.6%). None of the individuals in the healthy control group expressed CD13 or CD11b. The expression of CD13 and CD38 was significantly correlated with adverse results of complete blood count (P<0.05). CD11b expression was significantly associated with low platelet count (P=0.04). Expression of all studied markers (CD13, CD11b, CD38, and FMC7) was significantly correlated with the presence of 11q deletion (11qdel) (P=0.004, P<0.001, P=0.001, and P<0.001, respectively), diffuse pattern of bone marrow infiltration (P=0.005, 0.01, P<0.001, and P<0.001, respectively), and poor outcome (P=0.002, 0.01, P<0.001, and P<0.001, respectively). The expression of CD38 was also associated with the expression of CD13 and FMC7 (P=0.02 and P<0.001, respectively) and with the presence of trisomy 12 (P=0.01).
CD13 and CD11b expressions were found to be correlated with the standard adverse prognostic factors in CLL and with shorter survival, which questions their future possible role as prognostic markers in CLL. FMC7 and CD38 expression was also associated with unfavorable prognosis and poor outcome.
Keywords: chronic lymphocytic leukemia, myelomonocytic antigens, prognosis
|How to cite this article:|
Habashy DM, Barakat AF. Expression of myelomonocytic antigens in Egyptian B-cell chronic lymphocytic leukemia ( impact on prognosis). Egypt J Haematol 2012;37:207-12
|How to cite this URL:|
Habashy DM, Barakat AF. Expression of myelomonocytic antigens in Egyptian B-cell chronic lymphocytic leukemia ( impact on prognosis). Egypt J Haematol [serial online] 2012 [cited 2020 Jan 25];37:207-12. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/207/134966
| Introduction|| |
B-cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the western world. It is a disease affecting older individuals with an incidence that increases exponentially after the age of 50. Thus, as people continue to live longer, the overall prevalence of CLL will continue to rise 1. CLL is characterized by clonal proliferation of mature CD5+, CD19+, CD23+ B lymphocytes and weak surface expression of a monoclonal immunoglobulin (Ig) B-cell receptor. CLL cells accumulate in peripheral blood (PB), bone marrow (BM), lymph nodes, and spleen, permitting relatively easy diagnosis from the analysis of PB. The diagnostic criteria have been outlined by the International Workshop on CLL and include the presence of more than 5000/µl B lymphocytes in the PB 2. In the absence of cytopenias, lymphadenopathy, or organomegaly, patients who have less than 5000/µl CLL cells are defined as having a monoclonal B-cell lymphocytosis, an entity that may progress to frank CLL at a rate of ∼1–2% per year. Although rare familial forms of CLL have been described 3, the vast majority of patients have no known underlying predisposition, and, like most cancers, CLL is thought to arise as a result of acquired genetic mutations 4.
At the time of presentation, ∼25–50% of patients will be asymptomatic, and diagnosis is frequently made on routine blood analysis. B-cell CLL is a heterogenous disease and the majority of patients will have an indolent clinical course that does not require intervention for many years, followed by a progressive and often terminal phase lasting 1–2 years. Alternatively, some patients may progress rapidly from the time of diagnosis and suffer from complications such as autoimmune hemolytic anemia, thrombocytopenia, and infection. At some point in their clinical course, up to 5% of CLL cases will undergo a Richter’s transformation, whereby the CLL clone evolves into an aggressive high-grade lymphoma. Risk factors for transformation are poorly defined but include an elevated serum lactate dehydrogenase level, monoclonal gammopathy, progressive lymphadenopathy, systemic symptoms, and extranodal involvement 5.
Over the past 10–15 years, numerous studies have advanced our understanding of the biology of CLL. Many of these investigations have also led to the identification of various prognostic factors that can be used to stratify patients into risk categories. However, despite ongoing efforts to identify and characterize prognostic risk factors in CLL, it is unclear how these factors have affected routine clinical practice 4.
The differentiation antigens CD13, CD11c, and CD11b are known markers for myelomonocytic lineage. It was previously suggested that cross-lineage expression of myeloid-associated surface peptidase (CD13-aminopeptidase N) and/or cell adhesion molecules (CD11c-LeuCAMc and CD11b-LeuCAMb) may influence the biological and clinical behavior of chronic lymphoproliferative disorders of B cells 6.
Human CD38 is a cell surface molecule endowed with multiple functions. As a receptor, it regulates the activation of an intracellular signaling pathway, generally linked to lymphocyte activation and proliferation under physiological conditions 7. Data accumulating over a decade concur to define a model in which CD38 is a central element of a large supramolecular complex that includes surface signaling receptors, chemokine receptors, adhesion molecules, and matrix metalloproteases. Expression of CD38 within this supramolecular complex makes signal transduction as well as chemotaxis and homing more efficient, suggesting that the molecule is an integrator of proliferative and migratory signals. These data indicate that CD38 is not only a reliable disease marker but also a functional molecule in the CLL context 7.
The aim of this work was to detect the expression of CD13 and CD11b (myelomonocytic markers) in newly diagnosed CLL cases, correlate their expression with the standard prognostic factors and with patient outcome, and compare the expressions of these two markers in CLL patients with those in the healthy control group. Further, this study aimed to reveal the association between CD38 and FMC7 with the known prognostic factors of CLL and disease progression.
| Participants and methods|| |
This study was carried out on 30 CLL patients newly diagnosed according to the International Workshop on CLL criteria 2. The patients were attending the Hematology Oncology Unit of Mansoura University Hospitals. There were 23 male and seven female patients with a male-to-female ratio of 3.2 : 1, with a mean age of 59.6 years (9.8 years). Immunophenotypic analysis revealed the coexpression of CD19 and CD5 and expression of CD23 and CD20 in all studied cases. Ten healthy age-matched individuals were studied as the control group. They were seven male and three female individuals, with a male-to-female ratio of 2.3 : 1, with a mean age of 51.6 years (6.8 years). A thorough clinical and laboratory investigation was performed for all participants, including complete blood count (CBC), erythrocyte sedimentation rate, and immunophenotyping. Of the 30 CLL cases, 19 underwent BM aspirate smear film examination, 27 underwent trephine biopsy examination, and karyotyping and fluorescence in-situ hybridization analysis was carried out on 20 cases. Patients were followed up for 12 months to detect the outcome of the disease. In this study all participants were informed about the objectives and procedures of the study and gave their written consent.
A volume of 2 ml of PB was obtained on potassium ethylene diamine tetra-acetic acid (K2-EDTA) for CBC. Another 1 ml of PB or BM aspirate on sterile K2-EDTA was collected for flow cytometric studies. A measure of 1 ml of BM aspirate was collected in a sterile preservative-free heparin-coated vacutainer tube for karyotyping and fluorescence in-situ hybridization analysis. BM trephine biopsy samples were taken for histopathologic examination.
Flow cytometric analysis
The panel of monoclonal antibodies (MoAbs) used for the immunophenotypic diagnosis of CLL included CD5, CD19, CD23, CD79b, CD20, CD38, FMC7, CD10, CD103, CD123, surface κ and &lgr; light chains, and surface immunoglobulin M. CD13 [phycoerythrin (PE) conjugated] and CD11b [fluorescein isothiocyanate (FITC) conjugated] were evaluated after establishment of diagnosis. All MoAbs were provided by Immunotech, a Beckman Coulter company (Coulter Electronics Inc., Hialeah, Florida, USA).
The immunophenotyping staining procedure used was the standard ‘whole blood lysis’ technique, in which 50 μl of whole blood or BM sample (with leukocytic count adjusted to 5–10×103 cells/μl) was placed into polystyrene tubes and simultaneously stained with 5 μl of one FITC-labeled and one PE-conjugated anti-human monoclonal antibody or with their corresponding isotypic controls. After 15 min of incubation in the dark, at room temperature, 1.0 ml of laboratory-prepared ammonium chloride-based lysing solution was added and RBC lysis was allowed for 10 min at room temperature. Samples were washed once and resuspended in 0.5 ml of PBS. For surface light chain staining, the step of RBC lysis and washing with PBS was performed before adding the MoAbs. Staining with isotypic controls was performed to distinguish positive staining from autofluorescence and nonspecific antibody binding. Cells were analyzed on a Coulter EPICS XL flow cytometer using System II software (Beckman Coulter). The flow cytometer acquired a minimum of 10 000 cellular events; an electronic gate was set in a linear forward scatter/log side scatter histogram to determine the lymphoid cell population. A CD19 PE-cy5 conjugate was used in control samples only to separate B lymphocytes from total PB lymphocytes. Cells were considered positive for a marker when more than 20% of cells expressed that marker. The percentage of positive cells was defined as cells in the electronic gate that had FITC and/or PE fluorescence falling in the positive region determined by the corresponding isotypic control 8.
Statistical analysis of the data was performed using SPSS 15 (SPSS Inc., Chicago, Illinois, USA) software package under Windows 7 operating system. Qualitative data parameters were presented in the form of frequency and percentage. The normality of quantitative variables was tested using both the Shapiro–Wilk test and the Kolmogrov–Smirnov test. Central tendency of quantitative data parameters was presented in the form of mean and median, and measure of spread was presented as SD and 25th and 75th percentiles. Comparative analysis was performed using the Mann–Whitney U-test (Z value) for comparisons between two independent samples with a nonparametric distribution. Correlations among variables were determined by the Spearman rank correlation coefficient (&rgr;) or the Point biserial correlation (rpb) according to the nature of the studied variables. P value was assumed significant if less than 0.05. Graphic presentation of data was made using Excel 2010 software.
| Results|| |
The demographic and clinical data of the patient group are shown in [Table 1]. The laboratory data of the patient and control groups are shown in [Table 2]. A CD13-positive, CD11b-negative CLL case is shown in [Figure 1].
|Figure 1: A CD13-positive CD11b-negative chronic lymphocytic leukemia case.|
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|Table 1: Demographic and clinical characteristics and outcome of studied chronic lymphocytic leukemia patients|
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|Table 2: Laboratory parameters of studied chronic lymphocytic leukemia patients and controls|
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In CLL patients, CD13 was expressed in three cases (10%), whereas CD11b was expressed in four (13.3%). CD38 was expressed in 13 cases (43.3%) and FMC7 in 11 (36.6%) [Table 2]. All CD13-positive and CD11b-positive cases (100%) showed a diffuse pattern of BM infiltration. Eleven of 13 CD38-positive cases (84.6%) and nine of 11 FMC7-positive cases (81.8%) showed diffuse pattern of BM infiltration [Figure 2]. All CD13-positive and CD11b-positive cases died (100%); one patient among them expressed both markers. Ten of the 13 CD38-positive cases (76.9%) and eight of the 11 FMC7-positive cases (72.7%) died [Figure 3].
|Figure 2: Association between studied markers and pattern of bone marrow infiltration.|
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CBC parameters were significantly different between the patient and control groups (P⩽0.001). No significant difference in median percentage of expression of CD13 and CD11b was found between the two groups (P=0.177 and 0.396, respectively) [Table 3].
|Table 3: Comparison between the control and patient groups regarding laboratory findings at diagnosis|
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The expression levels of CD13 and CD38 were found to be significantly correlated with high total leukocytic count, low Hb level, and low platelet (PLT) count (P<0.05). Expression of CD11b was significantly correlated with low PLT count (P=0.04). The expression levels of all studied markers (CD13, CD11b, CD38, and FMC7) revealed significant correlations with the presence of 11qdel, diffuse pattern of BM infiltration, and shorter survival. Low expression levels of CD38 and FMC7 were detected in cases with 13qdel (P=0.008 and 0.03, respectively) [Table 4]. Expression of CD13 was significantly correlated with the expression of both CD11b and CD38 (P=0.02). Further, expression of CD38 correlated with that of FMC7 (P<0.001) and with the presence of trisomy 12 (P=0.01) [Table 4].
|Table 4: Correlations between all studied markers and demographic, clinical, and laboratory data and patient outcome|
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| Discussion|| |
Patients with CLL follow heterogenous clinical courses. Some survive for a long time without therapy, whereas others progress toward more advanced stages and die despite aggressive treatment 9. For a biological correlate to this diversity, there has been considerable interest in the phenotypic heterogeneity of B cells in CLL 10.
In this study, aberrant expressions of both CD13 and CD11b in CLL patients were examined. CD13 was expressed in 10% of cases, whereas CD11b was expressed in 13.3% of cases. These percentages were lower than those reported previously for CD13 (22, 17.1, and 30%, respectively) 6, 10, 11 and CD11b (21 and 20%, respectively) 6,12. In contrast, Tàssies et al. 12 detected a lower percentage for CD13 (4.8%). In the current work, no significant difference in median percentage of expressions of CD13 and CD11b was found between the patient and control groups. Previously, it was stated that the expression of myelomonocytic antigens on the control group did not exceed 4% of B lymphocytes 12. Expression of CD13 in the present study correlated significantly with adverse CBC parameters, diffuse pattern of BM infiltration, presence of adverse chromosomal abnormality (11qdel), and shorter survival. A statistically significant association between CD13 and clinical staging (Rai and Binet staging systems) was detected before 11.
A previous study revealed higher expression of CD11b (64.2%) and CD13 (50%) in CLL cases. It declared that the presence of myelomonocytic antigens on the surface of neoplastic B cells was frequently associated with a diffuse pattern of BM infiltration, a feature affecting prognosis independently of clinical stage 13. It was shown earlier that CD13-positive patients were 13.7-fold higher in number than CD13-negative cases presenting with diffuse pattern of BM infiltration 6,11.
Our study revealed a significant correlation between CD11b expression and low PLT count, diffuse pattern of BM infiltration, presence of adverse chromosomal abnormality (11qdel), and shorter survival. Association between CD11b and pattern of BM infiltration was detected previously 6. Tàssies et al. 12 detected an association of CD11b expression with a large number of lymphoid areas involved. They also reported death of seven of 21 CD11b-positive patients and eight of 84 CD11b-negative patients, which highlights an influence of CD11b on overall survival (OS).
Other investigators suggested that expression of myeloid-associated markers such as CD13, CD14, CD11c, and CD11b on neoplastic lymphocytes in CLL correlates with unfavorable prognosis 12–16. In addition, Ikematsu et al. 17 reported that CD13 and CD11b expression was restricted to patients with CD5-negative CLL, an immunological variant that is known for its poor prognosis. In contrast, Chang et al. 18 found no significant difference in the requirement for chemotherapy between CLL patients with high levels of expression of myeloid-associated markers and those without. In addition, high levels of expression of myeloid-associated markers showed no statistically significant impact on OS in those patients.
Expression of FMC7 in this work was shown in 36.6% of patients. This percentage was close to that mentioned before for small lymphocytic lymphoma/CLL cases (41%) 19. FMC7 in our study was also significantly correlated with the presence of adverse chromosomal abnormality (11qdel), diffuse pattern of BM infiltration, and shorter survival. It was stated before that the pattern of CD23±/FMC7+ may be expressed by a subgroup of CLL with trisomy 12, which is at greater risk for a more aggressive course 20,21. A previous study found that only 12% of CLL patients expressed FMC7. It declared that when CLL has transformed into CLL/prolymphocytic leukemia the percentage of FMC7+ cells increases, as does the expression of surface immunoglobulin, which provides a means of indicating patients with a potentially more aggressive clinical course and worthy of closer clinical follow up 22. Also, it was previously shown that CLL cases with high FMC7 and low CD23 expression was associated with a short survival 23.
In the present work, CD38 was expressed in 43.3% of patients and its expression was found to be significantly correlated with adverse CBC parameters, diffuse pattern of BM infiltration, presence of adverse chromosomal abnormality (11qdel, trisomy 12), and shorter survival. Its expression was also associated with CD13 and FMC7 expression. CD38 is considered an independent negative prognostic factor expressed by ∼1/3 of CLL patients 24. Many investigators have studied the association between CD38 expression and survival. A previous study stated that CD38 was expressed in 43% of CLL patients and those patients had significantly shorter survival times 25. Chang et al. 18 found that CLL patients with high levels of CD38 expression (defined as ≥30% of neoplastic lymphocytes expressing CD38) had a significantly poorer OS compared with those with low levels of CD38 expression (51 vs. 103 months). Previous studies also stated that patients with CD38 expression are more likely to need chemotherapy 18,25; however, no trial to date has shown that the CD38 expression status changes the management of CLL 4. It was stated before that levels of CD38 and myeloid-associated marker expression can change over the course of disease in B-cell CLL patients. Furthermore, the conversion from low-level to high-level expression of these markers may be associated with a change from an indolent to an aggressive clinical course 25,26.
The pattern of cell surface antigen expression was studied before in familial and sporadic CLL cases to determine whether unique identifiers of familial CLL could be detected. Detection of CD2 or CD13 expression in CLL suggested familial CLL and examination of family history for additional affected members. As for CD38, although the incidence of its expression was similar in familial and sporadic CLL cases (47 and 44%, respectively), the association with prognosis differed. There was a trend toward decreased survival in CD38-positive sporadic, but not familial, CLL patients 27.
With regard to CBC parameters, this study detected a significant difference between patient and control groups. Of the 30 studied CLL cases, 25 patients were anemic (13 of them had Hb level<10 g/dl, of whom eight died). Moreover, 16 cases were thrombocytopenic (11 of them had PLT count<100×109/l, of whom seven died). Anemia and thrombocytopenia are used in the Rai clinical staging system, together with lymphadenopathy and organomegaly, to establish five prognostic groups that can be used to predict median survival 4. Patients with thrombocytopenia or anemia or both, which is caused by extensive marrow infiltration and impaired production (Rai III/IV, Binet C), have a poorer prognosis compared with patients with immune cytopenias 28.
| Conclusion|| |
CD13 and CD11b expression was found to be associated with the standard adverse prognostic factors in CLL and with shorter survival, which questions their future possible role as prognostic markers in CLL. FMC7 and CD38 expression was also associated with unfavorable prognosis and poor outcome. Periodic monitoring of CD38 and, possibly, myeloid-associated markers may be necessary to fully assess the clinical/prognostic status of CLL patients. Additional studies on a wider scale are necessary to determine the most appropriate prognostic markers and methods for monitoring these patients.
The financial support and facilities offered by the Clinical Pathology Department, Hematology Unit, Faculty of Medicine, Ain Shams University, to carry out this work are greatly appreciated.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]