|Year : 2019 | Volume
| Issue : 1 | Page : 23-28
CD14 as a potential prognostic factor and Bcl-2 as a therapeutic target in egyptian B-Cell chronic lymphocytic leukemia patients
Mohamed Mahmoud El-Khawanky1, Mohamed Hanfy Morsy2, Amina Mohamed Elnaggar3, Ola Ali Hussin3, Mohga Mahmoud Zaewer3, Naglaa Ali Khalifa3
1 Department of Hematology, College of Medicine, Najranu Niversity, Najran, KSA
2 Department of Clinical Pathology, Al-Azhar School of Medicine, Al-Azhar University, Cairo, Egypt
3 Department of Clinical Pathology, College of Medicine, Zagazig University, Zagazig, Egypt
|Date of Web Publication||30-Apr-2019|
Dr. Mohamed Mahmoud El-Khawanky
Department of Hematology, College of Medicine, Najran University, Najran
Source of Support: None, Conflict of Interest: None
INTRODUCTION: B-cell chronic lymphocytic leukemia (B-CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40. B-CLL represents a neoplastic disorder caused primarily by defective programmed cell death and accompanied by a myriad of cellular and humoral immune defects.
AIM: This study aimed to assess Bcl-2 and CD14 expression in B-CLL patients and their study as probable prognostic and therapy targeting factors.
PATIENTS AND METHODS: In this study, we assessed Bcl-2 and cluster of differentiation (CD14) expression in a group of Egyptian patients with B-CLL. Forty B-CLL patients and 20 apparently healthy individuals served as the control group were included in this study.
STATISTICAL ANALYSIS: SPSS statistical software (IBM SPSS Inc., version 20, Chicago, Illinois, USA) was used for statistical analysis.
RESULTS: Aberrant expression of Bcl-2 protein appeared in all B-CLL patients (100%). Bcl-2 expression showed a highly positive correlation with total lymphocyte count and lymphocyte count (P =0.000 for both) and a positive correlation with lactate dehydrogenase (P = 0.044). The expression of myelomonocytic antigen “CD14” above the cutoff value 5 × 109/L was reported in 70% (28/40) of B-CLL patients, 55.6% (10/18) of the intermediate-risk group, and 81.8% (18/22) of high-risk group.
CONCLUSION: There was a significant increase in Bcl-2 protein and CD14 in B-CLL patients. Bcl-2 was highly increased in all patients and CD14 more observed in the high-risk group than that of the intermediate risk group.
Keywords: Apoptosis, B-cell chronic lymphocytic leukemia, Bcl-2, CD14, flow cytometry, immunophenotyping
|How to cite this article:|
El-Khawanky MM, Morsy MH, Elnaggar AM, Hussin OA, Zaewer MM, Khalifa NA. CD14 as a potential prognostic factor and Bcl-2 as a therapeutic target in egyptian B-Cell chronic lymphocytic leukemia patients. J Appl Hematol 2019;10:23-8
|How to cite this URL:|
El-Khawanky MM, Morsy MH, Elnaggar AM, Hussin OA, Zaewer MM, Khalifa NA. CD14 as a potential prognostic factor and Bcl-2 as a therapeutic target in egyptian B-Cell chronic lymphocytic leukemia patients. J Appl Hematol [serial online] 2019 [cited 2019 May 20];10:23-8. Available from: http://www.jahjournal.org/text.asp?2019/10/1/23/257469
| Introduction|| |
B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common forms of leukemia worldwide and Egypt., B-CLL is a lymphoproliferative disorder that rarely occurs under the age of 40 with increasing incidence by age. B-CLL is characterized by progressive expansion of malignant B-cell clone accompanied by a myriad of cellular and humoral immune defects.
B-CLL is a disease caused primarily by defects in apoptosis mechanisms. Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing Bcl-2. Bcl-2 family of pro- and anti-apoptotic proteins are key regulators of the apoptotic cascade. Of this family, Bcl-2 was the first identified and remains the best characterized. Aberrant expression of Bcl-2 is common in B-CLL, therefore, Bcl-2 is considered an attractive target for novel therapeutic agents.
In B-CLL patients, a novel lymphocyte population in peripheral blood has been expressing an antigen was detected with a monoclonal antibody directed against the human cluster of differentiation 14 (CD14) molecule. CD14 is a glycosyl-phosphatidylinositol-anchored membrane protein (mCD14) that is preferentially expressed on monocytes/macrophages. CD14 works as a receptor for lipopolysaccharide (LPS) and LPS-binding proteins that stimulate cells to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α).,
Although the definite function of the CD14 antigen in B-CLL is still an enigma, the expression of CD14 in B-CLL represents a novel prognostic factor and an important marker for predicting overall survival in B-CLL patients.
The aim of this study was to assess Bcl-2 and CD14 expression in B-CLL patients and their study as probable prognostic and therapy targeting factors.
| Patients and Methods|| |
This study was carried out on 60 participants. They were classified into 40 Egyptian patients diagnosed with B-CLL and 20 apparently healthy individuals served as the control group. The study was carried out at the Clinical Pathology and Internal Medicine (Oncology-Hematology unit) Departments in Zagazig University Hospitals, Egypt.
B-CLL patients with infectious diseases such as HIV, chronic malaria, and tuberculosis generally were excluded from the study.
The study has been approved by the local Ethics Committee, and informed consent was obtained from all the participants before the study was performed.
All patients were subjected to full clinical and laboratory examination, immunophenotyping by the BD FACScan flow cytometry (Bickton Dicknson) using CLL panel kit (DAKO): CD3, CD5, CD7, CD10, CD19, CD20, CD22, CD23, and HLA-DR. Cells were considered positive for a marker when > 20% of the cells expressed that marker. All participants were underwent for Bcl-2 and CD14 expression detection by flow cytometry.
Bcl-2 and CD14 assessment by flow cytometry.
Bcl-2 staining procedure
For each analysis, three tubes were labeled; tube “1” for leucogate, tube “2” for control, and tube “3” for Bcl-2. Up to 100 μL of the cell suspension was added to each tube. A volume of 100 μL of DAKO IntraStain reagent A (Fixation) was added to the tube “3” and vortex was done gently and incubated at room temperature for 15 min. One mL of cold (4°C) 70% methanol was added to the tube “3” and incubated in the dark for 60 min at 4°C. This would make the cell and nuclear membranes permeable. After incubation, the tube “3” was centrifuged at 1200 r.p.m. for 5 min at room temperature and the supernatant was aspirated. The tube was washed once with 2 mL of washing solution, then centrifuged at 1200 r.p.m. for 5 min at room temperature. The supernatant was aspirated. Ten μL was placed from the leucogate reagent into the tube “1” and 10 μL was placed from control reagent immunoglobulin G2b-Fluorescein isothiocyanate (IgG2b-FITC) into tube “2” and vortex was done thoroughly at low speed for 3 s. Ten μL of FITC conjugated Bcl-2 (Dako) were added to the tube “3” and vortex was done thoroughly at low speed for 3 s. The three tubes were incubated for 30 min at room temperature and protected from direct light during incubation.
CD14 staining procedure
For each analysis, three tubes were labeled; tube “1” for leucogate, tube “2” for control, and tube “3” for CD14. Up to 100 μL of the cell suspension was added to each tube. Ten μL were placed from the leucogate reagent into the tube “1”, 10 μL of a nonreactive monoclonal antibody (negative control) was placed from control reagent IgG1-PE into tube “2”, and the vortex was done thoroughly at low speed for 3 s. Ten μL of monoclonal mouse anti-human CD14 (Dako) and R-phycoerythrin-conjugated were placed into the tube “3” and vortex was done thoroughly at low speed for 3 s. The tubes were incubated for 20 min at room temperature (20°C–25°C) and protected from the direct light.
Lysing and washing procedure
Two mL of (1:10) diluted lysing solution were added to each tube. Immediate vortex was done thoroughly at low speed for 3 s and incubated for 10–12 min at room temperature in the dark. Immediately after incubation, tubes were centrifuged at 1200 r.p.m. for 5 min at room temperature and the supernatant was aspirated. Two mL of washing solution were added to each tube and the vortex was done. Centrifugation at 300 ×g for 5 min at room temperature was done, and the supernatant was discarded. All tubes were resuspended in 0.5 mL of FACs flow for each tube; vortex was done. The sample was ready for an analysis.
Gating and fluorescence analysis
Live data were acquiesced using CellQuest software. Cell subpopulations showing green fluorescence (FL1) intensities and red FL2 intensities that indicated positive staining for corresponding CD markers were identified by comparison of dual-parameter histograms to the corresponding negative control. The positive region for an individual sample was based on the nonimmune mouse IgG-FITC and IgG-PE stained samples, with the percentage of cells in the positive region of this negative control samples generally < 2% of total events.
| Results|| |
This study was carried out on 40 cases recently diagnosed as B-CLL. Their ages ranged from 35 to 77 years (mean 58.15 ± 10.46 years), they included 65% (26/40) of males and 35% (14/40) of females. Control group consisted of 20 apparently healthy persons; their ages ranged from 45 to 62 years (mean 53.6 ± 6.65 years), they included 60% (12/20) of males and 40% (8/20) of females.
Descriptive analysis of the demographic and laboratory characteristics of the studied groups is shown in [Table 1].
B-CLL patients showed a highly significant (P = 0.000) Bcl-2 expression (mean 79.9 ± 12.51%) in comparison with the control group (mean 6.8 ± 1.55%) [Table 1]. According to the modified Rai staging, all patients (100%) showed bcl-2 expression above the cutoff value (>10%) [Figure 1].
|Figure 1: Histogram of B-cell chronic lymphocytic leukemia patient showing positive Bcl-2 (74%) expression|
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In relation to clinical findings, Bcl-2 expression showed significant increased levels in patients with splenomegaly and hepatomegaly (P = 0.017 and 0.014 respectively) and without any significance with sex or lymphadenopathy (P = 0.349 and 0.146 respectively).
In addition, Bcl-2 showed a highly positive correlation with total lymphocyte count (TLC) and leucocyte count (P = 0.000 for both), a positive correlation with lactate dehydrogenase (LDH) (P = 0.044) and without any correlation with age, hemoglobin concentration, or platelets count [Table 2].
|Table 2: Bcl-2 and CD14 Correlation with age and laboratory parameters in B-cell-chronic lymphocytic leukemia group|
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There was a statistically significant (P = 0.03) high expression of CD14 in B-CLL patients (mean 16.49 ± 22.45%) in comparison with the control group (mean 0.14 ± 0.017%), with more expression incidence in high-risk B-CLL group 18/22 (81.8%) than in the intermediate risk group 10/18 (55.6%), according to the modified Rai staging and cutoff value 5 × 109/L [Table 3] and [Figure 2].
|Table 3: Incidence of CD14 positive B-cell-chronic lymphocytic leukemia patients according to the modified Rai staging and the cutoff value 5×109/l|
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|Figure 2: Histogram of B-cell chronic lymphocytic leukemia case showing positive CD14 (52%)|
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Regarding the clinical examination findings (lymphadenopathy, splenomegaly, and hepatomegaly), there was not any significant difference in CD14 expression among B-CLL patients.
In addition, CD14 did not show any significant correlation with other laboratory parameters (TLC, lymphocyte count, hemoglobin concentration, platelets count, LDH level, and Bcl-2) and age in B-CLL patients [Table 2].
| Discussion|| |
There are two contradictory paths, one trying to overcome the other, i.e. the dominating anti-apoptotic pathway where the disease emerged and represented by studying the expression of Bcl-2. The other is the apoptotic pathway, which was represented by studying CD14 expression.
B-CLL is characterized by a typical defect in apoptosis through the production of chemokines and cytokines that activate survival pathways such as NF-kB or PI3K/AKT and result in overexpression of key anti-apoptotic proteins such as Bcl-2 or through alterations in apoptosis regulators. Evading the apoptotic program is one of the hallmarks of tumorigenesis and represents an important mechanism in clinical resistance to therapies.
Apoptosis is a physiological cell death essential for the maintenance of homeostasis and the prevention of oncogenesis. The removal of apoptotic cell exhibits a range of synaptic receptors to mediate cell clearance by phagocytes., Of these receptors is CD14, which is a pattern recognition receptor that highly expressed in cells undergoing apoptosis.
The present work concerned with studying Bcl-2 and CD14 in Egyptian patients with B-CLL as anti-apoptotic and apoptotic markers, respectively with correlation the findings to the clinical and laboratory data.
Srinivas et al. detected the cutoff level of Bcl-2 above 10% as a percentage of positive cells.
We found a bright expression of Bcl-2 in all B-CLL cases (100%) with a highly significant difference (P = 0.000) in comparison with the control group, confirming the results of most studies.,, Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line that has been linked to prolonged survival of malignant B-cells expressing Bcl-2.
In the study, the mean levels of Bcl-2 protein expression were 79.9 ± 12.51%, whereas in control group was 6.8 ± 1.55%. Most B-CLL studies reported that B-CLL cells express relatively high amounts of Bcl-2. The mean levels of Bcl-2 protein expression were 60%, 76.3%, and 77% in different studies.,,
The mechanisms responsible for the high expression of Bcl-2 in most B-CLL patients remain mysterious. The t (8;21) translocation is considered the key mechanism for activation of the BCL2 gene. Other contributing mechanisms are the hypomethylation of Bcl-2 gene promoter region, deletion of endogenous microRNAs (miRs) such as miR-15a and miR-16-1 in B-CLL patients with 13q-deletion.
Bcl-2 showed a highly positive correlation with TLC and lymphocyte count (P = 0.000) and this matches the role of Bcl-2 in survival prolongation and B-lymphocytes accumulation in the blood and bone marrow. Moreover, there was a statistically significant relation between Bcl-2 expression and splenomegaly (P = 0.017), hepatomegaly (P = 0.014), and positive correlation with LDH (P = 0.044) due to B-cells accumulation and degradation.
Since all B-CLL patients showed overexpression of Bcl-2 protein, this makes Bcl-2 a good therapy target. Cervantes-Gomez et al. found that application of anti-Bcl-2 changing the therapeutic approach for B-CLL, particularly in relapsed or refractory CLL patients with the 17p deletion and showed a marked effectivity as monotherapy as well as in combination with cytotoxic chemotherapies.,
Understanding the presence and over expression of CD14 in B-CLL is still an enigma. It may contribute in disease elimination, indolence, or chronicity. CD14 receptor is a specific cell surface molecule highly expressed in cells undergoing apoptosis and contributing to apoptotic cell clearance.,
We observed a high frequency of CD14 antigen expression in B-CLL patients, confirming the results of some studies,, and opposing other reports.
CD14 promotes apoptotic cell clearance through inflammatory and noninflammatory manners. Of noninflammatory process is the enzymatic removal of membrane-bound CD14 by phosphatidylinositol-specific phospholipase C inducing noninflammatory cell apoptosis. Binding of LPS to CD14 initiates signal transduction through TLR-4 and results in the release of proinflammatory cytokines by macrophages (TNF-α, IL-1 β, IL-6, and IL-8). These cytokines trigger polymorphonuclear leukocytes migration from the blood to the site of injury, inducing CD14-dependent inflammatory process.
Patients with infectious diseases such as HIV, chronic malaria, and tuberculosis were generally excluded from the study, as increased levels of CD14 are associated with these diseases. The increased CD14 level associated with infectious diseases occurs either in soluble form secreted from the liver or monocytes or overexpressed on macrophage and neutrophils surfaces, while in this study, we detected CD14 expression on malignant lymphocytes of B-CLL patients which its expression is still an enigma.
In this study, 70% of B-CLL patients had CD14+ lymphocytes more than 5 × 109/L as a cutoff value. In a similar study, CD14+ cells using the same cutoff were detected in 44% of B-CLL cases, whereas the cutoff value 5 × 109/L CD14 positive cells showed the best discriminating power among patients with different clinical features.
Contrary to our findings, reported that CD14 expression in B-CLL in most cases was negative, whereas CD14 receptor is normally expressed on the monocyte-macrophage surface.
In this study, according to the modified Rai stages, there was a relatively significant expression of CD14+ cells above the cutoff value in the high-risk group 18/22 (81.8%) in comparison to the intermediate risk group 10/18 (55.6%). Callea et al. found a direct correlation between CD14 expression and advanced clinical stages, and the surface CD14 positivity was associated with shorter overall survival. We cannot correlate CD14 expression with low-risk patients as all cases of this study were in intermediate and high-risk stages.
Moreover, we refer the presence of smear cells in B-CLL as cells under apoptosis, which falsely known as artifacts produced by the lymphocytes damaged during the slide preparation, although they are rarely seen in other leukemias or lymphomas.
| Conclusion|| |
There was a significant increase in Bcl-2 and CD14 in B-CLL patients. Bcl-2 has been significantly increased in all patients, making it a good therapeutic target for treating B-CLL patients. CD14 was observed more frequently in high-risk group than the intermediate risk group, which makes it a potential prognostic factor.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]