|Year : 2019 | Volume
| Issue : 4 | Page : 1490-1495
CD62L expression in patients with chronic lymphocytic leukemia
Mohamed A Soliman1, Wafaa M. S. Radwan1, Enas A Elkhouly2, Sanaa S. M. Gebril3
1 Department of Clinical Pathology, Faulty of Medicine, Menoufia University, Shebin El Kom, Egypt
2 Department of Clinical Oncology, Faulty of Medicine, Menoufia University, Shebin El Kom, Egypt
3 Department of Clinical Pathology, Shebin Elkom Fever Hospital, Shebin El Kom, Egypt
|Date of Submission||24-Aug-2017|
|Date of Decision||17-Sep-2017|
|Date of Acceptance||26-Sep-2017|
|Date of Web Publication||31-Dec-2019|
Sanaa S. M. Gebril
Shebin El Kom City 32717, Menoufia Government
Source of Support: None, Conflict of Interest: None
The objective of this study was to evaluate CD62L expression in chronic lymphocytic leukemia (CLL) and its effect on survival of malignant B cells.
CLL is the most common adult leukemia and often presents as an early-stage disease. It could be stable for many years with no or minimal intervention. CD62L is one of the selectin family of adhesion molecules that plays an important role in the trafficking/homing of lymphocytes to the lymph node.
Patients and methods
This study was conducted on 35 patients with CLL and 15 age-matched and sex-matched healthy individuals as a control group. All patients were subjected to full history taking, clinical examination, and laboratory investigations. CD62L/CD19 expression on lymphocytes was measured for all the study participants using flow cytometry technique before and after cell culture.
CD62L/CD19 expression increased minimally in controls at day 7 compared with day 0 (mean ± SD = 33.9 ± 5.3 and 18.03 ± 3.4, respectively) (P < 0.001), whereas the diseased group showed marked elevation at day 7 than day 0 (mean ± SD = 90.7 ± 6.4 and 36.9 ± 17.2, respectively) (P < 0.001). CD62L/CD19 expression in patients was higher than the controls at day 0 (mean ± SD = 36.9 ± 17.2 and 18.03 ± 3.4, respectively) and showed increase of approximately threefolds at day 7 (mean ± SD: 90.7 ± 6.4 and 33.9 ± 5.3, respectively) (P < 0.001).
There is high expression of CD62L on lymphocytic cells in patients with CLL associated with increased survival of malignant B cells.
Keywords: B lymphocytes, chronic lymphocytic leukemia, l-selectin, lymph nodes
|How to cite this article:|
Soliman MA, Radwan WM, Elkhouly EA, Gebril SS. CD62L expression in patients with chronic lymphocytic leukemia. Menoufia Med J 2019;32:1490-5
|How to cite this URL:|
Soliman MA, Radwan WM, Elkhouly EA, Gebril SS. CD62L expression in patients with chronic lymphocytic leukemia. Menoufia Med J [serial online] 2019 [cited 2020 Apr 4];32:1490-5. Available from: http://www.mmj.eg.net/text.asp?2019/32/4/1490/274279
| Introduction|| |
Progressive gathering of monoclonal CD5+/CD19+ B lymphocytes in the peripheral blood, bone marrow (BM), and lymphatic tissue is characteristic of chronic lymphocytic leukemia (CLL). Early disease stage has survival of more than 10 years whereas the median survival in patients with advanced disease is ∼18 months to 3 years. Fludarabine, cyclophosphamide, and rituximab therapy has achieved complete responses. However, relapse is unavoidable; moreover, fludarabine, cyclophosphamide, and rituximab therapy is associated with significant toxicities . Tumor microenvironment is vital for proliferation, survival, metastasis, and angiogenesis in most malignancies including CLL .
Proliferation, survival, homing, and drug resistance in CLL have been attributed to several factors such as chemokines, cytokines, and direct cell–cell interactions mediated by adhesion molecules in the lymph node, spleen, and BM microenvironment, suggesting that these factors may represent important therapeutic targets .
High expression of l-selectin on human BM progenitor cells is an early marker of cells becoming committed to lymphoid differentiation . CD62L (l-selectin) attaches several ligands in the process of homing and migration such as CD34 . High expression of CD62L and chemokines on lymphocytes is essential for their migration to lymph nodes and BM . Once in the lymph nodes and BM, lymphocytes migrate into the proliferating centers by the power of chemokines such as CXCL12 .
This study was performed to evaluate the expression of CD62L in patients with CLL and its influence on the survival of malignant B cells.
| Patients and Methods|| |
Study population and selection of patients
This study was approved by the Ethical Committee of Faculty of Medicine, Menoufia University. Informed consent was obtained from every patient and controls. The study involved 35 patients with CLL and 15 age-matched and sex-matched healthy individuals as a control group.
All participants were submitted to the following:
- Clinical history taking and full physical examination
- Routine laboratory tests included the following:
- Complete blood count was performed using XT-1800i hematology analyzer (Sysmex, Kobe, Japan), which is based on fluorescent flow technology and hydrodynamic focusing
- Peripheral blood film
- Immunecytometry by FACSCalibur flowcytometer (BD Immunecytometry Systems, BD Bioscience, San Jose, California, USA)
- Liver functions and kidney function tests by Cobas Integra 400 autoanalyzer (Roche Diagnostics, Mannheim, Germany)
- Special laboratory investigations:
- CD62L expression by flow cytometry before and after cell culture
- Cell culture of mononuclear cells by Roswell Park Memorial Institute medium medium with 1% l-glutamine (EuroClone, Pero, Milan, Italy).
Collection of blood samples and mononuclear cell isolation
Venous blood samples (8 ml) were aseptically withdrawn in three tubes: one plain vacutainer tube (2 ml) and two EDTA vacutainer tubes, each of 3 ml, from all studied groups. The samples were processed within 2 h of collection for cell culture and immunecytometry using CD19 and CD62L monoclonal antibody (MoAbs).
Venous blood was collected in sterile collection tubes containing EDTA. The white blood cells were isolated by Ficoll gradient. A volume of 2 ml of Ficoll (Bio-Rad; Medical Diagnostics GmbH, Dreieich, Germany) was placed in a centrifuge tube and layered by 1 ml of EDTA blood sample on top placed very carefully ensuring that the blood and Ficoll do not mix under complete aseptic conditions, and then centrifuged at 1800 rpm for 20 min The cell suspension was washed three times in PBS with centrifugation for 5 min at 3200 rpm, and then the samples were adjusted to a final concentration of 106/ml by PBS for immunecytometry and 10 × 106/ml for cell culture.
Flow cytometry screening analysis of CD62L
MoAbs are designed to quantitatively determine the percentage of cells bearing these MoAbs within a population and quantitatively determine their density on cell surface.
CLL peripheral blood mononuclear cells (PBMCs) from 35 patients were screened for the expression of CD62L cell surface antigens directly after isolation from the patient (day 0) and after 7 days of high-density culture (day 7).
Overall, 100 μl of the washed suspension was stained with both MoAb anti-CD19 Fluorescein isothiocyanate (FITC) mouse monoclonal antihuman antibodies (Immunostep, SL. Salamanca, Spain) and anti-CD62L Phycoerythrin (PE) (20 μl of each MoAbs; Immunostep). After gentle mixing, cells were incubated for 20 min in refrigerator. Then the cells were washed twice with PBS, then the supernatant was discarded, and the cells were resuspended in residual buffer.
Data were acquired on a FACSCalibur flowcytometer (BD immunecytometry systems). The instrument setup was checked weekly using quality control windows beads (Flow Cytometry Standards Corporation, San Juan, Puerto Rico, USA).
Forward scatter and side scatter measurements were made using linear amplifiers, whereas fluorescence measurements were made with logarithmic amplifiers and flow cytometric two parameters dot plots and quadrant statistics were generated by cell quest software (Becton Dickinson immunecytometry systems, BD Life Sciences, San Jose, CA, 95131, USA).
Analysis was performed after manual gating around a mononuclear population on a forward scatter versus side scatter dot plot. The mononuclear cells were further evaluated as CD19+ and CD62L + cells. In the gated population, the percentages of positive cells were made by dual platform technique. Results were expressed as percentages of positive cells [Figure 1].
|Figure 1: Gating CD62L expression on B lymphocytes in patients and control group.|
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Chronic lymphocytic leukemia cell culture
Isolated PBMCs were resuspended in Roswell Park Memorial Institute medium-1640 medium with 1% l-glutamine (EuroClone), supplemented with 10% heat inactivated fetal calf serum (EuroClone), 100 U/ml penicillin G, and 100 mg/ml streptomycin (EuroClone) at a cell concentration of 10 × 106/ml. It should be noted that these cultures of PBMCs contain non-CLL cells derived from circulating white blood cells plus an adherent population of cells, which include nurse-like cells and endothelial cells. For reducing cell death, in vitro, CLL cells were cultured at high density and/or in the presence of accessory and nurse-like cells, to show the ability of the lymphocyte cells to express CD62L.
Data collected were tabulated and analyzed by statistical package of social science (SPSS, version 20; SPSS Inc., Chicago, Illinois, USA) on IBM personal computer. The following statistics were applied: descriptive statistics, e.g. percentage, mean, and SD and analytic statistics, e.g. χ2-test, Student's t-test, Mann–Whitney test, paired t-test, and Wilcoxon's test. P value of less than 0.05 was considered to be significant.
| Results|| |
The mean ± SD of hemoglobin and platelets in patient group was significantly lower than the control group. The mean ± SD of total leukocytic count and absolute lymphocyte in patient group was significantly higher than the control group. The mean ± SD of aspartate aminotransferase, alanine aminotransferase, total bilirubin, and direct bilirubin in patient group was higher than control group. However, there was no statistically significant difference between the studied groups regarding urea, creatinine level, and uric acid [Table 1].
|Table 1: Mean hemoglobin, platelet, total leukocytic count, absolute lymphocytes, and liver and kidney functions in the recruited participants|
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In patients with CLL, CD5+/CD19+ cells (total B cells) constituted more than 80% of the total cells in culture at day 0, and this was maintained at day 7, indicating persistence of the CLL population [Table 2].
|Table 2: Total B cells in the recruited participants in relation to diseased group regarding cell culture|
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The double-positive CD19/CD62L B cells were statistically highly significant, with marked elevation of approximately threefold increase in patient group from day 0 to day 7 (mean ± SD = 36.9 ± 17.2 and 90.7 ± 6.4, respectively) (P < 0.001) when compared with control group that showed minimal increase of less than onefold (mean ± SD = 18.03 ± 3.4 and 33.9 ± 5.3, respectively) (P < 0.001), which indicates increased CD62L expression is a feature of CLL cells rather than normal B cells [Table 3] and [Figure 2].
|Table 3: Expression of CD62L (l-selectin) on B cells in the recruited participants in relation to diseased group in relation to time|
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|Figure 2: Mean double-positive CD19/CD62L in the recruited participants (diseased cases vs. controls).|
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The mean florescence intensity (MFI) of CD62L on B cells was statistically highly significant with increased levels in patient group at day 0 and at day 7 (approximately onefold increase) (mean ± SD MFI = 249.2 ± 236.9 and 496.6 ± 340.6, respectively) (P < 0.001) when compared with control group that showed minimal increase (mean ± SD MFI = 207.8 ± 72.8 and 282.3 ± 78.3, respectively) (P < 0.05), which indicates increased MFI of CD62L expression is a feature of CLL cells rather than normal B cells [Table 4].
|Table 4: Mean florescence intensity of CD62L in the recruited participants in relation to diseased group before and after cell culture|
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| Discussion|| |
CLL was considered a homogeneous disease, in which mature B cells accumulate largely owing to a defective cell death, and also by proliferating cells . Later on, CLL has been recognized as a heterogeneous disease with notable diversity. Different characteristics in cell morphology, immunophenotype, cytogenetics, and molecular study have been identified .
Cellular adhesion molecules have an essential role in defining cellular shape and degree of contact with adjacent cells. Their role lies in generating and maintaining cell junctions, and they comprise an extensive cell–cell and cell–matrix network. Adhesion molecules' change in expression is involved in immune responses, lymphocyte homing, platelet aggregation, carcinoma metastasis, and embryo development, during which cell–cell interactions intervene the initial steps in lineage segregation . The selectins are heterophilic cellular adhesion molecules that bind fucosylated carbohydrates, e.g. mucins. CD62L is one of the three members of l-selectin family present on leukocytes 'lymphocytes'.
The surface molecule on B cells, CD62L, is known to play an important role in the trafficking/homing of lymphocytes to the lymph node. In this context, we postulated that the increase in the expression of CD62L is accompanied by profound survival of CLL cells, i.e. the increase in CD62L expression establishes its important role in different prosurvival stimuli provided to CLL cells by the microenvironment.
In this study, significantly higher levels of CD62L B-cell expression was demonstrated in patients with CLL compared with age-matched healthy control. This agrees with Purroy et al. , who suggested that CLL cells that reside in the BM and in secondary lymphoid tissues receive microenvironment survival and proliferative signals leading to persistence of residual disease after treatment. In their study, co-cultured primary CLL cells had many phenotypical features in common with circulating CLL cells, such as higher CD49d and CD62L expression and upregulation of ZAP-70 and CD38. The induced proliferation and chemoresistance in primary CLL cells by this co-culture system indicates aggressiveness and capability to interact with surrounding cells, respectively.
Downregulation of CD62L and CD184 surface expression was observed by Vlad et al.  but was opposed to the findings of Quiroga et al. , who showed that anti-IgM increases the expression of several adhesion molecules including CD62L. According to Quiroga et al. , these discrepancies can be because of using soluble anti-IgM, whereas Vlad et al.  used immobilized anti-IgM.
In addition, a study done by Rombout et al.  on CLL suggested that the method of B cell receptor (BCR) stimulation is of major importance concerning responsiveness of CLL cells in tumor microenvironment, whereas BCR pathway's genetic differences are less critical. They evaluated messenger RNA (mRNA) and protein (CD54, CD19, CD62L, and CD184) genes expression modulated by BCR triggering in immunoglobulin heavy chain variable region genes in mutated and unmutated CLL cells after stimulation using soluble or immobilized anti-IgM antibodies. Significant responses were induced in CD62L and CD184, mainly with immobilized anti-IgM.
Burgess et al.  stated that CD62L expression is associated with cell survival in vitro. They suggested that high CD62L and CXCR4 expression on lymphocytes is a requirement for its migration to lymph nodes and BM, whereas lower level of CD62L expression on CLL cells is associated with an impaired capability to migrate. They also stated that downregulation of CD62L and CXCR4 is a result of BCR activation. CD62L expression increased during in-vitro culture, and the blocking antibodies caused an increase in apoptosis suggesting a role for CD62L in cell survival, thus, inhibition of CD62L, by therapeutic antibodies, reduces CLL cell survival. CD62L antibody usage as treatment, in vitro, has overcome the prosurvival signals provided by stromal cells, which was particularly significant. They showed that CD62L was profoundly overexpressed on malignant B cells located within lymph nodes and the BM particularly in the proliferation centers (pseudo follicles).
Le Roy et al.  studied the antigen-driven signals involved in the pathogenesis and progression of CLL within the lymph node microenvironment, suggesting that, ex-vivo BCR stimulation causes instantaneous downregulation of CXCR4 and CD62L membrane expressions in CLL cells from patients at risk of disease progression only. Cells downregulating CXCR4 and CD62L in response to BCR triggering resulted in both a functional decrease in migration toward CXCL12 and adhesion to lymphatic endothelial cells.
A number of studies were carried out on CD62L expression in other hematological malignancies and other diseases. Renata et al.  suggested that most cases of Richter Syndrome were characterized by a loss/decrease of CD52 and CD62L and increased CD71 expression. Stenger  studied the association of T-cell CD62L expression and molecular response to tyrosine kinase inhibitor therapy in early chronic phase CML, whereas Feuerecker et al.  studied shedding of CD62L because of inflammation or immune dysfunction suggesting that l-selectin, or CD62L, plays a substantial role during inflammatory processes in leukocytic adherence to vascular endothelial cells as well as to platelets.
The expression of CD62L level can partially explain the increased cell survival associated with its higher levels.
In this study, there was no significant difference in CD62L expression between patients with organomegaly, organomegaly and lymphadenopathy, and those with no organomegaly.
CD62L is a double-edged sword. It is thought to act as a tumor promoter as well as tumor suppressor depending on the type of the tumor and/or other unclear factors. Its effect on cancer is accredited to both its inhibitory interaction on apoptosis and its capacity to counteract the activity of proliferative signals on stromal cells.
In conclusion, the present study identified CD62L as a cell surface marker associated with primary CLL cell survival. Despite CD62L was not examined in detail, its expression served to validate our approach, consistent with many of previously reported studies to play roles in CLL cell survival.
The outcome in CLL is still far from satisfaction with a significant scope for improvement by establishing the precise role of CD62L in the pathogenesis of CLL. However, in individual patient it allows more understanding of the molecular defect underlying the process of leukemogenesis and guide more informed therapy. CD62L modulating agents can be attractive option in treatment of patients with CLL.
| Conclusion|| |
From this study, we can conclude that: CD62L is a cell membrane surface marker associated with B malignant cell survival. Its expression increased significantly in patients with CLL predicting an important role in the survival, development, and progression of the disease, thus, CD62L may be considered a therapeutic target through its positive correlation with survival of malignant B cells especially in untreated patients.
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], [Table 4]