|Year : 2012 | Volume
| Issue : 4 | Page : 187-192
Role of adiponectin in chronic lymphocytic leukemia
Hosneia K. Akl1, Tarek M. Elgohary2
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||16-Feb-2012|
|Date of Acceptance||30-Mar-2012|
|Date of Web Publication||21-Jun-2014|
Hosneia K. Akl
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, P.O. Box 44519, Zagazig
Source of Support: None, Conflict of Interest: None
Assessing for clinical stage and searching for various new prognostic markers can be useful in better understanding chronic lymphocytic leukemia (CLL) pathogenesis and in deciding when to initiate therapy and how this therapy can be effective. Adiponectin is an abundant circulating adipokine that exhibits beneficial effects in the vasculature and has anti-inflammatory, antiproliferative, and proapoptotic properties.
Aim of the study
The aim of this study was to investigate the role of adiponectin in CLL pathogenesis and its relation to the well-established CLL prognostic factors.
Participants and methods
Forty-five individuals divided into two groups were included. Group I comprised 35 patients classified into three subgroups (Ia: the low-risk group; Ib: the intermediate-risk group; and Ic: the high-risk group). Group II comprised 10 apparently healthy age and sex-matched controls. In addition to routine laboratory investigations, β2-microglobulin (β2-MG) and adiponectin and angiogenin (ANG) were measured by immunoturbidimetry and ELISA techniques, respectively. ZAP-70 and CD38 expressions were assessed by flow cytometry.
Adiponectin was significantly lower in patients than in controls. It was significantly lower in subgroup Ic than in subgroup Ia (P<0.01), whereas the P-value was nonsignificant when Ib was compared with Ia and Ic. ANG was significantly higher in patients than in controls (P<0.001) and its level increased with clinical stage (Ia<Ib<Ic) with P-value less than 0.0001. Regarding β2-MG, it was higher in patients than in controls and the Ic subgroup had a significantly higher level compared with Ia and Ib subgroups (P<0.001) with no significant difference between these two subgroups. ZAP-70 expression was positive in 45.7% of the patients who had significantly lower adiponectin levels than those who were ZAP-70 expression negative (P<0.01). Forty percent of the patients were positive for CD38 expression with significantly lower adiponectin levels compared with CD38-negative patients (P<0.01). Adiponectin was inversely correlated to ANG, β2-MG, and percentages of ZAP-70-expressing and CD38-expressing cells.
These results support a key role for adiponectin in CLL, suggesting a possible promising therapeutic potential.
Keywords: adiponectin, CD38, chronic lymphocytic leukemia, β2-microglobulin, ZAP-70
|How to cite this article:|
Akl HK, Elgohary TM. Role of adiponectin in chronic lymphocytic leukemia. Egypt J Haematol 2012;37:187-92
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a disorder characterized by the relentless accumulation of CD5+ B lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs (lymph nodes and spleen) 1.
Angiogenesis, formation of new blood vessels from pre-existent ones, plays a fundamental role in the neoplastic process and is essential for the local progression and metastatic spread of solid tumors. This process is regulated by a delicate balance between proangiogenic and antiangiogenic factors 2. Switching on angiogenesis usually requires both upregulation of stimulators and downregulation of inhibitors 3.
The bone marrow microenvironment plays a crucial role in leukemogenesis. Recent studies suggest that its vascularity changes significantly during this process and that angiogenic factors are of major importance in leukemia 4.
Angiogenesis is involved in the pathogenesis of B-cell CLL, and a high microvascular density has been found to be associated with a poor prognosis 5.
Adiponectin, also named Acrp30, is a unique adipocyte-derived plasma protein (adipokine), which plays an important role in regulating fat and glucose metabolism; its serum levels correlate inversely with the occurrence of several diseases as well as with the risk of developing multiple types of cancers 6,7.
Although adiponectin has been suggested as one of the regulators of angiogenesis, its precise role in this process is still not clear 8. However, its negative regulatory effect was reported by Bråkenhielm et al. 9, which was supported recently by the finding by Man et al. 10.
With the aim to evaluate the role of adiponectin in the pathogenesis of B-CLL, its serum level was assessed and correlated to clinical and some standard biological prognostic markers.
| Participants and methods|| |
This study was carried out on 45 individuals divided into two groups:
Group I (the patient group): This group included 35 untreated B-CLL patients (19 men and 16 women with a mean age of 58±9 years) newly diagnosed on clinical, morphologic, and immunophenotypic bases and staged according to the Binet staging system 11 into three subgroups:
Group Ia (stage A): This subgroup included 14 patients of the low-risk category.
Group Ib (stage B): This subgroup included 10 patients of the intermediate-risk category.
Group Ic (stage C): This subgroup included 11 patients of the high-risk category.
Group II (the control group): This group included 10 apparently healthy individuals (six men and four women with a mean age of 56±7 years). All studied individuals were subjected to the following:
(1) Full history taking.
(2) Thorough clinical examination, especially for organomegaly and lymphadenopathy.
(3) Laboratory investigations including the following:
- Complete blood count using Sysmex SF 3000, (Sysmex Corporation, Kobe, Japan) with examination of Leishman-stained films.
- Bone marrow aspiration (for some patients).
- Liver and kidney function tests using a Dimension autoanalyzer (Dade Behring, Miami, Florida, USA).
- β2-microglobulin (β2-MG) determination: a commercial immunoturbidimetric assay kit (Tina-quant β2-microglobulin, Roche, Roche Diagnostics, Meylan, France) was used for quantitative determination of serum β2-MG on a Roche/Hitachi Cobas C system (Cobas C701, Roche Diagnostics).
- Immunophenotyping (for patients only) with the whole-blood lysis technique using FACscan flow cytometry (Becton-Dickinson, San Jose, California, USA). Samples were stained with the following FITC-labeled or PE-labeled panel of monoclonal antibodies: CD19, CD20, CD22, CD23, CD5, CD38, CD79a, and FMC7. Staining with PE-labeled monoclonal antibodies against intracellular ZAP-70 was preceded by fixation/permeabilization procedures (IntraPrep kit, Coulter Electronics Inc., Hialeah, Florida, USA). Negative isotype-matched controls were used to determine nonspecific binding. At least 10 000 cells/sample were acquired; an appropriate leukemic cell gate-based method on both forward and side scatter or CD45 expression and side scatter was selected and analyzed by Cell Quest software (Becton Dickinson). The cutoff point of positivity for leukemic cells was greater than or equal to 20%.
- Serum angiogenin (ANG) determination using a commercially available ELISA kit (Quantikine; R&D Systems, Minneapolis, Minnesota, USA). Standards and samples were pipetted into ANG monoclonal antibody-precoated wells.
- After washing unbound substances, an enzyme-linked antibody specific for ANG was added. After another wash, substrate was added and the intensity of the color developed in proportion to the amount of bound ANG was measured 12.
- Serum adiponectin determination with the ELISA technique using Human Adiponectin Quantikine (R&D Systems). Calibration was carried out with adiponectin standards. Optical densities at 450 nm were measured. A standard curve was created by plotting the logarithm of the mean absorbance of each standard versus the logarithm of its concentration.
Data were statistically analyzed using SPSS (version15) (SPSS Inc., Chicago, Illinois, USA) and presented in the form of number, percentage, range, median, and mean±SD. The Student t-test, the Mann–Whitney test, and the Kruskal–Wallis test were used. A simple Pearson’s correlation coefficient was applied to test the relationship between adiponectin and other laboratory parameters. A P-value less than or equal to 0.05 was considered significant.
| Results|| |
Results are summarized, analyzed, and presented in [Table 1], [Table 2], [Table 3], [Table 4] and [Table 5] and [Figure 1], [Figure 2], [Figure 3] and [Figure 4].
|Figure 1: Significant inverse correlation between adiponectin and angiogenin (ANG) concentrations.|
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|Figure 2: Significant inverse correlation between adiponectin and β2-MG) concentration.|
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|Figure 3: Significant inverse correlation between adiponectin concentration and percentage of ZAP-70-expressing cells.|
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|Figure 4: Significant inverse correlation between adiponectin concentration and percentage of CD38-expressing cells.|
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|Table 1: Comparison between laboratory findings of patients and those of controls|
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|Table 2: Comparison between patient subgroups and controls regarding some laboratory findings|
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|Table 3: Comparison between ZAP-70-positive and ZAP-70-negative cases regarding adiponectin|
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|Table 4: Comparison betweenCD38-positive and CD38-negative cases regarding adiponectin|
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|Table 5: Correlation between adiponectin and other laboratory parameters in the patient group|
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[Table 1] shows the comparison of laboratory findings between patients and controls. There were significantly reduced Hb level, platelet count, and adiponectin level and increased β2-MG and ANG in patients compared with controls (P<0.001; for platelets P<0.01).
Comparison between laboratory findings of the patients’ subgroups is presented in [Table 2]. There was significant reduction in Hb concentration in Ic compared with Ia and Ib subgroups (P<0.001 and P<0.05, respectively) and in Ib compared with the Ia subgroup (P<0.05). There was significant reduction in platelet count in the Ic subgroup compared with the Ia and Ib subgroups (P<0.001) and in the Ib subgroup compared with Ia (P<0.01). There was significant reduction in adiponectin level (P<0.01) when Ic was compared with Ia; however, P-value was nonsignificant when Ib was compared with Ia and Ic. Significant elevation in ANG level was found in patients compared with controls (P<0.001); its level also increased with clinical stage (Ia<Ib<Ic) (P<0.001). β2-MG showed statistically significant elevation in Ic compared with Ia and Ib (P<0.001) with no significant difference between Ia and Ib subgroups. The percentage of ZAP-70-positive cells was significantly increased with Binet staging (Ia<Ib<Ic) (P<0.001). Also, there was an increase in the percentage of CD38-positive cells with Binet staging (Ia<Ib<Ic), which was significant with P-value less than 0.001 when Ic was compared with Ia and Ib but was nonsignificant when Ib was compared with Ia.
Flow cytometric analysis of ZAP-70 revealed positive expression in 16/35 (45.7%) patients. There was significant reduction in adiponectin level in ZAP-70-positive compared with ZAP-70-negative cases (P<0.01) [Table 3].
Of 35 patients, 14 (40%) were positive for CD38 expression with statistically significant lower adiponectin level compared with CD38-negative patients (P<0.01) [Table 4].
Correlation studies: Adiponectin showed significant negative correlation with ANG, β2-MG concentration, percentage of ZAP-70-expressing cells, and percentage of CD38-expressing cells with r=−0.61, P<0.001; r=−85, P<0.001; r=−97, P<0.0001; and r=−0.43, P<0.05, respectively [Table 5] and [Figure 1], [Figure 2], [Figure 3] and [Figure 4].
| Discussion|| |
B-CLL is a malignancy of progressive accumulation of well-differentiated monoclonal CD5-positive B lymphocytes and represents a frequently occurring form of leukemia 13.
Adiponectin is an adipocytokine produced in significant amounts by adipocytes, which are the most abundant stromal cells in human adult bone marrow 14. It functions as an insulin-sensitizing hormone and also possesses anti-inflammatory, antiproliferative, and proapoptotic properties 9,15–17. By induction of apoptosis through activation of the caspase cascade in vascular endothelial cells, adiponectin may play a negative regulatory role in angiogenesis 7.
As an increasing body of evidence supports the existence of increased tissue site angiogenesis in CLL and several angiogenic factors are involved in its pathogenesis 5, 18, this study investigated the potential role of adiponectin, correlated to serum ANG, in CLL pathogenesis and whether it has a prognostic significance by evaluating its relation to some standard prognostic markers.
In the present study adiponectin serum levels were significantly lower in patients than in controls. An association between adiponectin and B-CLL was recorded in the study by Avcu et al. 19, who reported that adiponectin levels in CLL patients were lower than in controls. Also, Dalamaga et al. 13 reported that adiponectin tended to be lower in cases than in controls, although there was no significant difference, whereas no difference could be found by Molica et al. 5,14 in adiponectin between cases and controls either at the circulating level or at the gene expression level, which was examined in purified B cells from patients and controls looking for the cellular source of adiponectin, which was found to be homogenously expressed at low levels, indicating that adiponectin production relies on adipocytes in bone marrow fat. In contrast, they observed that adiponectin receptors (adipoR1 and adipoR2) are highly expressed in B cells of CLL, suggesting that the effect of adiponectin on leukemic cells is specific and receptor mediated. Molica et al. 5,14 carried out studies on cases of Binet stage A only, whereas this study was carried out on B-CLL of different Binet stages with statistically significant lower adiponectin levels in Binet stage C than in stage A, which may suggest an association between adiponectin level and CLL clinical staging. Adiponectin has been inversely related to risk of acute myeloid leukemia, myeloproliferative disorders, and multiple myeloma; in addition, it was demonstrated that it could inhibit lymphopoiesis 20–24. It has been suggested that adiponectin plays a protective role as a tumor suppressor through an array of mechanisms, including inhibition of growth factors, inhibition of the proliferative signaling pathway, and induction of apoptosis by downregulation of bcl2, antiapoptotic gene upregulated in B-CLL, and upregulation of other factors such as Bax, which favor apoptosis in cell models of different tumors 9, 13, 25–27. Induction of apoptosis in vascular endothelial cells may be the way through which angiogenesis is negatively regulated by adiponectin.
In the current study, adiponectin serum level was inversely correlated to ANG, a potent inducer of angiogenesis 28, which had a significantly higher level in our cases than in controls. This was in keeping with the report of Molica et al. 5 demonstrating the antiangiogenic role of adiponectin in CLL and showing that in pathological situations angiogenesis may be triggered not only by overproduction of proangiogenic factors but also by downregulation of angiogenesis inhibitors.
β2-MG showed a significantly higher level in patients compared with controls and in subgroup Ic compared with subgroups Ia and Ib. β2-MG, provided the patient has normal renal function, becomes elevated in patients with aggressive disease. Moreover, the relative level has been found to correlate with the kinetics of tumor progression and/or tumor burden 29.
DNA analysis has distinguished two major types of CLL with different survival times: ZAP-70-positive CLL with shorter average survival and ZAP-70-negative CLL with longer survival 1. ZAP-70 positivity was found in 45.7% of the cases of this study with significantly lower adiponectin level compared with ZAP-70-negative cases. This inverse correlation is in agreement with the results of Molica et al. 5, 14, whose study demonstrated that lower circulating levels of adiponectin were more frequent among early CLL patients with aggressive phenotype as defined by higher expression of ZAP-70. As it has been suggested that the primitive signaling pathway that utilizes ZAP-70 is maintained when B cells are transformed at early stages of development and as adiponectin has antiproliferative and proapoptotic roles 13, 30, the relationship between them seems to be accepted.
CD38, which is a marker of disease aggression in B-CLL 31, was found to be significantly expressed by cells in 40% of the cases of this study who had lower adiponectin levels compared with cases with CD38-nonexpressing cells. In accordance with this result, Molica et al. 5,14 reported that patients with higher levels of adiponectin had a more favorable biological profile.
| Conclusion|| |
The results of this study suggest that serum adiponectin levels are altered in patients with CLL and show an inverse correlation with the most useful CLL prognostic markers ZAP-70 and CD38. Larger studies are recommended to further understand the role of this adipokine and how clinicians can benefit from its angiogenesis modulatory and proapoptotic properties, as its secretion by bone marrow adipocytes might represent a possible promising drug target in the field of hematology.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]