|Year : 2014 | Volume
| Issue : 4 | Page : 246-252
Murine double minute-2 expression in B-cell chronic lymphocytic leukemia: correlation with apoptosis and disease outcome
Ashraf M El-Hefni1, Tarek A El-Gohary2, Naglaa A Khalifa2
1 Hematology & Oncology Unit, Department of Internal Medicine, Zagazig University, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Egypt
|Date of Submission||20-Oct-2014|
|Date of Acceptance||21-Nov-2014|
|Date of Web Publication||25-Mar-2015|
Ashraf M El-Hefni
Hematology & Oncology Unit, Department of Internal Medicine, Faculty of Medicine, Zagazig University, Zagazig 44519
Source of Support: None, Conflict of Interest: None
Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of quiescent monoclonal CD5+ B cells, which arise from undefined defects in apoptotic cell death. murine double minute-2 (MDM2) oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53 and block p53-mediated transactivation and apoptosis.
Aim of this work The aim of the study was to evaluate the overexpression of MDM2 in B-CLL patients and correlate it with apoptosis using annexin-V expression and disease outcome.
Patients and methods This study was conducted on 55 patients including 45 patients with typical B-CLL; their ages ranged from 55 to 72 years, with a mean age of 58.55 ± 3.9 years. Ten age-matched and sex-matched apparently healthy individuals were included as the control group. The diagnosis of B-CLL was based on clinical, hematological, immunophenotypic, and cytogenetic criteria. Annexin-V and MDM2 oncoprotein expression was assessed by flow cytometric analysis.
Results Our study revealed that MDM2 was overexpressed in 53% of B-CLL cases and it was significantly elevated in comparison with the control group (P < 0.001). There was an inverse correlation between MDM2 overexpression and apoptosis (P < 0.001). The mean level of MDM2 expression was significantly reduced after therapy compared with the pretreatment level in the responder group (P < 0.001), whereas there was no significant difference (P>0.05) in the nonresponder group. The expression of MDM2 significantly correlated with the percentage of CD38 expression, LDH level, and B2M level (P < 0.001, P < 0.001, and P < 0.001, respectively) and patients who had responded to treatment showed a significant increase in the level of annexin-V (P < 0.001). MDM2 expression, percentage of CD38 expression, and LDH and B2M levels were significantly higher in the high-risk patients compared with low-risk and intermediate-risk patients (P < 0.001, P < 0.01, P < 0.05, and P < 0.001, respectively).
Conclusion MDM2 expression was inversely correlated with apoptosis, with decreased MDM2 expression in responder patients.
Keywords: Annexin-V, B-cell chronic lymphocytic leukemia, murine double minute-2
|How to cite this article:|
El-Hefni AM, El-Gohary TA, Khalifa NA. Murine double minute-2 expression in B-cell chronic lymphocytic leukemia: correlation with apoptosis and disease outcome. Egypt J Haematol 2014;39:246-52
|How to cite this URL:|
El-Hefni AM, El-Gohary TA, Khalifa NA. Murine double minute-2 expression in B-cell chronic lymphocytic leukemia: correlation with apoptosis and disease outcome. Egypt J Haematol [serial online] 2014 [cited 2019 Dec 11];39:246-52. Available from: http://www.ehj.eg.net/text.asp?2014/39/4/246/153969
| Introduction|| |
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by progressive accumulation of monoclonal CD5+ B cells arrested in the G 0 /G 1 phase of the cell cycle. Most B-CLL cells in the circulation are nondividing or slowly dividing and their accumulation has been suggested to result from a decreased rate of cell mortality rather than from increased proliferation ,. CLL is primarily caused by defects in apoptosis, slow accumulation of lymphocytes, and not being able to 'die properly'. The key genes regulating apoptosis and cell cycle in B-CLL lymphocytes are p53, ATM, murine double minute-2 (MDM2), Bcl-2/Bax, caspase-3, CDK-inhibitor p27, and cyclins D2 and D3. The relationship between some of these genes and standard therapy is studied for prospective therapeutic alternatives that result from new molecular-genetic findings .
The MDM2 proto-oncogene is located on chromosome 12 (q14-q15). This region contains many genes thought to be involved in the control of cell growth. MDM2 is amplified and frequently overexpressed in a variety of human tumors, including leukemia. Overexpression of MDM2 in either transformed normal cells or neoplastic cells enhances the tumorigenic potential and resistance to apoptosis . MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain (transactivation domain), by inhibiting p53 acetylation, by promoting nuclear export of p53, and by promoting proteasomal degradation of p53. The by promoting proteasomal degradation most important action of these mechanisms is to and interact directly with the tumor suppressor p53 block p53-mediated transactivation and apoptosis . Although MDM2 finally degrades p53 in the nucleus to cytoplasm, MDM2 must localize to the ansactivatingrbind p53, and repress p53-mediated transactivation and apoptotic activities. When MDM2 sequestrated in the cytoplasm by the tumor suppressor PTEN, its p53 binding and inhibitory functions are blocked . It has been suggested that MDM2 309G polymorphisms contribute to the risk of developing B-CLL and that an unfavorable SNP309 G/G genotype is associated with a gene-dosage-dependent increase in MDM2 expression .
Annexin-V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine, a membrane component normally localized to the internal face of the cell membrane. Early in the apoptotic pathway, molecules of phosphatidylserine are translocated to the outer surface of the cell membrane where annexin-V can readily bind them .
CD38 is a transmembrane glycoprotein that plays an important role in lymphocyte proliferation. It is important in the regulation of intracellular calcium and protects lymphocytes against apoptosis as it regulates the expression of BCL2 . CD38 expression of tumor cells was significantly associated with a nonmutated variable region of immunoglobulin heavy chain and ZAP-70 (protein tyrosine kinase). ZAP-70 was found to play an important role in CD38 signaling . CD38 has been identified as an important prognostic factor in B-CLL .
b2-microglobulin (the HLA class I light chain) serves the essential immunological function of presenting antigen to CD8+ T lymphocytes. Tumor cells may present tumor-specific antigen to T cells through these molecules, but many tumors show a loss or downregulation of HLA class I expression and this may serve as an immune escape mechanism .
Serum lactate dehydrogenase (LDH) is commonly increased in patients with hematopoietic malignancies and has been shown to be a prognostic factor in patients with non-Hodgkin's lymphoma and chronic lymphatic leukemia .
This work was conducted to evaluate the overexpression of MDM2 proteins in B-CLL and to correlate it with apoptosis via annexin-V expression, other prognostic variables, and disease outcome.
| Patients and methods|| |
This study was carried out between 2011 and 2013 at the hematology and oncology unit, internal medicine department, and clinical pathology departments, Faculty of medicine, Zagazig University (Zagazig, Egypt). It included 55 patients divided into two groups: the control group and the patient group.
The control group comprised 10 apparently healthy volunteers; six were male and four were female and their ages ranged from 48 to 66 years, with a mean of 53.55 ± 6.65 years.
The patient group comprised 45 patients with CLL; 24 were male and 21 were female and their ages ranged from 55 to 72 years, with a mean of 58.55 ± 3.9 years.
The National Cancer Institute-sponsored working group (NCI-WG) revised guidelines were used for the diagnosis and treatment of our patients .
Indications to start treatment
The indications for treatment included the presence of systemic symptoms, bulky lymphadenopathy, increasing organomegaly, high WBC count with a lymphocyte doubling time of less than 12 months, presence of anemia or thrombocytopenia due to BM infiltration, and repeated infection.
The patients were given FC (30 mg/m 2 fludarabine IV D1-3 and 300 mg/m 2 cyclophosphamide IV D1-3).
Response criteria: NCI-WG 
The National Cancer Institute-sponsored group guidelines for CLL were used for assessing the response to treatment as follows.
Complete response(CR): patients were reported to show CR when there were no symptoms, physical examination was normal, lymphocytes were less than or equal to 4 × 10 3 /ml, neutrophils were at least 1.5 × 10 3 /ml, HB was at least 11 gm/dl, PLTs were more than 100 × 10 3 /ml, and BM biopsy showed fewer than 30% lymphocytes with no nodules.
Partial response (PR): patients were reported to have PR if physical examination of the LN ± liver + spleen showed a reduction of 50% or more in cancerous cells, along with one or more of the following: neutrophil count at least 1.5 × 10 3 /ml, HB level at least 11 gm/dl or at least 50% improvement, and PLT greater than 100 × 10 3 /ml.
Progressive disease (PD): patients were reported to have PD when physical examination (LN, liver, spleen) showed a 50% or higher increase in cancerous cells or a new disease site was identified or there was an increase of 50% or more of circulating lymphocytes or on development of Richter's syndrome.
Stable disease (SD): patients with any condition other than the ones mentioned above were reported to have SD.
All patients were subjected to the following:
- Thorough history taking and clinical examination, laying stress on lymphadenopathy, hepatomegaly, and splenomegaly.
- Complete blood picture (cell-Dyn 1700).
- Bone marrow aspiration and biopsy.
- Immunophenotyping of peripheral blood by Becton Dickenson flow cytometry (Becton Dickinson, San Diago, California, USA) using a wide panel of FITC-, PE-, and PreCp-conjugated monoclonal antibodies including CD19, CD20, CD22, CD23, HLA-DR, CD10, CD5, CD3, CD7, and k and l light chains and CD38. Positivity for CD38 was defined as the presence of more than 20% positive cells.
- Direct Coombs tests .
- Serum B2-microglobulin by ELISA.
- Serum LDH determination on automated clinical chemistry analyzer (ADVIA 1650, Siemens, Tarrytown, NY, USA).
Special laboratory investigation
Estimation of MDM2 expression and lymphocyte apoptosis was carried out with annexin-V before and 6 months after therapy using FACScan flow (Becton Dickinson) cytometry.
A volume of 2 ml of venous blood was drawn into a EDTA tube for complete evaluation of the blood picture and for performing the direct Coombs test and 3 ml of heparinized blood was taken for flow cytometric immunophenotyping. In addition, 2 ml of venous blood was collected and left to clot. Serum was separated for estimation of LDH and B2M.
Heparinized blood was diluted with PBS. A volume of 4 ml of the diluted blood was carefully layered over 2 ml of Ficoll-hypaque (Sigma, Sigma-Aldrich, St. Louis, USA).
The tube was centrifuged for 20 min at 1500 rpm at room temperature. The mononuclear layer was carefully collected with a Pasteur pipette and transferred into a new tube. Cells were washed twice in PBS by centrifugation at 3000 rpm for 10 min at 4°C. The cell pellet was then resuspended in 2 ml PBS and the final cell suspension was adjusted to 5 × 10 6 cells/ml.
Detection of murine double minute-2 by flow cytometry 
Anti-human MDM2 (clone SMP14) was a purified mouse monoclonal antibody conjugated with fluorescein isothiocyanate isomer (FITC). A volume of 50 ml of the cell suspension was taken into two tubes labeled '1' and '2' (test and control). A measure of 10 ml of the negative control and 10 ml of anti-human MDM2 reagents was then added, respectively. The tubes were vortexed and then incubated at 4°C for 30 min and protected from exposure to light. After incubation, the tubes were washed with 2 ml PBS each and centrifuged at 1500 rpm for 5 min. The supernatant was then aspirated, leaving 50 ml of fluid (cell pellet). The cell pellet was resuspended in 500 ml PBS and the cells were then ready for flow cytometric analysis.
Flow cytometric acquisition and analysis was performed with a FACScan flow cytometer using Cell-Quest software (BD Biosciences, San Jose, CA, USA). A sample was considered positive when there was a clear shift in the fluorescence axis compared with that produced by the isotypic control and when more than 15% of the cells stained with the monoclonal antibody anti-MDM2.
Assay of apoptosis 
To quantify the percentage of cells undergoing apoptosis, we used annexin-V-FITC (catalog no. TA4683 from R&D Systems, MN, USA). Briefly, cells were washed twice with cold PBS and then resuspended in 1 × binding buffer; a concentration of 1 × 10 6 cells/l.5 ml annexin-V-FITC and 10 ml propidium iodide was added to 100 ml of the suspended cells. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. At the end of incubation, 400 ml binding buffer was added and the cells were analyzed immediately by flow cytometry.
Data were analyzed with the statistical package for social science computer program (version 16.0; SPSS Inc., Chicago, Illinois, USA). Numerical data were expressed as mean ± SD and qualitative data were expressed as frequency and percentage. Comparison of quantitative data between two groups was made using the Student t-test (P < 0.05; significant), whereas comparison between patients in the same group before and after treatment was made using the paired t-test. Correlation between variables was determined using Spearman correlation.
| Results|| |
The clinical and laboratory data of the studied groups were illustrated and the levels of MDM2 and annexin-V were found to be significantly higher in B-CLL patients when compared with healthy controls (P < 0.001) [Table 1].
MDM2 expression above the cutoff value of 15% was reported in 24/45 (53%) patients. Twenty-three patients responded to treatment and 12 showed no response [Table 2].
|Table 2: Distribution of the chronic lymphocytic leukemia patients according to their clinical outcome|
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Of 45 patients, 35 received the planned treatment and 10 of them achieved CR (29%), 13 achieved PR (37%) with an overall response of 66%, and 12 patients showed no response to treatment (SD+PD: 34%); there was no indication to start treatment in the remaining five patients [Table 3].
The mean MDM2 and annexin-V levels were significantly different among nontreated patients, responders, and nonresponders. Notably, only 23 of the 35 patients responding to treatment had overexpression of MDM2 above the cutoff value [Table 3].
There was a highly significant decrease in MDM2 overexpression and increase of annexin-V overexpression in B-CLL patients after treatment among responders, whereas there was no significant difference among nonresponders [Table 4] and [Table 5].
|Table 4: Murine double minute-2 and annexin-V before and after therapy in nonresponder patients (n = 12)|
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|Table 5: Murine double minute-2 and annexin-V before andafter therapy in responder patients (n=23)|
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When correlation was applied to test the significance between MDM2 expression and annexin-V as well as other prognostic variables, a highly significant inverse correlation was detected indicating that apoptosis of the cell decreased with MDM2 overexpression, whereas a significant positive correlation was observed between MDM2 overexpression and other poor prognostic variables such as CD38, LDH, the absolute lymphocytic count, and B2-microglobulin [Table 6].
|Table 6: Correlation between the levels of murine double minute-2 and other prognostic variables|
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MDM2 overexpression was significantly frequent in high-risk patients (Rai stages III and IV, high LDH levels, high B2-microglobulin, and CD38 overexpression) compared with low-risk and intermediate-risk patients [Table 7], [Figure 1],[Figure 2] and [Figure 3].
|Figure 2: MDM2 expression. FITC, fluorescein isothiocyanate; MDM2, murine double minute-2.|
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|Table 7: Comparison between low-risk and intermediate-risk versus high-risk patients regarding MDM2, annexin-V, and other prognostic variables|
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| Discussion|| |
B-CLL is characterized by accumulation of quiescent monoclonal CD5+ B cells, which arise from undefined defects in apoptotic cell death. Investigations of the molecular mechanisms involved in resistance and sensitivity to drug-induced and radiation-induced apoptosis in these cells offer new therapeutic strategies for the treatment of CLL . The overexpression of the MDM2 gene, and the protein product of which binds to and inactivates p53, suppresses the ability of p53 to activate genes responsible for repair and/or apoptosis. MDM2 overexpression, which may be p53 dependent or independent, has been found to bind several other proteins that function to regulate cell cycle progression, including the E2F-1/DP1 transcription factor complex and the retinoblastoma tumor suppressor protein, which were additional targets of MDM2. Thus, MDM2 was proposed to have a p53-independent role in promoting cell cycle progression from G1 to S phase. Changes in the level of MDM2 can disturb the control of the cell cycle and contribute to oncogenesis .
In our study, MDM2 overexpression above the cutoff value of 15% was reported in 24/45 (53%) patients and there was a significant difference in MDM2 expression between healthy controls and CLL patients. These results were partially consistent with those of Konikova and Kusenda , who observed abnormal expression of the MDM2 protein in 76% of B-CLL patients.
Watanabe et al.  found that the MDM2 gene expression in patients with B-CLL was higher than that in patients with normal B cells. They hypothesized that B cells that have levels more than 10-fold higher than those of controls are considered tumorigenic, and MDM2 in patients who showed twofold or five-fold higher might played a role so they speculated that the observed abnormal expression of the MDM2 gene might result from aberration in some undetermined regions that control the expression, or from aberrations in some undefined protein that regulate the expression of MDM2 gene. Johnston et al.  measured the expression of MDM2 mRNA and found a 20-fold increased level in patients with wildtype p53, which was not observed in cells having a p53 mutation, postulating that p53 increases MDM2 expression, which then might bind to and inactivate p53.
Huang et al.  found expression of MDM2 in all of the CLL samples examined using northern blot and they ehsuggested that MDM2 might have played a role in the pathogenesis of CLL and helped explain how abnormalities of chromosome 12 were related to CLL as the MDM2 gene had been mapped to this chromosome. Further, Quesnel et al.  found that most of the patients with an overexpression of the MDM2 gene had unfavorable cytogenetic abnormalities, poor prognostic features, poor response to chemotherapy, and short survival.
There was no significant relation between MDM2 expression before and after therapy in nonresponders, this might be attributed to greater stability of the MDM2 protein, inhibition of degradation by proteosomes, or due to p53 mutation .
MDM2 overexpression was significantly more frequent in advanced rather than earlier stages of B-CLL. These results were similar to those of Watanabe et al.  and Konikova and Kusenda , who found that MDM2 overexpression was significantly more frequent in patients at advanced clinical stages. They suggested that MDM2 could have a role through the p53 pathway, as it might also promote neoplastic growth by mechanisms other than inactivation of the p53 protein.
Significant decrease in MDM2 expression after therapy in responders who had overexpressed MDM2 might be attributed to induction of cell apoptosis. These results were not in agreement with those of Johnston et al. , who predicted that the cellular levels of MDM2 were not predictors of clinical sensitivity in CLL but an increase in MDM2 levels after drug treatment was an indicator of p53 function in these cells.
The results of Haidar et al.  were also in contrast to our results, as they reported that there was no statistically significant correlation of MDM2 protein overexpression with clinical disease stage and history of previous chemotherapy for CLL.
An inverse correlation between apoptosis and MDM2 expression was detected, which might be attributed to the fact that overexpressed MDM2 cells had poor induction to apoptosis and those patients with low levels had higher response for apoptosis after chemotherapy, which might be attributed to DNA damage and subsequent alterations in the pathway of p53 to maintain genomic stability .
In the current study, there was a statistically significant association between MDM2 overexpression and absolute lymphocyte count. Regarding the other routine prognostic factors, it was found that serum LDH was significantly higher in patients compared with controls and significant increase was found in the high-risk group as compared with the low-risk and intermediate-risk group. These results are in agreement with those of Criel et al. . There was a significant positive correlation between LDH level and MDM2 expression as well.
There was a significant positive correlation between serum B2M and MDM2 overexpression in patients and a significant increase was found in the high-risk group when compared with the low-risk and intermediate-risk group as well as the control group.
Melillo et al.  and Shanafelt et al.  found a close relationship between B2M and cell mass in B-CLL, which correlated with disease stage and the extent of bone marrow infiltration. They also reported that there was significant reduction in B2M levels after treatment, whereas its increase reflects CLL relapse. Further, they observed that B2M reflected the total burden of malignant cells mainly in MM and B-CLL; in other lymphoproliferative disorders it provided less prognostic information.
In our study there was significant difference in CD38 between patients and controls. These results were consistent with those of Ibrahim et al. , who reported that patients with CD38+ had significantly aggressive disease regardless of their clinical stage. Also significant increment was found in the high-risk group when compared with low-risk and intermediate-risk groups. These results were consistent with those of Ghia et al. . The high expression of CD38 was found in 24% (6/25) of patients who had poor response to therapy. Domingo et al.  found that 19% of CLL patients had CD38+ expression and reported that CD38 expression identifies a subgroup of B-CLL patients with aggressive clinical presentation and worse outcome and its expression was probably associated with other prognostic factors. The feasibility of determining this parameter makes it easily reproducible and adds prognostic information at diagnosis to aid the prediction of the clinical course and outcome of B-CLL. Also there was a significant positive correlation between CD38 and MDM2 expression.
Matrai  reported that cell surface expression of CD38 in CLL was recognized recently as a marker of PD and poor outcome. In contrast to traditional staging systems, CD38 was able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other prognostic factors, helps identify patients who might benefit from early and more intensive therapy ,.
A single nucleotide polymorphism at position 309 in the promoter region of MDM2 leading to increased expression of MDM2 and attenuated function of p53 was identified as an additional independent risk factor in B-CLL. Targeting MDM2-p53 interactions might emerge as a successful treatment strategy for B-CLL .
| Conclusion|| |
MDM2 expression inversely correlates with apoptosis. Patients responding to treatment showed a significant reduction in MDM2 expression and a significant increase in annexin-V. Future studies evaluating MDM2-p53 interactions as a potential therapeutic target in B-CLL patients are warranted.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]