|Year : 2013 | Volume
| Issue : 4 | Page : 155-159
MDM2 promoter SNP309 mutation and HS1 expression in chronic lymphocytic leukemia patients ( exploring their assumed prognostic value)
Ahmed M.L. Bedewy1, Waleed R. El-Bendary2, Lubna M. Ibrahim3
1 Department of Hematology, Medical Research Institute, Alexandria University, Alexandria, Egypt
2 Department of Clinical Pathology, Military Medical Academy, Cairo, Egypt
3 Department of Human Genetics, Medical Research Institute, Alexandria University, Alexandria, Egypt
|Date of Submission||19-Mar-2013|
|Date of Acceptance||26-Mar-2013|
|Date of Web Publication||19-Jun-2014|
Ahmed M.L. Bedewy
MD, Department of Hematology, Medical Research Institute, Alexandria University, 8 Mahmoud Younes Street, Smouha, 21615 Alexandria
Source of Support: None, Conflict of Interest: None
Chronic lymphocytic leukemia (CLL) is one of the most common forms of leukemia and thus research is required to identify cost-effective and efficient prognostic markers.
The aim of the study was to identify the significance of MDM2 promoter SNP309 mutation and hematopoietic lineage cell-specific protein 1 (HS1) expression in the prognosis of CLL.
Patients and methods
Forty-eight newly diagnosed Egyptian CLL patients were clinically evaluated and assessed for MDM2 promoter SNP309 mutation, HS1 expression, ZAP-70 expression, 17p13 deletion, and 13q14 deletion. Thirty-one patients received chemotherapy, and the type of response to chemotherapy was observed.
The results showed that MDM2 promoter SNP309 mutation was associated with cytogenetic abnormalities, the need to receive chemotherapy, and the response to fludarabine/cyclophosphamide in treated patients. HS1 expression was not associated with any of the studied CLL characteristics at diagnosis. Moreover, no association was found between HS1 expression and the need to receive chemotherapy during the study period. However, a significant association between HS1 expression and the response to fludarabine/cyclophosphamide was found.
The study concluded that, despite the opposing researchers’ reports, MDM2 promoter SNP309 mutation and HS1 expression are candidate prognostic markers for CLL patients awaiting a large-scale evaluation.
Keywords: chronic lymphocytic leukemia, hematopoietic lineage cell-specific protein 1, MDM2 promoter SNP309
|How to cite this article:|
Bedewy AM, El-Bendary WR, Ibrahim LM. MDM2 promoter SNP309 mutation and HS1 expression in chronic lymphocytic leukemia patients ( exploring their assumed prognostic value). Egypt J Haematol 2013;38:155-9
|How to cite this URL:|
Bedewy AM, El-Bendary WR, Ibrahim LM. MDM2 promoter SNP309 mutation and HS1 expression in chronic lymphocytic leukemia patients ( exploring their assumed prognostic value). Egypt J Haematol [serial online] 2013 [cited 2020 Jan 24];38:155-9. Available from: http://www.ehj.eg.net/text.asp?2013/38/4/155/134783
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemias worldwide 1. An important characteristic of CLL is its variable clinical course. Many patients have been observed to survive for years without treatment, whereas another group of patients face high levels of morbidity and mortality despite aggressive treatment 2. These two groups can be classified on the basis of the IgVH mutation. The IgVH (immunoglobulin heavy chain variable region) mutation indicates longer survival, whereas CLL patients without the IgVH mutation have lower chances of survival. However, assessment of the IgVH mutation is very expensive and thus extensive research is being carried out to develop techniques for assessment of surrogate markers such as ZAP-70 3. Cytogenetic abnormalities are best detected by fluorescent in-situ hybridization and affect the prognosis variably 4.
A distinct hypothesis has been discussed by the researchers that correlates the action of the p53 tumor suppressor pathway and MDM2 promoter mutation with the development and progression of CLL. The MDM2 protein is a nuclear phosphoprotein that directly binds to p53 and prevents p53-mediated transactivation. Furthermore, overexpression of MDM2 can lead to complete inhibition of p53 function 5. The absence of p53 functioning enables damaged cells to escape the cell cycle checkpoint control and progress as oncogenic cells.
In-depth research has shown that a single-nucleotide polymorphism (SNP309) in the first intron of the promoter region of MDM2 gene can lead to such an oncogenic cascade. The T to G change of this polymorphism enhances binding of the transcription factor Sp1 6. High-affinity binding of Sp1 increases the expression of MDM2 mRNA and thus the protein. High levels of MDM2 protein thus lead to attenuation of the p53 response pathway and affect the early development of cancer and drug sensitivity 7. Recent studies have indicated that MDM2 SNP309 is a risk modifier in various cancers including CLL. However, the impact of the gene on CLL is still conflicting.
Hematopoietic lineage cell-specific protein 1 (HS1) is a 79 kDa intracellular protein that is expressed exclusively in hematopoietic cells. In B lymphocytes, HS1 becomes highly tyrosine phosphorylated following receptor engagement 8, in parallel with the activation of Syk-related and Src-related tyrosine kinases 9. HS1 is believed to act as a key molecule in cell signal transduction following the B cell receptor engagement 10. A role of HS1 in the process of clonal expansion and deletion induced by antigen–receptor interaction in B and T lymphocytes has been described 11. Moreover, HS1 acts as a central regulator of the cytoskeleton and may play an important role in CLL cell trafficking and tissue invasion 12. As the relevance of the MDM2 SNP309 polymorphism and HS1 expression as unfavorable prognostic markers remains arguable, here we have investigated the prognostic role of this SNP in our Egyptian CLL cohort.
| Patients and methods|| |
Forty-eight newly diagnosed CLL patients were selected for the study during the period from January 2010 to October 2012. Informed consents were obtained from the patients before blood sample collection. Maintenance of confidentiality was assured for all the patients. CLL disease conditions in all the patients were confirmed by observing clinical presentations, blood and bone marrow morphology, direct Coombs test, and immunophenotyping by flow cytometry. The clinical staging system of Binet was adopted. All the patients were assessed for an indication to receive chemotherapy according to the guidelines of the International Workshop on CLL at diagnosis 13. Patients who did not have an indication to start chemotherapy at presentation were followed up monthly during the study period.
The Fludarabine/Cyclophosphamide regimen 14 was provided to indicated patients and the response to therapy was evaluated after six courses according to the guidelines of the International Workshop on CLL 13.
Determination of cytogenetic aberration
Both del 17p13 and del 13q14 were assessed in the participants of the present study with interphase-fluorescence in-situ hybridization performed on peripheral blood lymphocytes using Repeat-free Poseidon Probe ON DLEU (13q14)/p53 (17p13) (Product No. KBI-10113; Kreatech, Amsterdam, the Netherlands), according to the manufacturer’s instructions.
Genotyping for MDM2 promoter mutation
Mononuclear cell preparations obtained from the peripheral blood samples of the patients were stored at −80°C and were then used to isolate genomic DNA. The Genomic DNA Purification Kit (Thermo Scientific, Chicago, Illinois, USA) was used to isolate genomic DNA. To identify the MDM2 SNP309 genotype in the 48 patients, PCR-restriction fragment length polymorphism (PCR-RFLP) was performed.
The MDM2 promoter region was amplified by PCR using the primer pair F (5′-CGGGAGTTCAGGGTAAAGGT-3′) and R (5′-AGCAAGTCGGTGCTTACCTG-3′). As a result, the 352 base pair (bp) product was amplified. The entire assay was performed using 20 ng of genomic DNA, 10 μmol/l of each primer, 1 U of Thermo-StartTaq DNA Polymerase (Thermo Scientific, Chicago, Illinois, USA), 10 mmol/l dNTP, and 25 mmol/l MgCl2. After an enzyme activation step at 94°C for 10 min, PCR was performed for 45 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, and was concluded with a final extension step of 72°C for 10 min 15.
For RFLP analysis, 7 μl of the 352 bp PCR product was digested with MspA1I restriction enzyme according to the manufacturer’s instructions (Promega, Madison, Wisconsin, USA). Digestion products were resolved on 2% ethidium bromide-stained agarose gels. SNP309 introduces a new MspA1I restriction enzyme site. The wild-type product (TT) is digested into three bands (233, 88, 31 bp), the SNP309 homozygous product (GG) i1s digested into four bands (187, 88, 46, 31 bp), and the heterozygous product (TG) yields a combination of five bands (233, 187, 88, 46, 31 bp) 15.
Real-time PCR (RQ-PCR) for HS1 mRNA
Cellular RNA was extracted from EDTA-anticoagulated fresh blood samples using the RNeasy Mini Kit (Qiagen, Valencia, California, USA) with the volume of sample adjusted to contain 5–10×106 white blood cells per reaction. The real-time reverse transcriptase PCR was performed on StepOne (Applied Biosystems, California, USA) using the DyNAmo SYBR Green 2-Step qRT-PCR Kit (Thermo Scientific, Middletown, Virginia, USA) for HS1 and GAPDH as a housekeeping gene. The primers and cycling conditions were as described previously 10. Threshold cycles (Ct) were obtained for the HS1 and GAPDH for each case and then ΔCt was calculated. The same assay was performed on peripheral blood samples from 20 healthy individuals and the relative expression of HS1 was calculated according to the 2ΔΔCt method 16.
The SPSS program for Windows was used for all the statistical analyses. Any result was considered statistically significant if P was less than or equal to 0.05.
| Results|| |
The study population included 24 men and 24 women, median age 57.5 years. Out of the 48 patients, 17 had A stage CLL disease, whereas the others had either B or C stage disease. A nondiffuse pattern of infiltration in the bone marrow trephine biopsy was detected in 31 patients, whereas diffuse infiltration was observed in 17 patients. Direct Coombs test was positive in only four patients. Sixteen patients were positive for ZAP-70. Fourteen patients in the study population showed 13q14 deletion, 10 patients showed 17p13 deletion, and the remaining 24 patients had neither 13q14 deletion nor 17p13 deletion. The G/G genotype for the MDM2 gene was found in 20.8% of the studied patients, whereas 41.7% presented the G/T genotype and 37.5% of patients had the T/T genotype [Table 1].
|Table 1: MDM2 promoter SNP309 and HS1 expression in relation to other studied parameters in chronic lymphocytic leukemia patients|
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The polymorphic variants of the MDM2 promoter mutation were significantly associated with 17p13 deletion (P<0.001) and 13q14 deletion (P=0.006), with the G/G genotype associated with absence of the 17p13 deletion and absence of the 13q14 deletion. No significant association was found between MDM2 polymorphic variants and age, sex, pattern of infiltration in the bone marrow trephine biopsy, Binet’s clinical stage, and ZAP-70 expression [Table 1].
Thirty-one patients out of the studied cohort showed at least one indication to receive chemotherapy, whereas the other 17 patients did not. Eighty-eight percent of patients with a diffuse pattern of bone marrow involvement were indicated to receive chemotherapy (P<0.001). Eleven of 16 patients with ZAP-70 positivity (69%) (P=0.004) and nine of 10 patients with 17p13 deletion (90%) (P<0.001) showed an indication to start therapy during the study period, whereas 43% of 13q14 deletion-positive patients were treated (P<0.001) [Table 2].
|Table 2: Studied parameters in relation to the need to start chemotherapy during the study period|
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The association between the MDM2 polymorphic variants and the need to start chemotherapy was highly significant, with 90% of G/G patients requiring chemotherapy, whereas only 58% of G/T and T/T patients did. However, no significant association was found between the HS1 expression and the need to start chemotherapy [Table 2].
In the treated patients, the response to the chemotherapeutic protocol provided was evaluated; 23 patients (74%) responded (achieved complete or partial remission) and eight patients (26%) failed to respond (stable or progressive disease). Patients younger than 60 years old were more likely to be responders than those 60 years of age or older (P=0.043). The nondiffuse pattern of bone marrow involvement was significantly associated with response to chemotherapy (P=0.043). ZAP-70 expression and 17p13 deletion were associated with failure to respond to chemotherapy (P=0.021 and P<0.001, respectively). All the 13q14 deletion-positive patients were responders to chemotherapy [Table 3].
|Table 3: Studied parameters in relation to the response to chemotherapy during the study period|
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Only one-third of patients with the G/G MDM2 genotype responded to chemotherapy, whereas 91% of G/T and T/T patients responded to chemotherapy (P<0.001). Among patients without the 17p13 deletion, the MDM2 promoter SNP309 genotype was significantly associated with the type of response to chemotherapy (P=0.029), with all patients who had the T/T genotype achieving complete remission. However, among the 17p13 deletion-positive patients, no association was found between the MDM2 genotype and the type of response to chemotherapy [Figure 1]. HS1 expression was significantly higher in nonresponders than in responders (P=0.004) [Table 3].
|Figure 1: MDM2 promoter SNP309 genotypes in relation to the type of response to chemotherapy among patients with no 17p deletion (P=0.029) (a) and patients who had the 17p deletion (P=0.510) (b).|
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| Discussion|| |
The present work shows the demographic data, clinical stages, and established prognostic markers (pattern of infiltration in bone marrow trephine biopsy, ZAP-70 expression, and cytogenetic abnormalities, namely 17p13 deletion and 13q14 deletion) in a cohort of 48 newly diagnosed CLL patients. In addition, genotyping of MDM2 promoter SNP309 and HS1 mRNA expression were studied in order to investigate their value in the prognosis of CLL.
The MDM2 genotype was not significantly associated with age, sex, Binet’s stage, pattern of bone marrow infiltration, or ZAP-70 expression, whereas it was significantly associated with the cytogenetic abnormalities studied. Forty percent of patients with 17p13 deletion had the MDM2 G/G genotype, whereas only 15.8% of patients without 17p13 deletion had this genotype. In terms of 13q14 deletion, only 7% of patients with this deletion had the MDM2 G/G genotype, whereas 26% of patients without the 13q14 deletion had this genotype.
Several published studies have evaluated the prognostic value of MDM2 promoter SNP309 in CLL patients. Dong et al. 17 found no association between MDM2 promoter SNP309 polymorphism and age of CLL Chinese patients at diagnosis, sex, stage, ZAP-70 expression, or 17p13 deletion. However, 17p13 deletion was detected in 10% of their cohort, whereas in the present study, 17p13 deletion was present in 21% of patients. Moreover, they did not report data on 13q14 deletion.
Another research group, namely, Kaderi et al. 18, carried out their research on a Swedish cohort of CLL patients. They found no association between any particular MDM2 SNP309 genotype and Binet’s stage, IGHV mutational status, or recurrent genomic aberrations. Hence, they argue against the use of the MDM2 SNP309 as a prognostic marker in CLL. Moreover, Zenz et al. 19 studied the MDM2 SNP309 genotype in 617 German patients with CLL. They did not find an effect of the polymorphism on age of disease onset, course, or outcome. Consequently, they suggested that the MDM2-SNP 309 has no influence on disease characteristics in CLL.
In our study, no significant association was found between HS1 expression and any of the disease characteristics studied. In contrast to this, Butrym et al. 11 described a significant correlation between HS1 expression and patients’ age at diagnosis and stage among other characteristics that did not include cytogenetic abnormalities, whereas they reported no significant correlation of HS1 expression with ZAP-70 expression. In addition, Frezzato et al. 10 reported an association between high HS1 expression and 17p13 deletion among other markers and between low HS1 expression and 13q14 deletion, whereas no significant association was found between HS1 and ZAP-70 expression. An explanation for the contrasting findings lies in the method of evaluation of HS1 expression. Frezzato et al. 10 used a method to report the relative expression of HS1 that is different from the 2ΔΔCt method. Moreover, Butrym and his colleagues evaluated HS1 protein, not mRNA, expression by semiquantitative western blotting.
In terms of the need to receive chemotherapy during the study period, this was significantly associated with the MDM2 promoter SNP309 mutation G/G genotype. In agreement with this, Gryshchenko et al. 20 found an association between MDM2 promoter SNP309 G genotype and shorter treatment-free survival in their Austrian CLL patients. However, the studies of Kaderi et al. 18 and Zenz et al. 19 reported no association between this polymorphism and course of CLL in their Swedish and German cohorts, respectively. Actually, we find the attribution of these controversial results to ethnic differences or size of the studied cohort untrue. However, upcoming work can unravel the truth behind the MDM2 promoter SNP309 in CLL.
In the present study, HS1 expression was not significantly associated with the need to receive chemotherapy during the study period. In contrast with this finding, Scielzo et al. 21 showed that HS1 significantly influences the tissue invasion and infiltration in CLL, supported by its regulatory role in cytoskeleton remodeling, thus controlling lymphocyte trafficking and homing, which can directly affect the stage of the disease and consequently the need to receive chemotherapy. It is noteworthy that Scielzo and his colleagues assayed HS1 expression at the protein level, whereas the present work assayed HS1 mRNA. However, it might be suggested that investigation of a cutoff value of HS1 expression on the basis of larger scale studies can unveil significant associations.
In the present study, MDM2 promoter mutation G/G genotype and high HS1 expression were associated significantly with failure to achieve response to chemotherapy along with age older than 60 years at diagnosis, diffuse pattern of marrow infiltration, ZAP-70 expression, presence of 17p13 deletion, and absence of 13q14 deletion. Moreover, the association between the MDM2 promoter mutation and the response to chemotherapy was still significant among patients who lacked the 17p13 deletion. This proves that the effect of the MDM2 promoter mutation is robust and is not a bystander effect of the 17p deletion. To the best of our knowledge, the association of MDM2 promoter SNP309 mutation and HS1 mRNA expression with the response to fludarabine and cyclophosphamide in CLL has not been reported by other researchers. Nevertheless, for MDM2 promoter SNP309, this association complemented the prognostic value of it. However, this was the only significant association of HS1 denoting prognostic value in the present study.
As reported above, further research is needed to precisely judge the value of the MDM2 promoter SNP309 polymorphism and HS1 expression in CLL.
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[Table 1], [Table 2], [Table 3]