• Users Online: 577
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 38  |  Issue : 4  |  Page : 155-159

MDM2 promoter SNP309 mutation and HS1 expression in chronic lymphocytic leukemia patients ( exploring their assumed prognostic value)


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 Submission19-Mar-2013
Date of Acceptance26-Mar-2013
Date of Web Publication19-Jun-2014

Correspondence Address:
Ahmed M.L. Bedewy
MD, Department of Hematology, Medical Research Institute, Alexandria University, 8 Mahmoud Younes Street, Smouha, 21615 Alexandria
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000434286.41257.dd

Rights and Permissions
  Abstract 

Background

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.

Aim

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.

Results

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.

Conclusion

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 Aug 7];38:155-9. Available from: http://www.ehj.eg.net/text.asp?2013/38/4/155/134783


  Introduction Top


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 Top


Patients

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.

Statistical analysis

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 Top


Study cohort

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

Click here to view


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

Click here to view


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

Click here to view


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).

Click here to view



  Discussion Top


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.[21]

 
  References Top

1.Sellner L, Dietrich S, Dreger P, Glimm H, Zenz T.Can prognostic factors be used to direct therapy in chronic lymphocytic leukemia?Curr Hematol Malig Rep2012;7:3–12.  Back to cited text no. 1
    
2.Berkova A, Pavlistova L, Babicka L, Houskova L, Tajtlova J, Balazi P, et al..Combined molecular biological and molecular cytogenetic analysis of genomic changes in 146 patients with B-cell chronic lymphocytic leukemia.Neoplasma2008;55:400–408.  Back to cited text no. 2
    
3.Trojani A, Montillo M, Nichelatti M, Tedeschi A, Colombo C, Veronese S, et al..ZAP-70, IgVh, and cytogenetics for assessing prognosis in chronic lymphocytic leukemia.Cancer Biomark2010;6:1–9.  Back to cited text no. 3
    
4.Smoley SA, Van Dyke DL, Kay NE, Heerema NA, Dell’ Aquila ML, Dal Cin P, et al..Standardization of fluorescence in situ hybridization studies on chronic lymphocytic leukemia (CLL) blood and marrow cells by the CLL Research Consortium.Cancer Genet Cytogenet2010;203:141–148.  Back to cited text no. 4
    
5.Shi D, Gu W.Dual roles of MDM2 in the regulation of p53: ubiquitination dependent and ubiquitination independent mechanisms of MDM2 repression of p53 Activity.Genes Cancer2012;3:240–248.  Back to cited text no. 5
    
6.Knappskog S, Lønning PE.Effects of the MDM2 promoter SNP285 and SNP309 on Sp1 transcription factor binding and cancer risk.Transcription2011;2:207–210.  Back to cited text no. 6
    
7.Iwakuma T, Lozano G.MDM2, an introduction.Mol Cancer Res2003;1:993–1000.  Back to cited text no. 7
    
8.Kahner BN, Dorsam RT, Mada SR, Kim S, Stalker TJ, Brass LF, et al..Hematopoietic lineage cell-specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.Blood2007;110:2449–2456.  Back to cited text no. 8
    
9.Hao S, August A.Actin depolymerization transduces the strength of B-cell receptor stimulation.Mol Biol Cell2005;16:2275–2284.  Back to cited text no. 9
    
10.Frezzato F, Gattazzo C, Martini V, Trimarco V, Teramo A, Carraro S, et al..HS1, a Lyn kinase substrate, is abnormally expressed in B-chronic lymphocytic leukemia and correlates with response to fludarabine-based regimen.PLoS One2012;7:e39902.  Back to cited text no. 10
    
11.Butrym A, Majewski M, Dzietczenia J, Kuliczkowski K, Mazura G.High expression of hematopoietic cell specific Lyn substrate-1 (HS1) predicts poor survival of B-cell chronic lymphocytic leukemia patients.Leuk Res2012;36:876–880.  Back to cited text no. 11
    
12.Davids MS, Burger JA.Cell trafficking in chronic lymphocytic leukemia.Open J Hematol2012;3S13.  Back to cited text no. 12
    
13.Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al..Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines.Blood2008;111:5446–5456.  Back to cited text no. 13
    
14.Kipps TJLichtman MA, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT.Chronic lymphocytic leukemia and related diseases.William’s Hematology2010:8th ed..Newyork:McGraw-Hill.  Back to cited text no. 14
    
15.Wang G, Firoz EF, Rose A, Blochin E, Christos P, Pollens D, et al..MDM2 expression and regulation in prostate cancer racial disparity.Int J Clin Exp Pathol2009;2:353–360.  Back to cited text no. 15
    
16.Livak KJ, Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2−[INCREMENT][INCREMENT]Ct method.Methods2001;25:402–408.  Back to cited text no. 16
    
17.Dong HJ, Fang C, Fan L, Zhu DX, Wang DM, Zhu HY, et al..MDM2 promoter SNP309 is associated with an increased susceptibility to chronic lymphocytic leukemia and correlates with MDM2 mRNA expression in Chinese patients with CLL.Int J Cancer2012;130:2054–2061.  Back to cited text no. 17
    
18.Kaderi MA, Mansouri M, Zainuddin N, Cahill N, Gunnarsson R, Jansson M, et al..Lack of association between the MDM2 promoter polymorphism SNP309 and clinical outcome in chronic lymphocytic leukemia.Leuk Res2010;34:335–339.  Back to cited text no. 18
    
19.Zenz T, Häbe S, Benner A, Kienle D, Döhner H, Stilgenbauer S.The MDM2-309 T/G promoter single nucleotide polymorphism does not alter disease characteristics in chronic lymphocytic leukemia.Haematologica2008;93:1111–1113.  Back to cited text no. 19
    
20.Gryshchenko I, Hofbauer S, Stoecher M, Daniel PT, Steurer M, Gaiger A, et al..MDM2 SNP309 is associated with poor outcome in B-cell chronic lymphocytic leukemia.J Clin Oncol2008;26:2252–2257.  Back to cited text no. 20
    
21.Scielzo C, Bertilaccio MT, Simonetti G, Dagklis A, ten Hacken E, Fazi C, et al..HS1 has a central role in the trafficking and homing of leukemic B cells.Blood2010;116:3537–3546.  Back to cited text no. 21
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Patients and methods
Results
Discussion
Introduction
Patients and methods
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed684    
    Printed23    
    Emailed0    
    PDF Downloaded84    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]