|Year : 2019 | Volume
| Issue : 4 | Page : 1485-1489
Tumor suppressor p53 gene codon 72 polymorphism and imatinib cytogenetic response in chronic myeloid leukemia
Khaled Abd Almoamen Khalifa, Enas S Essa, Wafaa M Shehata Radwan, Enas A Elkholy, Yasmin A. H Sadek Younis
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||12-Feb-2016|
|Date of Decision||06-Apr-2016|
|Date of Acceptance||11-Apr-2016|
|Date of Web Publication||31-Dec-2019|
Yasmin A. H Sadek Younis
Department of Clinical Pathology, Menoufia University, Shebein El Kom, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
To study p53 codon 72 polymorphism in relation to cytogenetic response to imatinib treatment in patients with chronic myeloid leukemia (CML).
P53 polymorphism involves the substitution of an arginine for a proline at codon position 72. Many studies have investigated a genetic link between this variation and response to treatment in cancer.
Patients and methods
This study was conducted on 54 CML patients presented to the Clinical Oncology Department, Menoufia University during the period from June 2013 to April 2015. They were classified according to their cytogenetic response to imatinib therapy into 40 CML patients, cytogenetic responders to imatinib and 14 CML patients who are cytogenetic nonresponders to imatinib. Patients were genotyped for p53 codon 72 polymorphism using PCR. Follow up of the patients should be done after 3, 6, 9, 12, and 18 months after diagnosis, and was done by complete blood count, conventional cytogenetic, and fluorescence in-situ hybridization.
Age, sex, hematologic, and cytogenetic response to imatinib in CML patients did not differ significantly among p53 codon 72 genotypes (arg/arg, arg/pro, and pro/pro) (P = 0.44, P = 0.45, and P = 0.11, respectively). P53 codon 72 polymorphism did not significantly alter the risk to imatinib cytogenetic unresponsiveness (P = 0.9221).
It could be concluded that p53 codon 72 polymorphism is not associated with imatinib unresponsiveness in CML.
Keywords: chronic myeloid leukemia, imatinib, p53 codon 72, polymorphism
|How to cite this article:|
Khalifa KA, Essa ES, Shehata Radwan WM, Elkholy EA, Sadek Younis YA. Tumor suppressor p53 gene codon 72 polymorphism and imatinib cytogenetic response in chronic myeloid leukemia. Menoufia Med J 2019;32:1485-9
|How to cite this URL:|
Khalifa KA, Essa ES, Shehata Radwan WM, Elkholy EA, Sadek Younis YA. Tumor suppressor p53 gene codon 72 polymorphism and imatinib cytogenetic response in chronic myeloid leukemia. Menoufia Med J [serial online] 2019 [cited 2020 Mar 29];32:1485-9. Available from: http://www.mmj.eg.net/text.asp?2019/32/4/1485/274282
| Introduction|| |
Chronic myeloid leukemia (CML) is a malignancy of the hematopoietic stem cell, characterized by the presence of Philadelphia chromosome and/or BCR-ABL fusion gene. DNA damage in the hematopoietic precursor cell is the essential prerequisite for the development of leukemia and the body has developed a series of mechanisms aimed at preventing such damage . Advances in understanding the biology of leukemia pathogenesis and prognosis have not been matched with clinical improvements. Novel agents and treatment approaches are required .
Imatinib has been used to treat CML. Its mechanism of action is based on the inhibition of the chimeric oncoprotein BCR-ABL tyrosine kinase, inhibiting pathways related to the malignant transformation . Some patients develop resistance or are intolerant to imatinib treatment and new options for second-line treatment are required to be available .
P53 has many mechanisms of anticancer function, and plays a role in apoptosis, genomic stability, and inhibition of angiogenesis. P53 polymorphism is a proline (Pro) to arginine (Arg) substitution in amino acid residue 72 of human p53 in normal and transformed cells and this polymorphism lies in the proline-rich domain of p53 protein, which is implicated in the apoptosis response to the DNA-damaging agents . Also, the Arg variant has been shown to possess greater apoptotic potential than the Pro variant in vitro . P53 gene polymorphism has been shown to play a role in drug resistance in some diseases as in chronic lymphocytic leukemia; the response rate to chemotherapy (chlorambucil, fludarabine, or combination chemotherapy) was very low in patients with a p53 mutation as compared with patients without p53 mutation . The presence of p53 mutations in breast cancer treated with an anthracycline leads to a reduced response rate to chemotherapy . In this study, we aimed to assess the relation between p53 codon 72 polymorphism and cytogenetic response to imatinib treatment in CML patients.
| Patients and Methods|| |
This study was conducted on 54 CML patients presented to the Clinical Oncology Department, Menoufia University during the period from June 2013 to April 2015. They were classified according to their cytogenetic response to imatinib therapy into 40 CML patients who are cytogenetic responders to imatinib and 14 CML patients who are cytogenetic nonresponders to imatinib. The patients were genotyped for p53 codon 72 polymorphism using the PCR. The study protocol was approved by the ethics committee in Menoufia University. Informed consents were taken from the patients before the beginning of the study. Patients started their treatment by taking a once daily administration of 400 mg of imatinib mesylate as a first-line therapy and were monitored according to recommendations provided by the European Leukemia Net 2013. Follow up of the patients should be done after 3, 6, 9, 12, and 18 months after diagnosis, and was done by complete blood count, conventional cytogenetics, and fluorescence in-situ hybridization which were obtained from CML patients records. Fluorescence in-situ hybridization was used to assess cytogenetic response to imatinib therapy in CML patients.
p53 codon 72 genotyping assay
DNA was extracted from the EDTA-treated blood sample using the Thermo Scientific Gene JET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Isolated DNA was amplified using PCR.
The 141 and 177 base pair fragments encompassing the arginine and proline polymorphic site in the p53 region was amplified using the following primers. Forward: 5\-TCC CCC TTG CCG TCC-3/5/-GCC AGA GGC TGC TCC CCC-3/5\ CTG GTG CAG GGG CCACGC3\5/CGT GAC AGT CAC AGA CCT3/. PCR cycling conditions included an initial denaturation step at 95°C for 10 min, followed by 35 cycles, each cycle consisted of denaturation at 94°C for 1 min, annealing at 60°C for the arg allele and 54°C for the pro allele for 1 min, extension at 72°C for 1 min, and final extension of 5 min at 72°C. PCR mix without the DNA sample was used to ensure contamination-free PCR product. The products were visualized on 2% agarose gel electrophoresis [Figure 1] and [Figure 2].
|Figure 1: A representative agarose gel picture for p53 codon 72 proline allele (171 bp) in lanes 1, 2, 3, 5, 6. Lane 4: DNA ladder.|
Click here to view
|Figure 2: A representative agarose gel picture for p53 codon 72 arginine allele (144 bp) in lanes 1, 5, 6, 7. Lane 4: DNA ladder.|
Click here to view
Results were collected, tabulated, and statistically analyzed by the Statistical Package for the social sciences, Version 20 (SPSS Inc., Chicago, Illinois, USA).
| Results|| |
In this study we investigated 54 CML patients as regards age and sex; 25 (46.3%) men and 29 (53.7%) women with male/female = 1:1.16, their age ranged between 20and 78 years (mean ± SD, 44.11 ± 13.20) [Table 1].
|Table 1: Chronic myeloid leukemia patient characteristics during 18 months of therapy|
Click here to view
As regards age, CML patients were divided according to age into: those who were more than 40 years (26 patients, 48.1%) and those who were less than 40 years (28 patients, 51.9%) [Table 1]. This classification is used for the prognosis of CML patients. These results are based on Camelo-Santos paper; 2013. Bad prognostic factors include age of CML patients being more than 40 years, increased number of blast cells, increased size of the spleen, and bone marrow fibrosis.
As regards the three genotypes, 10 CML patients were homogeneous for proline (pro/pro) (18.5%), 13 CML patients were homogeneous for arginine (arg/arg) (24.1%), and 31 CML patients were heterogeneous for proline and arginine (arg/pro) (57.4%) [Table 2].
Comparing carriers of the three genotypes (pro/pro, arg/arg, and arg/pro) regarding age and sex, there was no significant difference in age and sex among the three genotypes (P = 0.44 and 0.45, respectively) [Table 2].
Comparing carriers of the three genotypes (pro/pro, arg/arg, and arg/pro) regarding cytogenetic responses to imatinib treatment there was no significant difference among the three genotypes (P = 0.11) [Table 3].
|Table 3: Hematological and cytogenetic responses to imatinib treatment among p53 codon 72 genotypes|
Click here to view
CML patients were classified according to their cytogenetic response to imatinib therapy into 40 (74.1%) CML patients who are responders to imatinib and 14 (25.9%) CML patients who are nonresponders to imatinib [Table 4].
|Table 4: p53 codon 72 genotypes distribution between cytogenetic responder and nonresponders to imatinib treatment|
Click here to view
There was no significant difference in p53 codon 72 genotype distribution between CML patients who were cytogenetic responders to imatinib and those who were cytogenetic nonresponders. (P = 0.16 for arg/pro vs. arg/arg, P = 0.28 for pro/pro vs. arg/arg and arg/pro, and P = 0.25 for arg/pro and pro/pro vs. arg/arg) [Table 4].
There was no significant difference in p53 codon 72 arg and pro allele distribution between CML patients who were cytogenetic responders to imatinib and those who were cytogenetic nonresponders (P = 0.9221) [Table 5].
|Table 5: p53 codon 72 alleles distribution between cytogenetic responders and nonresponders to imatinib treatment|
Click here to view
| Discussion|| |
This work aimed to determine the relation between genetic polymorphism in p53 codon 72 and cytogenetic response to imatinib in CML.
This study was carried out on 54 patients with CML, 25 (46.3%) men and 29 (53.7%) women. Their age ranged between 20 and 78 years with mean ± SD = 44.11 ± 13.20.
In our study, of the total number of 54 CML patients 46.3% were male patients with female predominance. In contrast to our results in Camelo-Santos et al.  and Mir et al.  study there was CML male patient predominance (55.7 and 60%, respectively) from a total number of 85 and 100 patients, respectively. This difference could be attributed to the sample size.
As regards age, CML patients were divided into: those who were more than 40 years (26 patients, 48.1%) and those who were less than 40 years (28 patients, 51.9%) for follow up and prognosis of CML patients. These results are based on Camelo-Santos et al. 
In the present study, we first investigated the association of the p53 codon 72 polymorphism with clinical parameters (age at diagnosis, sex, hematological, and cytogenetic responses to imatinib treatment) in CML patients.
In this study, we found no significant association between the codon 72 polymorphism of p53 gene, age and sex. These results indicate that the p53 codon 72 polymorphism does not affect age at diagnosis or sex of CML patients. These results are in agreement with Mir et al.  and Asim et al. 
In our study, there was no significant difference in genotype distribution between cytogenetic responders and nonresponders to imatinib. These results were in contrast to Mir et al.  who found that there was significant difference in genetic distribution between cytogenetic responders and nonresponders to imatinib (P = 0.012).
Regarding allele frequencies (arg and pro alleles) in responders and nonresponders to imatinib, our results revealed no significant difference between both groups (P = 0.9221).
Wendel et al.  found that p53 mutations are associated with progression to imatinib resistance in some patients with CML.
| Conclusion|| |
P53 polymorphism is not associated with imatinib cytogenetic unresponsiveness in the studied CML patients. This suggests that this polymorphism is not involved in failure to imatinib therapy in CML.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nowell PC. Discovery of the Philadelphia chromosome: a personal perspective. J Clin Investig 2007; 117
Anguille S, Van Tendeloo VF, Berneman ZN. Leukemia-associated antigens and their relevance to the immunotherapy of acute myeloid leukemia. Leuk J 2012; 26
Kerkela R, Graz Atallah E, Kantarjian H. In reply to cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Cancer Res 2006; 25
Bhamidipati PK, Kantarjian H, Cortes J. Management of imatinib-resistant patients with chronic myeloid leukemia. Ther Adv Hematol 2013; 4
Baptiste N, Friedlander P, Chen X, Prives C. The proline-rich domain of p53 is required for cooperation with anti-neoplastic agents to promote apoptosis of tumor cells. Oncogene 2002; 21
Dumont P, Leu JI, Della Prieta AC, George DL, Murphy M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat Genet 2003; 33
Wattel E, Preudhomme C, Bernard H, Michael V, Bruno Q, Isabelle D, et al
. P53gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDRlMDR3 gene expression. Blood 2015; 82
Anelli A, Brentani RR, Gadelha AP, Amorim D, Albuquerque A, Soares F. Correlation of p53 status with outcome of neo adjuvant chemotherapy using paclitaxel and doxorubicin in stage IIIB breast cancer. Ann Oncol 2003; 14
Camelo-Santos J, Adriana B, Elisangela S, Lidia AG. Arginine homozygosity in codon 72 of p53 correlates with failure to imatinib response in chronic myeloid leukemia. Biomed Pharmacother 2013; 67
Mir R, Zuberi M, Ahmad I, Javid J, Yadav P. Biological and clinical implications of exon 8 P53 (R282W) gene mutation in relation to development and progression of chronic myeloid leukaemia patients in India population. J Cell Sci Ther 2013; 4
Asim M, Leena B, Mohamed A, Elshazali W. Genotyping of P53 gene exon 4 Codon 72 in Sudanese patients with myeloid leukaemia. Blood J 2014; 2
Wendel HG, Stanchina E, Cepero E, Ray S, Emig M, Fridman JS, et al
. Loss of p53 impedes the anti leukemic response to BCR-ABL inhibition. Proc Natl Acad Sci USA 2006; 103
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]