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Polymorphism of TP53 codon 72 showed no association with breast cancer in Iranian women
Cancer Genetics and Cytogenetics 173 (2007) 38e42
Polymorphism of TP53 codon 72 showed no association with breast cancer in Iranian women Baharak Khadanga, Mohammad Javad Fattahia, Abdolrasol Taleib, Alamtaj Samsami Dehaghanic, Abbas Ghaderia,d,* a
Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, P.O. Box 71345-1798, Shiraz, Iran b Department of Surgery, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran Received 5 May 2006; received in revised form 31 August 2006; accepted 7 September 2006
Abstract
Breast cancer is the most common female malignancy worldwide. Despite the high incidence of sporadic cases, the rate of familial breast cancer is low. The tumor suppressor gene TP53 (alias p53), located on chromosome 17, has been involved in various malignancies. Mutations in codon 72 of TP53 have been studied in breast cancer and most solid tumors. For study of polymorphisms and allele frequency, 221 female patients with sporadic breast cancer and 205 healthy blood donors as control group were recruited. DNA from peripheral blood mononuclear cells was extracted and amplified using allele-specific polymerase chain reaction. Frequency of homozygotic arginine at codon 72 was 37.6% in patients and 36.6% in controls, for homozygotic proline it was 13.1 and 19.5%, and for heterozygotic Arg/Pro it was 49.3 and 43.9%, respectively. No significant difference was found between patients and controls regarding allele frequencies. Mutation in codon 72 of TP53 gene was not associated with breast cancer in Iranian patients. Ó 2007 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is a malignant proliferation of epithelial cells lining the ducts or lobules of the breast and is the most common malignancy in women [1], accounting for about one third of all cancers in women worldwide [2]. Despite the high incidence of sporadic cases, the rate of familial breast cancer is low [1]. The tumor suppressor gene TP53 (alias p53), located on chromosome band 17p13.1, is one of the most mutated genes; it is found in ~50% of human cancers, including cervical, prostate, gastric, breast, endometrial, hepatocellular, and ovarian carcinomas [3e12]. TP53 controls numerous downstream targets, resulting in gene transcription, DNA repair, cell cycle arrest, and apoptosis [13]. The gene comprises 11 exons. More than 90% of mutations in breast cancer occur in exons 5e9, which encode the DNA binding domain of p53 protein [14]. At least 10 polymorphisms are described in this gene; the most
* Corresponding author. Tel.: þ98-711-2303687; fax: þ98-7112304952. E-mail address: [email protected] (A. Ghaderi). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.09.010
commonly studied one is at codon 72, which encodes either arginine (CGC) or proline (CCC) [15]. Hui et al. [10] investigated the association of TP53 codon 72 polymorphisms with susceptibility to hepatocellular carcinoma in a Chinese population and reported an increased risk of hepatocellular carcinoma in hepatitis B surface antigenenegative (HBs-negative) subjects with the proline allele. Zehbe et al. [16] reported a higher risk of cervical carcinoma in patients harboring the TP53 arginine 72 variant. In India, the Arg/Pro genotype in patients with lung cancer was associated with early progression of the disease, compared with Arg/Arg carriers [17]. According to a metaanalysis conducted by Matakidou et al. [18], however, there was no relationship between TP53 codon 72 polymorphisms and risk of lung cancer. Dumont et al. [19] suggested that the TP53 proline 72 variant is associated with increased risk of cancer due to a decreased ability to induce apoptosis. A strong association between the Arg/Arg genotype and breast cancer was reported in Turkish patients [20]. Similarly, Langerød et al. [21] reported a growth advantage of breast carcinoma cells with the arginine 72 allele in
B. Khadang et al. / Cancer Genetics and Cytogenetics 173 (2007) 38e42
39
Fig. 1. PCR amplification of the TP53 codon 72 Arg allele (141 bp) and Pro allele (177 bp). The result of amplification for Pro homozygotic (lanes 1 and 2), Arg homozygotic (lane 3 and 4), and Arg/Pro heterozygotic (lanes 5 and 6) genotypes are indicated. The reaction in lane 1 (Pro) and lack of reaction in lane 2 (Arg) indicate that the first individual is Pro homozygotic, and the lack of reaction in lane 3 (Pro) and the existence of a PCR-amplification Arg band in lane 4 indicate that individual 2 is Arg homozygotic. The reaction in both lanes (5 and 6) indicates that individual 3 is heterozygotic.
a Norwegian population, and Papadakis et al. [22] reported the Arg/Arg genotype as a risk factor for breast cancer in a Greek population. In contrast, Tommiska et al. [23] did not observe any association between TP53 codon 72 variants and breast cancer risk. The present study was designed to investigate TP53 gene codon 72 polymorphism and allele frequency in patients with sporadic breast cancer and healthy individuals in southern Iran. 2. Materials and methods 2.1. Patients A total of 221 female patients with sporadic breast cancer (age range, 25e80; mean, 47 years) were included, referred from the Surgery and Gynecology Departments of Shiraz Medical School. Control cases were 205 healthy blood donors with no signs and symptoms of autoimmune disease or malignancy (age range, 23e72; mean, 45.3 years). 2.2. DNA preparation and polymerase chain reaction method Blood samples were collected and genomic DNA was extracted by means of the salting method from blood lymphocytes. Amplification of genomic DNA was performed
by allele-specific polymerase chain reaction (PCR). Two sets of primers (forward, F; reverse, R) were used to amplify the arginine and proline alleles, as follows: Arg F: TCC CCC TTG CCG TCC CAA Arg R: CTG GTG CAG GGG CCA CGC Pro F: GCC AGA GGT TGC TCC CCC Pro R: CGT GCA AGT CAC AGA CTT Each set of primers was added to a separate tube with a total volume of 25 mL, containing 0.3 mmol/L dNTPs, 1.5 mmol/L MgCl2, 2 U Taq polymerase, and 1 buffer (20 mmol/L Tris-HCl, pH 8.4, and 50 mmol/L KCl). Amplification was performed at 35 cycles with a touch-down program: denaturation at 94 C for 30 seconds, annealing at 68e62 C for 10 cycles and 62e58 C for the remaining 25 cycles, with extension at 72 C for 30 seconds in each cycle. The PCR product was 177 bp for the Pro allele and 141 bp for the Arg allele (Fig. 1).
2.3. Statistical analysis Data regarding age, tumor type, invasion, metastasis, menopause status, age of diagnosis, presence of estrogen (ER) and progesterone (PR) receptors, and lymph node involvement were recorded. Results were analyzed with
B. Khadang et al. / Cancer Genetics and Cytogenetics 173 (2007) 38e42
40
Table 1 Frequencies of TP53 genotype among patient and control groups Genotype
Patients, no. (%)
Controls, no. (%)
Arg/Arg Pro/Pro Arg/Pro Total
83 (37.6) 29 (13.1) 109 (49.3) 221
75 (36.6) 40 (19.5) 90 (43.9) 205
SPSS software (version 11.5 for Windows software); c2 test was used to compare the results.
3. Results
Neither estrogen receptor (ER) expression (P 5 0.94) nor progesterone receptor (PR) expression (P 5 0.19) expression was associated with presence of a specific genotype in patients. No correlation was found between histological type of tumor and genotype frequency (P 5 0.53). Patients were divided into two groups based on the age of diagnosis, those under 35 years as early and those over 35 years as late, with still no difference in the prevalence of genotypes between two groups. The same result was achieved considering premenopausal and postmenopausal cases (P 5 0.9).
4. Discussion
A total of 221 female patients with sporadic breast cancer were studied, with a mean age of 48.7 years. Of these patients, 169 (88.5%) had infiltrative ductal carcinoma and 20 (10.5%) had medullary carcinoma; 2 patients (1%) had other types of breast cancer. Of the 221 patients, 58.6% had lymph node involvement, 1.8% had distant metastasis, and 77% had pretumoral invasion (vascular, lymphatic, or preneural). No genotype differed significantly (P 5 0.2) between patients and controls (Table 1). The Arg/Arg genotype appeared in 37.6 and 36.6% of the patients and controls, respectively; the Pro/Pro genotype in 13.1 and 19.5%; and the Arg/Pro genotype in 49.3 and 43.9%. Likewise, allele frequencies did not differ significantly (P 5 0.9) between patients and controls (Table 2). The Arg allele frequency was 62.2 and 58.5% in patients and controls, respectively; the corresponding Pro allele frequency was 37.8 and 41.5%. In terms of tumorenodeemetastasis (TNM) clinical staging, 42 patients (18.2%) were in stage 0 or I, 152 patients (68.7%) were in stage II, and 29 patients (13.1%) were in stage III or IV. Ten (35.7%) patients with stage III or IV disease had Arg/Arg genotype, 1 (3.6%) had Pro/Pro genotype, and 17 (60.7%) had the Arg/Pro genotype (P 5 0.308), indicating no statistically significant effect of allele on progression in clinical stage of the disease. Of the patients with lymph node involvement, 44 patients (34.1%) had the Arg/Arg genotype, 16 (12.2%) had Pro/Pro, and 70 (53.7%) had the heterozygous genotype, revealing no statistically significant relationship between genotype and lymph node involvement (P 5 0.15). The same was true considering genotype frequency and distant metastasis. Table 2 Allelic frequencies of TP53 codon 72 among patients and control groups Allele
Patients, no. (%)
Controls, no. (%)
Arg Pro Total
275 (62.2) 167 (37.8) 442
240 (58.5) 170 (41.5) 410
Several studies have shown an association between polymorphisms at codon 72 and human malignancies. We investigated the allele and genotype frequencies of TP53 codon 72 in 221 female patients with sporadic breast cancer and 205 healthy individuals from southern Iran. No significant difference in the frequency of genotypes and alleles was found between patients and controls. In addition, no significant association was detected between prevalence of alleles and genotypes and tumor type, clinical stage of tumor, lymph node involvement, metastasis, age of diagnosis, menopause status, and ER and PR expression. Our results are in agreement with similar studies on bladder cancer [24], but are contrary to some other reports [25e28]. Prevalence of this polymorphism in cervical [29e32], lung [33,34], colon [35], bladder [36,37], and skin [38] carcinoma have previously been investigated. There are few reports of studies assessing the TP53 codon 72 polymorphisms in breast cancer, with inconsistent results [39e41]. Some investigators reported a difference in the frequency of the Arg allele between breast cancer patients and controls [39,42], and others propose Pro allele as a risk factor for breast cancer [40,43]. Tommiska et al. [23] suggested that codon 72 polymorphism, particularly the Pro/Pro genotype, is an independent prognostic factor in patients with breast cancer and provided evidence that patients harboring this genotype will have a reduced survival. A report from China indicated an association between HPV oncogenic strains and breast cancer, with evidence that human papilloma virus (HPV) E6 oncoprotein more readily targets the Arg allele than Pro allele for degradation of p53 protein [44]. In light of that finding, Makni et al. [45] reported a correlation between HPV associated cervical cancer and Arg homozygosity of TP53 codon 72. Contrary to these findings, our previous study [46] revealed no association between HPV oncogenic strains in patients with cervical cancer, a result which is explained by the low frequency of HPV oncogenic strains in our population [46]. In conclusion, the data of the present study show that TP53 gene codon 72 polymorphism is not associated with increased risk of breast cancer in southern Iranian patients.
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This finding may be a reflection the low frequency of oncogenic strains of HPV in our population [46].
Acknowledgments This work financially was supported by Shiraz Institute for Cancer Research (Grant No: ICR-82-94) References [1] Berns EM, van Staveren IL, Look MP, Smid M, Klijn JGM, Foekens JA. Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer. Br J Cancer 1998;77:1130e6. [2] Goldhirsch A. Breast cancer. In: Cavalli F, Hansen H, Kaye SB, editors. Textbook of medical oncology. London: Martin Dunitz, 2000. pp. 137e83. [3] Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49e53. [4] Wu MT, Chen MC, Wu DC. Influences of lifestyle habits and p53 codon 72 and p21 codon 31 polymorphisms on gastric cancer risk in Taiwan. Cancer Lett 2004;205:61e8. [5] Powell BL, Staveren IL, Roosken P, Grieu F, Berns EM, Iacopetta B. Associations between common polymorphisms in TP53 and p21 WAF1/Cip1 and phenotypic features of breast cancer. Carcinogenesis 2002;23:311e5. [6] Keshava C, Frye BL, Wolff MS, McCanlies EC, Weston A. Waf-1 (p21) and p53 polymorphisms in breast cancer. Cancer Epidemiol Biomarkers Prev 2002;11:127e30. [Erratum in: Cancer Epidemiol Biomarkers Prev 2004;13:1682]. [7] Roh JW, Kim JW, Park NH, Song YS, Park IA, Park SY, Kang SB, Lee HP. p53 and p21 genetic polymorphisms and susceptibility to endometrial cancer. Gynecol Oncol 2004;93:499e505. [8] Huang SP, Wu WJ, Chang WS, Wu MT, Chen YY, Chen YJ, Yu CC, Wu TT, Lee YH, Huang JK, Huang CH. p53 codon 72 and P21 codon 31 polymorphisms in prostate cancer. Cancer Epidemiol Biomarkers Prev 2004;13:2217e24. [9] Lee SA, Kim JW, Roh JW, Choi JY, Lee KM, Yoo KY, Song YS, Kang D. Genetic polymorphisms of GSTM1, p21, p53 and HPV infection with cervical cancer in Korean women. Gynecol Oncol 2004;93:14e8. [10] Zhu ZZ, Cong WM, Zhu GS, Liu SF, Xian ZH, Wu WQ, Zhang XZ, Wang YH, Wu MC. Association of p53 codon 72 polymorphism with genetic susceptibility to hepatocellular carcinoma in a Chinese population. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2005;22:632e5. [11] Wang Y, Kringen P, Kristensen GB, Holm R, Baekelandt MM, Olivier M, Skomedal H, Hainaut P, Trope CG, Abeler VM, Nesland JM, Borresen Dale AL, Helland A. Effect of the codon 72 polymorphisms (c.215 GOC, p.Arg 72 Pro) in combination with somatic sequences variants in TP53 gene on survival in patients with advanced ovarian carcinoma. Hum Mutat 2004;24:21e34. [Erratum in: Hum Mutat 2006;27:209e10]. [12] Hogdall EV, Hogdall CK, Christensen L, Glud E, Blaakaer J, Bock JE, Vuust J, Norgaard-Pedersen B, Kjaer SK. Distribution of p53 codon 72 polymorphisms in ovarian tumour patients and their prognostic significance in ovarian cancer patients. Anticancer Res 2002;22:1859e64. [13] Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 2003;10:431e42. [14] Bautista S, Theillet C. p53 mutations in breast cancer, incidence and relations to tumor aggressiveness and evolution of the disease. Pathol Biol (Paris) 1997;45:882e92. [15] Donehower LA. p53 guardian and suppressor of longevity? Exp Gerontol 2005;40:7e9.
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[16] Zehbe I, Voglino G, Wilander E, Genta F, Tommasino M. Codon 72 polymorphism of p53 and its association with cervical cancer. Lancet 1999;354:218e9. [17] Jain N, Singh V, Hedau S, Kumar S, Daga MK, Dewan R, Murthy NS, Husain SA, Das BC. Infection of human papillomavirus type 18 and p53 codon 72 polymorphism in lung cancer patients from India. Chest 2005;128:3999e4007. [18] Matakidou A, Eisen T, Houlston RS. TP53 polymorphisms and lung cancer risk a systemic review and meta-analysis. Mutagenesis 2003;18:377e85. [19] Dumont P, Leu JI, Della Pietra AC, George DL, Murphy M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat Genet 2003;33:357e65. [20] Buyru N, Tigli H, Dalay N. p53 codon 72 polymorphism in breast cancer. Oncol Rep 2003;10:711e4. [21] Langerød A, Bukholm IR, Brega˚ard A, Lønning PE, Andersen TI, Rognum TO, Meling GI, Lothe RA, Børresen-Dale AL. The TP53 codon 72 polymorphism may affect the function of TP53 mutations in breast carcinomas but not in colorectal carcinomas. Cancer Epidemiol Biomarkers Prev 2002;11:1684e8. [22] Papadakis EN, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of breast cancer. Mol Cell Biol Res Commun 2000;3:389e92. [23] Tommiska J, Eerola H, Heinonen M, Salonen L, Kaare M, Tallila J, Ristimaki A, von Smitten K, Aittomaki K, Heikkila P, Blomqvist C, Nevanlinna H. Breast cancer patients with p53 Pro72 homozygous genotype have a poorer survival. Clin Cancer Res 2005;11: 5098e103. [24] Toruner GA, Ucar A, Tez M, Cetinkaya M, Ozen H, Ozcelik T. p53 codon 72 polymorphism in bladder cancer: no evidence of association with increased risk of invasiveness. Urol Res 2001;29:393e5. [25] Santos AM, Sousa H, Catarino R, Pinto D, Pereira D, Vasconcelos A, Matos A, Lopes C, Medeiros R. TP53 codon 72 polymorphism and risk for cervical cancer in Portugal. Cancer Genet Cytogenet 2005;159:143e7. [26] Agorastos T, Masouridou S, Lambropoulos AF, Chrisafi S, Miliaras D, Pantazis K, Constantinides TC, Kotsis A, Bontis I. p53 codon 72 polymorphism and correlation with ovarian and endometrial cancer in Greek women. Eur J Cancer Prev 2002;13:277e80. [27] Leiros GJ, Galliano SR, Sember ME, Kahn T, Schwarz E, Eiguchi K. Detection of human papillomavirus DNA and p53 codon 72 polymorphism in prostate carcinomas of patients from Argentina. BMC Urol 2005;5:15. [28] Siddique MM, Balram C, Fiszer-Maliszewska L, Aggarwal A, Tan A, Tan P, Soo KC, Sabapathy K. Evidence for selective expression of the p53 codon 72 polymorphs: implications in cancer development. Cancer Epidemiol Biomarkers Prev 2005;14:2245e52. [29] Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L. Role of p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393:229e34. [30] Crook T, Ludwig RL, Marston NJ, Willkomm D, Vousden KH. Sensitivity of p53 lysine mutants to ubiquitin-directed degradation targeted by human papillomavirus E6. Virology 1996;217:285e92. [31] Madeleine MM, Shera K, Schwartz SM, Daling JR, Galloway DA, Wipf GC, Carter JJ, McKnight B, McDougall JK. The p53 Arg 72 Pro polymorphism, human papillomavirus, and invasive squamous cell cervical cancer. Cancer Epidemiol Biomarkers Prev 2000;9: 225e7. [32] Tenti P, Vesentini N, Rondo Spaudo M, Zappatore R, Migliora P, Carnevali L, Ranzani GN. p53 codon 72 polymorphism does not affect the risk of cervical cancer in patients from northern Italy. Cancer Epidemiol Biomarkers Prev 2000;9:435e8. [33] Wang YC, Lee HS, Chen SK, Yang SC, Chen CY. Analysis of K-ras gene mutations in lung carcinomas: correlation with gender, histological subtypes, and clinical outcome. J Cancer Res Clin Oncol 1998;124:517e22.
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[34] Pierce LM, Sivaraman L, Chang W, Lum A, Donlon T, Seifried A, Wilkens LR, Lau AF, Le Marchand L. Relationships of TP53 codon 72 and HRAS1 polymorphisms with lung cancer risk in an ethnically diverse population. Cancer Epidemiol Biomarkers Prev 2000;9: 1199e204. [35] Sayhan N, Yazici H, Budak M, Bitisik O, Dalay N. p53 codon 72 genotypes in colon cancer: association with human papillomavirus infection. Res Commun Mol Pathol Pharmacol 2001;109:25e34. [36] Kuroda Y, Tsukino H, Nakao H, Imai H, Katoh T. p53 codon 72 polymorphism and urothelial cancer risk. Cancer Lett 2003;189:77e83. [37] Soulitzis N, Sourvinos G, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism and its association with bladder cancer. Cancer Lett 2002;179:175e83. [38] Dokianakis DN, Koumantaki E, Billiri K, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of HPV-associated non-melanoma skin cancers in immunocompetent hosts. Int J Mol Med 2000;5:405e9. [39] Wang-Gohrke S, Rebbeck TR, Besenfelder W, Kreienberg R, Runnebaum IB. p53 germline polymorphisms are associated with an increased risk for breast cancer in German women. Anticancer Res 1998;18:2095e9. [40] Sjalander A, Birgander R, Hallmans G, Cajander S, Lenner P, Athlin L, Beckman G, Beckman L. p53 polymorphisms and haplotypes in breast cancer. Carcinogenesis 1996;17:1313e6.
[41] Papadakis EN, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of breast cancer. Mol Cell Biol Res Commun 2000;3:389e92. [42] Suspitsin EN, Buslov KG, Grigoriev MY, Ishutkina JG, Ulibina JM, Gorodinskaya VM, Pozharisski KM, Berstein LM, Hanson KP, Togo AV, Imyanitov EN. Evidence against involvement of p53 polymorphism in breast cancer predisposition. Int J Cancer 2003;103: 431e3. [43] Weston A, Pan CF, Ksieski HB, Wallenstein S, Berkowitz GS, Tartter PI, Bleiweiss IJ, Brower ST, Senie RT, Wolff MS. p53 haplotype determination in breast cancer. Cancer Epidemiol Biomarkers Prev 1997;6:105e12. [44] Li T, Lu ZM, Guo M, Wu QJ, Chen KN, Xing HP, Mei Q, Ke Y. p53 codon 72 polymorphism (C/G) and the risk of human papillomavirus-associated carcinomas in China. Cancer 2002;95:2571e6. [45] Makni H, Franco EL, Kaiano J, Villa LL, Labrecque S, Dudley R, Storey A, Matlashewski G. p53 polymorphism in codon 72 and risk of human papillomavirus-induced cervical cancer: effect of inter-laboratory variation. Int J Cancer 2000;87:528e33. [46] Farjadian S, Asadi E, Doroudchi M, Dehaghani AS, Tabei SZ, Kumar VP, Ghaderi A. High risk HPV types in southern Iranian patients with cervical cancer. Pathol Oncol Res 2003;9: 121e5.
Polymorphism of TP53 codon 72 showed no association with breast cancer in Iranian women Baharak Khadanga, Mohammad Javad Fattahia, Abdolrasol Taleib, Alamtaj Samsami Dehaghanic, Abbas Ghaderia,d,* a
Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, P.O. Box 71345-1798, Shiraz, Iran b Department of Surgery, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran Received 5 May 2006; received in revised form 31 August 2006; accepted 7 September 2006
Abstract
Breast cancer is the most common female malignancy worldwide. Despite the high incidence of sporadic cases, the rate of familial breast cancer is low. The tumor suppressor gene TP53 (alias p53), located on chromosome 17, has been involved in various malignancies. Mutations in codon 72 of TP53 have been studied in breast cancer and most solid tumors. For study of polymorphisms and allele frequency, 221 female patients with sporadic breast cancer and 205 healthy blood donors as control group were recruited. DNA from peripheral blood mononuclear cells was extracted and amplified using allele-specific polymerase chain reaction. Frequency of homozygotic arginine at codon 72 was 37.6% in patients and 36.6% in controls, for homozygotic proline it was 13.1 and 19.5%, and for heterozygotic Arg/Pro it was 49.3 and 43.9%, respectively. No significant difference was found between patients and controls regarding allele frequencies. Mutation in codon 72 of TP53 gene was not associated with breast cancer in Iranian patients. Ó 2007 Elsevier Inc. All rights reserved.
1. Introduction Breast cancer is a malignant proliferation of epithelial cells lining the ducts or lobules of the breast and is the most common malignancy in women [1], accounting for about one third of all cancers in women worldwide [2]. Despite the high incidence of sporadic cases, the rate of familial breast cancer is low [1]. The tumor suppressor gene TP53 (alias p53), located on chromosome band 17p13.1, is one of the most mutated genes; it is found in ~50% of human cancers, including cervical, prostate, gastric, breast, endometrial, hepatocellular, and ovarian carcinomas [3e12]. TP53 controls numerous downstream targets, resulting in gene transcription, DNA repair, cell cycle arrest, and apoptosis [13]. The gene comprises 11 exons. More than 90% of mutations in breast cancer occur in exons 5e9, which encode the DNA binding domain of p53 protein [14]. At least 10 polymorphisms are described in this gene; the most
* Corresponding author. Tel.: þ98-711-2303687; fax: þ98-7112304952. E-mail address: [email protected] (A. Ghaderi). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.09.010
commonly studied one is at codon 72, which encodes either arginine (CGC) or proline (CCC) [15]. Hui et al. [10] investigated the association of TP53 codon 72 polymorphisms with susceptibility to hepatocellular carcinoma in a Chinese population and reported an increased risk of hepatocellular carcinoma in hepatitis B surface antigenenegative (HBs-negative) subjects with the proline allele. Zehbe et al. [16] reported a higher risk of cervical carcinoma in patients harboring the TP53 arginine 72 variant. In India, the Arg/Pro genotype in patients with lung cancer was associated with early progression of the disease, compared with Arg/Arg carriers [17]. According to a metaanalysis conducted by Matakidou et al. [18], however, there was no relationship between TP53 codon 72 polymorphisms and risk of lung cancer. Dumont et al. [19] suggested that the TP53 proline 72 variant is associated with increased risk of cancer due to a decreased ability to induce apoptosis. A strong association between the Arg/Arg genotype and breast cancer was reported in Turkish patients [20]. Similarly, Langerød et al. [21] reported a growth advantage of breast carcinoma cells with the arginine 72 allele in
B. Khadang et al. / Cancer Genetics and Cytogenetics 173 (2007) 38e42
39
Fig. 1. PCR amplification of the TP53 codon 72 Arg allele (141 bp) and Pro allele (177 bp). The result of amplification for Pro homozygotic (lanes 1 and 2), Arg homozygotic (lane 3 and 4), and Arg/Pro heterozygotic (lanes 5 and 6) genotypes are indicated. The reaction in lane 1 (Pro) and lack of reaction in lane 2 (Arg) indicate that the first individual is Pro homozygotic, and the lack of reaction in lane 3 (Pro) and the existence of a PCR-amplification Arg band in lane 4 indicate that individual 2 is Arg homozygotic. The reaction in both lanes (5 and 6) indicates that individual 3 is heterozygotic.
a Norwegian population, and Papadakis et al. [22] reported the Arg/Arg genotype as a risk factor for breast cancer in a Greek population. In contrast, Tommiska et al. [23] did not observe any association between TP53 codon 72 variants and breast cancer risk. The present study was designed to investigate TP53 gene codon 72 polymorphism and allele frequency in patients with sporadic breast cancer and healthy individuals in southern Iran. 2. Materials and methods 2.1. Patients A total of 221 female patients with sporadic breast cancer (age range, 25e80; mean, 47 years) were included, referred from the Surgery and Gynecology Departments of Shiraz Medical School. Control cases were 205 healthy blood donors with no signs and symptoms of autoimmune disease or malignancy (age range, 23e72; mean, 45.3 years). 2.2. DNA preparation and polymerase chain reaction method Blood samples were collected and genomic DNA was extracted by means of the salting method from blood lymphocytes. Amplification of genomic DNA was performed
by allele-specific polymerase chain reaction (PCR). Two sets of primers (forward, F; reverse, R) were used to amplify the arginine and proline alleles, as follows: Arg F: TCC CCC TTG CCG TCC CAA Arg R: CTG GTG CAG GGG CCA CGC Pro F: GCC AGA GGT TGC TCC CCC Pro R: CGT GCA AGT CAC AGA CTT Each set of primers was added to a separate tube with a total volume of 25 mL, containing 0.3 mmol/L dNTPs, 1.5 mmol/L MgCl2, 2 U Taq polymerase, and 1 buffer (20 mmol/L Tris-HCl, pH 8.4, and 50 mmol/L KCl). Amplification was performed at 35 cycles with a touch-down program: denaturation at 94 C for 30 seconds, annealing at 68e62 C for 10 cycles and 62e58 C for the remaining 25 cycles, with extension at 72 C for 30 seconds in each cycle. The PCR product was 177 bp for the Pro allele and 141 bp for the Arg allele (Fig. 1).
2.3. Statistical analysis Data regarding age, tumor type, invasion, metastasis, menopause status, age of diagnosis, presence of estrogen (ER) and progesterone (PR) receptors, and lymph node involvement were recorded. Results were analyzed with
B. Khadang et al. / Cancer Genetics and Cytogenetics 173 (2007) 38e42
40
Table 1 Frequencies of TP53 genotype among patient and control groups Genotype
Patients, no. (%)
Controls, no. (%)
Arg/Arg Pro/Pro Arg/Pro Total
83 (37.6) 29 (13.1) 109 (49.3) 221
75 (36.6) 40 (19.5) 90 (43.9) 205
SPSS software (version 11.5 for Windows software); c2 test was used to compare the results.
3. Results
Neither estrogen receptor (ER) expression (P 5 0.94) nor progesterone receptor (PR) expression (P 5 0.19) expression was associated with presence of a specific genotype in patients. No correlation was found between histological type of tumor and genotype frequency (P 5 0.53). Patients were divided into two groups based on the age of diagnosis, those under 35 years as early and those over 35 years as late, with still no difference in the prevalence of genotypes between two groups. The same result was achieved considering premenopausal and postmenopausal cases (P 5 0.9).
4. Discussion
A total of 221 female patients with sporadic breast cancer were studied, with a mean age of 48.7 years. Of these patients, 169 (88.5%) had infiltrative ductal carcinoma and 20 (10.5%) had medullary carcinoma; 2 patients (1%) had other types of breast cancer. Of the 221 patients, 58.6% had lymph node involvement, 1.8% had distant metastasis, and 77% had pretumoral invasion (vascular, lymphatic, or preneural). No genotype differed significantly (P 5 0.2) between patients and controls (Table 1). The Arg/Arg genotype appeared in 37.6 and 36.6% of the patients and controls, respectively; the Pro/Pro genotype in 13.1 and 19.5%; and the Arg/Pro genotype in 49.3 and 43.9%. Likewise, allele frequencies did not differ significantly (P 5 0.9) between patients and controls (Table 2). The Arg allele frequency was 62.2 and 58.5% in patients and controls, respectively; the corresponding Pro allele frequency was 37.8 and 41.5%. In terms of tumorenodeemetastasis (TNM) clinical staging, 42 patients (18.2%) were in stage 0 or I, 152 patients (68.7%) were in stage II, and 29 patients (13.1%) were in stage III or IV. Ten (35.7%) patients with stage III or IV disease had Arg/Arg genotype, 1 (3.6%) had Pro/Pro genotype, and 17 (60.7%) had the Arg/Pro genotype (P 5 0.308), indicating no statistically significant effect of allele on progression in clinical stage of the disease. Of the patients with lymph node involvement, 44 patients (34.1%) had the Arg/Arg genotype, 16 (12.2%) had Pro/Pro, and 70 (53.7%) had the heterozygous genotype, revealing no statistically significant relationship between genotype and lymph node involvement (P 5 0.15). The same was true considering genotype frequency and distant metastasis. Table 2 Allelic frequencies of TP53 codon 72 among patients and control groups Allele
Patients, no. (%)
Controls, no. (%)
Arg Pro Total
275 (62.2) 167 (37.8) 442
240 (58.5) 170 (41.5) 410
Several studies have shown an association between polymorphisms at codon 72 and human malignancies. We investigated the allele and genotype frequencies of TP53 codon 72 in 221 female patients with sporadic breast cancer and 205 healthy individuals from southern Iran. No significant difference in the frequency of genotypes and alleles was found between patients and controls. In addition, no significant association was detected between prevalence of alleles and genotypes and tumor type, clinical stage of tumor, lymph node involvement, metastasis, age of diagnosis, menopause status, and ER and PR expression. Our results are in agreement with similar studies on bladder cancer [24], but are contrary to some other reports [25e28]. Prevalence of this polymorphism in cervical [29e32], lung [33,34], colon [35], bladder [36,37], and skin [38] carcinoma have previously been investigated. There are few reports of studies assessing the TP53 codon 72 polymorphisms in breast cancer, with inconsistent results [39e41]. Some investigators reported a difference in the frequency of the Arg allele between breast cancer patients and controls [39,42], and others propose Pro allele as a risk factor for breast cancer [40,43]. Tommiska et al. [23] suggested that codon 72 polymorphism, particularly the Pro/Pro genotype, is an independent prognostic factor in patients with breast cancer and provided evidence that patients harboring this genotype will have a reduced survival. A report from China indicated an association between HPV oncogenic strains and breast cancer, with evidence that human papilloma virus (HPV) E6 oncoprotein more readily targets the Arg allele than Pro allele for degradation of p53 protein [44]. In light of that finding, Makni et al. [45] reported a correlation between HPV associated cervical cancer and Arg homozygosity of TP53 codon 72. Contrary to these findings, our previous study [46] revealed no association between HPV oncogenic strains in patients with cervical cancer, a result which is explained by the low frequency of HPV oncogenic strains in our population [46]. In conclusion, the data of the present study show that TP53 gene codon 72 polymorphism is not associated with increased risk of breast cancer in southern Iranian patients.
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This finding may be a reflection the low frequency of oncogenic strains of HPV in our population [46].
Acknowledgments This work financially was supported by Shiraz Institute for Cancer Research (Grant No: ICR-82-94) References [1] Berns EM, van Staveren IL, Look MP, Smid M, Klijn JGM, Foekens JA. Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer. Br J Cancer 1998;77:1130e6. [2] Goldhirsch A. Breast cancer. In: Cavalli F, Hansen H, Kaye SB, editors. Textbook of medical oncology. London: Martin Dunitz, 2000. pp. 137e83. [3] Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49e53. [4] Wu MT, Chen MC, Wu DC. Influences of lifestyle habits and p53 codon 72 and p21 codon 31 polymorphisms on gastric cancer risk in Taiwan. Cancer Lett 2004;205:61e8. [5] Powell BL, Staveren IL, Roosken P, Grieu F, Berns EM, Iacopetta B. Associations between common polymorphisms in TP53 and p21 WAF1/Cip1 and phenotypic features of breast cancer. Carcinogenesis 2002;23:311e5. [6] Keshava C, Frye BL, Wolff MS, McCanlies EC, Weston A. Waf-1 (p21) and p53 polymorphisms in breast cancer. Cancer Epidemiol Biomarkers Prev 2002;11:127e30. [Erratum in: Cancer Epidemiol Biomarkers Prev 2004;13:1682]. [7] Roh JW, Kim JW, Park NH, Song YS, Park IA, Park SY, Kang SB, Lee HP. p53 and p21 genetic polymorphisms and susceptibility to endometrial cancer. Gynecol Oncol 2004;93:499e505. [8] Huang SP, Wu WJ, Chang WS, Wu MT, Chen YY, Chen YJ, Yu CC, Wu TT, Lee YH, Huang JK, Huang CH. p53 codon 72 and P21 codon 31 polymorphisms in prostate cancer. Cancer Epidemiol Biomarkers Prev 2004;13:2217e24. [9] Lee SA, Kim JW, Roh JW, Choi JY, Lee KM, Yoo KY, Song YS, Kang D. Genetic polymorphisms of GSTM1, p21, p53 and HPV infection with cervical cancer in Korean women. Gynecol Oncol 2004;93:14e8. [10] Zhu ZZ, Cong WM, Zhu GS, Liu SF, Xian ZH, Wu WQ, Zhang XZ, Wang YH, Wu MC. Association of p53 codon 72 polymorphism with genetic susceptibility to hepatocellular carcinoma in a Chinese population. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2005;22:632e5. [11] Wang Y, Kringen P, Kristensen GB, Holm R, Baekelandt MM, Olivier M, Skomedal H, Hainaut P, Trope CG, Abeler VM, Nesland JM, Borresen Dale AL, Helland A. Effect of the codon 72 polymorphisms (c.215 GOC, p.Arg 72 Pro) in combination with somatic sequences variants in TP53 gene on survival in patients with advanced ovarian carcinoma. Hum Mutat 2004;24:21e34. [Erratum in: Hum Mutat 2006;27:209e10]. [12] Hogdall EV, Hogdall CK, Christensen L, Glud E, Blaakaer J, Bock JE, Vuust J, Norgaard-Pedersen B, Kjaer SK. Distribution of p53 codon 72 polymorphisms in ovarian tumour patients and their prognostic significance in ovarian cancer patients. Anticancer Res 2002;22:1859e64. [13] Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 2003;10:431e42. [14] Bautista S, Theillet C. p53 mutations in breast cancer, incidence and relations to tumor aggressiveness and evolution of the disease. Pathol Biol (Paris) 1997;45:882e92. [15] Donehower LA. p53 guardian and suppressor of longevity? Exp Gerontol 2005;40:7e9.
41
[16] Zehbe I, Voglino G, Wilander E, Genta F, Tommasino M. Codon 72 polymorphism of p53 and its association with cervical cancer. Lancet 1999;354:218e9. [17] Jain N, Singh V, Hedau S, Kumar S, Daga MK, Dewan R, Murthy NS, Husain SA, Das BC. Infection of human papillomavirus type 18 and p53 codon 72 polymorphism in lung cancer patients from India. Chest 2005;128:3999e4007. [18] Matakidou A, Eisen T, Houlston RS. TP53 polymorphisms and lung cancer risk a systemic review and meta-analysis. Mutagenesis 2003;18:377e85. [19] Dumont P, Leu JI, Della Pietra AC, George DL, Murphy M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat Genet 2003;33:357e65. [20] Buyru N, Tigli H, Dalay N. p53 codon 72 polymorphism in breast cancer. Oncol Rep 2003;10:711e4. [21] Langerød A, Bukholm IR, Brega˚ard A, Lønning PE, Andersen TI, Rognum TO, Meling GI, Lothe RA, Børresen-Dale AL. The TP53 codon 72 polymorphism may affect the function of TP53 mutations in breast carcinomas but not in colorectal carcinomas. Cancer Epidemiol Biomarkers Prev 2002;11:1684e8. [22] Papadakis EN, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of breast cancer. Mol Cell Biol Res Commun 2000;3:389e92. [23] Tommiska J, Eerola H, Heinonen M, Salonen L, Kaare M, Tallila J, Ristimaki A, von Smitten K, Aittomaki K, Heikkila P, Blomqvist C, Nevanlinna H. Breast cancer patients with p53 Pro72 homozygous genotype have a poorer survival. Clin Cancer Res 2005;11: 5098e103. [24] Toruner GA, Ucar A, Tez M, Cetinkaya M, Ozen H, Ozcelik T. p53 codon 72 polymorphism in bladder cancer: no evidence of association with increased risk of invasiveness. Urol Res 2001;29:393e5. [25] Santos AM, Sousa H, Catarino R, Pinto D, Pereira D, Vasconcelos A, Matos A, Lopes C, Medeiros R. TP53 codon 72 polymorphism and risk for cervical cancer in Portugal. Cancer Genet Cytogenet 2005;159:143e7. [26] Agorastos T, Masouridou S, Lambropoulos AF, Chrisafi S, Miliaras D, Pantazis K, Constantinides TC, Kotsis A, Bontis I. p53 codon 72 polymorphism and correlation with ovarian and endometrial cancer in Greek women. Eur J Cancer Prev 2002;13:277e80. [27] Leiros GJ, Galliano SR, Sember ME, Kahn T, Schwarz E, Eiguchi K. Detection of human papillomavirus DNA and p53 codon 72 polymorphism in prostate carcinomas of patients from Argentina. BMC Urol 2005;5:15. [28] Siddique MM, Balram C, Fiszer-Maliszewska L, Aggarwal A, Tan A, Tan P, Soo KC, Sabapathy K. Evidence for selective expression of the p53 codon 72 polymorphs: implications in cancer development. Cancer Epidemiol Biomarkers Prev 2005;14:2245e52. [29] Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L. Role of p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393:229e34. [30] Crook T, Ludwig RL, Marston NJ, Willkomm D, Vousden KH. Sensitivity of p53 lysine mutants to ubiquitin-directed degradation targeted by human papillomavirus E6. Virology 1996;217:285e92. [31] Madeleine MM, Shera K, Schwartz SM, Daling JR, Galloway DA, Wipf GC, Carter JJ, McKnight B, McDougall JK. The p53 Arg 72 Pro polymorphism, human papillomavirus, and invasive squamous cell cervical cancer. Cancer Epidemiol Biomarkers Prev 2000;9: 225e7. [32] Tenti P, Vesentini N, Rondo Spaudo M, Zappatore R, Migliora P, Carnevali L, Ranzani GN. p53 codon 72 polymorphism does not affect the risk of cervical cancer in patients from northern Italy. Cancer Epidemiol Biomarkers Prev 2000;9:435e8. [33] Wang YC, Lee HS, Chen SK, Yang SC, Chen CY. Analysis of K-ras gene mutations in lung carcinomas: correlation with gender, histological subtypes, and clinical outcome. J Cancer Res Clin Oncol 1998;124:517e22.
42
B. Khadang et al. / Cancer Genetics and Cytogenetics 173 (2007) 38e42
[34] Pierce LM, Sivaraman L, Chang W, Lum A, Donlon T, Seifried A, Wilkens LR, Lau AF, Le Marchand L. Relationships of TP53 codon 72 and HRAS1 polymorphisms with lung cancer risk in an ethnically diverse population. Cancer Epidemiol Biomarkers Prev 2000;9: 1199e204. [35] Sayhan N, Yazici H, Budak M, Bitisik O, Dalay N. p53 codon 72 genotypes in colon cancer: association with human papillomavirus infection. Res Commun Mol Pathol Pharmacol 2001;109:25e34. [36] Kuroda Y, Tsukino H, Nakao H, Imai H, Katoh T. p53 codon 72 polymorphism and urothelial cancer risk. Cancer Lett 2003;189:77e83. [37] Soulitzis N, Sourvinos G, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism and its association with bladder cancer. Cancer Lett 2002;179:175e83. [38] Dokianakis DN, Koumantaki E, Billiri K, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of HPV-associated non-melanoma skin cancers in immunocompetent hosts. Int J Mol Med 2000;5:405e9. [39] Wang-Gohrke S, Rebbeck TR, Besenfelder W, Kreienberg R, Runnebaum IB. p53 germline polymorphisms are associated with an increased risk for breast cancer in German women. Anticancer Res 1998;18:2095e9. [40] Sjalander A, Birgander R, Hallmans G, Cajander S, Lenner P, Athlin L, Beckman G, Beckman L. p53 polymorphisms and haplotypes in breast cancer. Carcinogenesis 1996;17:1313e6.
[41] Papadakis EN, Dokianakis DN, Spandidos DA. p53 codon 72 polymorphism as a risk factor in the development of breast cancer. Mol Cell Biol Res Commun 2000;3:389e92. [42] Suspitsin EN, Buslov KG, Grigoriev MY, Ishutkina JG, Ulibina JM, Gorodinskaya VM, Pozharisski KM, Berstein LM, Hanson KP, Togo AV, Imyanitov EN. Evidence against involvement of p53 polymorphism in breast cancer predisposition. Int J Cancer 2003;103: 431e3. [43] Weston A, Pan CF, Ksieski HB, Wallenstein S, Berkowitz GS, Tartter PI, Bleiweiss IJ, Brower ST, Senie RT, Wolff MS. p53 haplotype determination in breast cancer. Cancer Epidemiol Biomarkers Prev 1997;6:105e12. [44] Li T, Lu ZM, Guo M, Wu QJ, Chen KN, Xing HP, Mei Q, Ke Y. p53 codon 72 polymorphism (C/G) and the risk of human papillomavirus-associated carcinomas in China. Cancer 2002;95:2571e6. [45] Makni H, Franco EL, Kaiano J, Villa LL, Labrecque S, Dudley R, Storey A, Matlashewski G. p53 polymorphism in codon 72 and risk of human papillomavirus-induced cervical cancer: effect of inter-laboratory variation. Int J Cancer 2000;87:528e33. [46] Farjadian S, Asadi E, Doroudchi M, Dehaghani AS, Tabei SZ, Kumar VP, Ghaderi A. High risk HPV types in southern Iranian patients with cervical cancer. Pathol Oncol Res 2003;9: 121e5.