|Year : 2013 | Volume
| Issue : 3 | Page : 202-206
Allele frequencies of the epidermal growth factor receptors polymorphism R521K in colorectal cancer patients and healthy subjects indicate a risk-reducing effect of K521 in Syrian population
Ola Haj Mustafa1, Abdul Rezzak Hamzeh2, Lina Ghabreau3, Nizar Akil3, Ala-Eddin Almoustafa4, Amal Alachkar5
1 Department of Pharmacology, University of Aleppo, Aleppo, Syria
2 Department of Biochemistry, Faculty of Pharmacy, University of Aleppo, Aleppo, Syria
3 Department of Pathology, Faculty of Medicine, University of Aleppo, Aleppo, Syria
4 Department of Oncology, Faculty of Medicine, McGill University, Montrexal, Canada; Research Cancer Centre of the Syrian Society Against Cancer, Aleppo, Syria
5 Department of Pharmacology, University of Aleppo, Aleppo, Syria; Department of Pharmacology, School of Medicine, University of California, Irvine, CA 92617, USA
|Date of Web Publication||20-Mar-2013|
355 Med Surge II, Department of Pharmacology, School of Medicine, University of California, Irvine, CA92617, USA
Source of Support: This work was supported by the University of Aleppo., Conflict of Interest: None
Background: Colorectal cancer contributes heavily to cancer morbidity and mortality worldwide. Numerous therapies are currently in use, including monoclonal antibodies against cellular components involved in tumorigenesis such as epidermal growth factor receptors (EGFRs). Studies showed the polymorphism [R521K] GàA in the EGFR gene to be involved in both colorectal cancer susceptibility and clinical response to therapeutics (e.g., Cetuximab). Aim: We aimed at uncovering allele frequencies of this polymorphism among Syrian colorectal cancer patients and healthy individuals. Materials and Methods: Forty-seven patients with colorectal cancer were included in a case-control study along with 48 healthy subjects, all native Syrians. Individuals were genotyped using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and results were statistically analyzed to elucidate significant differences between the two groups. Results: Allele frequencies were 40.4% (G/G), 57.4% (G/A) and 2.1% (A/A) in colorectal cancer patients and 41.6% (G/G), 43.7% (G/A) and 14.5% (A/A) in healthy subjects. The A/A genotype was significantly lower in colorectal cancer patients than in the control group. Conclusions: Homozygosity for the A allele is linked to reducing the risk of developing colorectal cancer in Syrian patients. The lower prevalence of (A/A) locally may predict sub-optimal rates of clinical response to Cetuximab compared with populations with higher frequencies of the A allele. Larger scale investigations are needed for a stronger conclusion.
Keywords: Colorectal cancer, Epidermal growth factor receptor, Polymorphism, R521K, Syrian
|How to cite this article:|
Mustafa OH, Hamzeh AR, Ghabreau L, Akil N, Almoustafa AE, Alachkar A. Allele frequencies of the epidermal growth factor receptors polymorphism R521K in colorectal cancer patients and healthy subjects indicate a risk-reducing effect of K521 in Syrian population. North Am J Med Sci 2013;5:202-6
|How to cite this URL:|
Mustafa OH, Hamzeh AR, Ghabreau L, Akil N, Almoustafa AE, Alachkar A. Allele frequencies of the epidermal growth factor receptors polymorphism R521K in colorectal cancer patients and healthy subjects indicate a risk-reducing effect of K521 in Syrian population. North Am J Med Sci [serial online] 2013 [cited 2019 Nov 12];5:202-6. Available from: http://www.najms.org/text.asp?2013/5/3/202/109189
| Introduction|| |
Colorectal cancer (CRC) is one of the most prevalent cancers worldwide, with high metastasis and poor survival rates. CRC clearly follows the multistage model; initially affecting normal mucosa and finishing up as an invasive adenocarcinoma, with transitional stages including dysplastic lesions, adenoma and in situ adenocarcinoma. ,,, This sequence of events happens as a result of the accumulation of genetic aberrations, which ultimately leads to dysfunctional changes in cell proliferation, differentiation and apoptosis. ,, Eventually, such imbalance between the rates of cell growth and apoptosis results in the development and progression of CRC.
The genomic picture of CRCs is highly complex, with up to 70 mutations in well-established cancer genes being found to be involved in tumor initiation and progression.  One of the most extensively studied genes in various cancers is the one encoding epidermal growth factor receptor (EGFR). EGFR, a transmembrane glycoprotein, is a member of the human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases. The timely activation of certain tyrosine kinases initiates transduction cascades of several signaling pathways, including ras/raf/MAPK (Mitogen-activated protein kinases) and phosphatidylinositol-3-kinase, which are involved in gene expression, cell growth, cell proliferation, angiogenesis and apoptosis. ,,,, The central role of EGFR signaling in cell growth and survival implies that alterations in the function of EGFR may have a substantial impact in the development and progression of cancer.
A considerable mass of published work provided evidence for the role of EGFR abnormalities in several types of cancer including those of the lung, colon, head and neck, pancreas, breast, ovary, bladder, kidney and gliomas. ,,,,,,, In fact, the activity of EGFR was shown to be elevated in most solid tumors. In response to this data, attention was directed toward EGFR variants, and indeed it was shown that many EGFR polymorphisms play a significant role in the development and prognosis of CRC. ,,,, One of these is R521K, or R497K according to an older nomenclature, which is a functional polymorphism in EGFR that arose from a G-to-A transition, resulting in an Arg to Lys substitution. Substituting arginine by lysine in codon 521 on the border between extracellular subdomains III and IV of EGFR causes a reduction in the activity of the receptor. This may be attributed to attenuating ligand binding and ligand-induced EGFR signaling.  Moreover, it has been reported that the variant R521K can predict a better response to the anticancer drug Cetuximab in combination with irinotecan. 
This study aims to investigate allele frequencies of the functional polymorphism R521K in EGFR in order to find potential associations between these frequencies and CRC in the Syrian population. This regionally unprecedented screening is an important beginning to many feasibility studies focusing on introducing anti-EGFR antibodies (e.g., Cetuximab) into therapeutic protocols for treating Syrian CRC patients. This assessment may assist local health authorities in setting up their policies with regard to procuring Cetuximab and similar medications.
| Materials and Methods|| |
This case-control study was conducted on a group of 47 operated-on patients with confirmed diagnosis of colon cancer. The control group consisted of 48 healthy subjects with no history of cancer. Patients were recruited from the University Hospital in Aleppo, Syria. All subjects were native Syrians and of the same ethnicity (Arabs). Blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) tubes and anonymously coded and stored. Informed consent was obtained from patients and healthy subjects. The study was approved by the Ethical Committee at the University of Aleppo.
DNA isolation and PCR
Genomic DNA was extracted from 200 μl of venous blood using a spin column format kit (Fermentase, Lithuania). The isolation of DNA was carried out according to the manufacturer's instructions.
The R521K (G < A) polymorphism of the EGFR gene was genotyped using a previously described PCR-RFLP method using the restriction enzyme MvaI.  The PCR reaction was carried out in a total volume of 20 μl, containing 4 μl of genomic DNA as template, 0.4 μM of each primer (synthesized by VBC-Biotech, Austria), 2 μM of PCR buffer, 0.4 μM of each dNTP, 2 μM of MgCl 2 , 1X Taq buffer and 1 unit of Taq DNA Polymerase (Fermentase, Lithuania). PCR amplification was carried out in a MasterCycler® thermal cycler (Eppendorf, Germany), with an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 57°C for 30 s and 72°C for 10 s, and a final extension at 72°C for 5 min.
The sequences of the forward and reverse primers were: 5'-TGCTGTGACCCACTCTGTCT-3' and 5'- CCAGAAGGTTGCACTTGTCC-3', respectively.
The PCR product (155 bp) was digested with MvaI restriction enzyme (FastDigest® Fermentase) at 37°C for 10 min. This restriction enzyme recognizes the sequence CC/WGG. The amplicon with (G) is cleaved twice by the enzyme, rendering three fragments (38, 50 and 67 bp), whereas the A variant is cleaved only once (38 and 117 bp).
Digestion products were separated by electrophoresis on 2% Nusieve ethidium bromide-stained agarose gels (TopVision Agarose, Fermentas, Lithuania) and visualized under UV light. Analyses by sex and age were skipped due to the small sample size.
Standard contingency table analysis and Chi-square test were used to assess the association of the genotype and allele frequencies for the EGFR R521K polymorphism with CRC. Risk value to CRC was estimated by calculating the odds ratios with 95% confidence intervals. P values < 0.05 were considered statistically significant.
| Results|| |
The R521K polymorphism was genotyped in CRC patients and healthy subjects. The results are summarized in [Table 1], which showed that the frequencies of the G/G, G/A and A/A genotypes in healthy subjects were 41.6% ( n = 20), 43.7% ( n = 21), and 14.5% ( n = 7), respectively. In the colorectal patients' group, the frequencies of the G/G, G/A and A/A genotypes were 40.4% ( n = 19), 57.4% ( n = 27) and 2.1% ( n = 1). Data analysis indicates that the A/A genotype frequency is significantly different between patient and control groups (χ2 = 4.05, df = 1, P = 0.04). The frequencies of the G and A alleles were 63.5% ( n = 61) and 36.4% ( n = 35), respectively, in the control group and 69.1% ( n = 65) and 30.85% ( n = 29), respectively, in CRC patients. No significant difference in the allele frequency was observed between the two groups (χ2 = 0.14, df = 1, P = 0.7, and χ2 = 0.33, df = 1, P = 0.56 for the G and A alleles, respectively).
|Table 1: R521K genotype and allele frequencies in patients with colorectal Syrian cancer patients and in healthy subjects|
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| Discussion|| |
The EGFR polymorphic variant (142285 G > A) results in an arginine (R)/lysine (K) substitution in codon 521 in the extracellular domain of EGFR. This substitution is associated with attenuation in the affinity and ligand binding to the EGFR, which leads to reduced activation of downstream tyrosine kinase activity in downstream mitogenic pathways. ,
In line with these observations, the R521K polymorphism is speculated to be linked with less susceptibility to cancer, and indeed several studies confirmed better prognostic features in more than one type of cancer. ,,,, In CRC, there are a number of studies that highlighted the association between G/A or A/A and decreased tumor recurrence, lower probability of subsequent metastasis and longer survival in post-surgery CRC patients. The R521K polymorphism of EGFR was also associated with better response and survival in CRC patients treated with 5-FU and oxaliplatin-based chemotherapy, Cetuximab as single agent or in combination with irinotecan and with FOLFOX-4. ,,,,,,,
However, only one study showed an association of this polymorphism with a decreased risk of CRC. 
The aim of the current study was to reveal local allele frequencies of the non-synonymous EGFR variant R521K. Such data is critical to evaluate the worth of using this polymorphism as a biomarker for susceptibility to CRC and to predict the clinical response to drugs such as Cetuximab in the Syrian population. The latter point is highly relevant to address the feasibility of using the new anti-EGFR drug Cetuximab in the treatment of Syrian CRC patients. This can be based on the findings that certain alleles of this polymorphism correlate with a higher response rate and a favorable prognosis in Cetuximab-treated CRC patients.
In this study, we assessed R521K polymorphism genotype frequencies in Syrian colorectal patients and compared these frequencies with those from a representative control sample of the Syrian population.
Importantly, our results showed that R521K genotype frequencies in the CRC cohort were significantly different from those observed in the control cohort. Our results are consistent with the findings of an Italian study that suggested a negative correlation between R521K and susceptibility to CRC.  However, our data differed from the latter study because it showed that only A/A genotype was significantly different between the two groups. This discrepancy could be explained by the ethnic differences that may have an impact on susceptibility to cancer. Our results showed for the first time that allelic distribution of this polymorphism in the Syrian population was distinct, and remarkably different, from other ethnic groups [Table 2].
Compared with other populations, the prevalence of A/A genotype was remarkably higher in healthy Syrian subjects than in Europeans, African Americans and Sub-Sahara Africans, but significantly lower than that of Asians [Table 2] (Single Nucleotide Polymorphism (SNP) database, ncbi.nlm.nih.gov). It is well established that the ethnic differences may have a considerable influence on susceptibility to cancer as well as on clinical response profiles to chemotherapy. ,,, Based on the findings that this polymorphism influences the anticancer activity of Cetuximab, possibly by enhancing the anticancer effect of Cetuximab, our results and previous findings in different ethnic groups suggest that Syrian patients might have poorer outcome to Cetuximab-based treatment than Asian CRC patients. However, more cross-ethnic studies on a larger scale are required to confirm this line of reasoning.
In conclusion, our study demonstrates for the first time that one genotypic variant of R521K polymorphism, namely the A/A, confers a protective advantage in relation to CRC in the Syrian population. Indeed, our data confirmed the negative correlation between the A/A variant and susceptibility to CRC. Finally, considering only the R521K polymorphism, especially the very low frequency of A/A in Syrian patients as a predicting factor of Cetuximab treatment, it appears that the use of Cetuximab might not produce a very good outcome in Syrian CRC patients. This presents a weak case for introducing Cetuximab by health authorities in the Syrian Arab Republic and studies with larger sample sizes are called for to reach the best possible conclusion in this regard.
| Acknowledgment|| |
This work was supported by the University of Aleppo, Syria.
| References|| |
|1.||Pritchard CC, Grady WM. Colorectal cancer molecular biology moves into clinical practice. Gut 2011;60:116-29. |
|2.||Nagawa H, Muto T. Molecular biology in the development of colorectal cancer. Gan To Kagaku Ryoho 1999;26:2147-53. |
|3.||Mulcahy HE, O'Donoghue DP. Molecular biology. Setting the stage in colorectal cancer? Gut 1993;34:1476-7. |
|4.||Finlay GJ. Genetics, molecular biology and colorectal cancer. Mutat Res 1993;290:3-12. |
|5.||Giarnieri E, Midiri G, Cirolla VA, Covotta A, Covotta L, Avitto FM, et al. From molecular biology to new treatment approaches to colorectal cancer: Basic research, experimental trials and surgical implications. G Chir 2003;24:109-14. |
|6.||Cascinu S, Zaniboni A, Scartozzi M, Meriggi F. Molecular biology for stage II colorectal cancer: The jury is still out. J Clin Oncol 2007;25:2861. |
|7.||Vecchione L, Jacobs B, Normanno N, Ciardiello F, Tejpar S. EGFR-targeted therapy. Exp Cell Res 2011;317:2765-71. |
|8.||Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001;37:S3-8. |
|9.||Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49-139. |
|10.||Citri A, Yarden Y. EGF-ERBB signalling: Towards the systems level. Nat Rev Mol Cell Biol 2006;7:505-16. |
|11.||Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489-501. |
|12.||Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006;7:606-19. |
|13.||Campiglio M, Ali S, Knyazev PG, Ullrich A. Characteristics of EGFR family-mediated HRG signals in human ovarian cancer. J Cell Biochem 1999;73:522-32. |
|14.||Suwa T, Ueda M, Jinno H, Ozawa S, Kitagawa Y, Ando N, et al. Epidermal growth factor receptor-dependent cytotoxic effect of anti-EGFR antibody-ribonuclease conjugate on human cancer cells. Anticancer Res 1999;19:4161-5. |
|15.||Todd R, Wong DT. Epidermal growth factor receptor (EGFR) biology and human oral cancer. Histol Histopathol 1999;14:491-500. |
|16.||Huang Y, Yang J, Zhou Q, Zhao Y. Expression of EGFR in human lung cancer tissues. Zhongguo Fei Ai Za Zhi 1998;1:98-100. |
|17.||Harari PM, Huang SM. Head and neck cancer as a clinical model for molecular targeting of therapy: Combining EGFR blockade with radiation. Int J Radiat Oncol Biol Phys 2001;49:427-33. |
|18.||Wallerand H, Cai Y, Wainberg ZA, Garraway I, Lascombe I, Nicolle G, et al. Phospho-Akt pathway activation and inhibition depends on N-cadherin or phospho-EGFR expression in invasive human bladder cancer cell lines. Urol Oncol 2010;28:180-8. |
|19.||Tímár J, Kopper L, Bodrogi I. Molecular pathology and targeted therapy of clear cell renal cancer. Magy Onkol 2006;50:309-14. |
|20.||Viana-Pereira M, Lopes JM, Little S, Milanezi F, Basto D, Pardal F, et al. Analysis of EGFR overexpression, EGFR gene amplification and the EGFRvIII mutation in Portuguese high-grade gliomas. Anticancer Res 2008;28:913-20. |
|21.||Milano G, Etienne-Grimaldi MC, Dahan L, Francoual M, Spano JP, Benchimol D, et al. Epidermal growth factor receptor (EGFR) status and K-Ras mutations in colorectal cancer. Ann Oncol 2008;19:2033-8. |
|22.||Gonçalves A, Esteyries S, Taylor-Smedra B, Lagarde A, Ayadi M, Monges G, et al. A polymorphism of EGFR extracellular domain is associated with progression free-survival in metastatic colorectal cancer patients receiving cetuximab-based treatment. BMC Cancer 2008;8:169. |
|23.||Spindler KL, Nielsen JN, Ornskov D, Brandslund I, Jakobsen A. Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer. Acta Oncol 2007;46:1113-7. |
|24.||Spindler KL, Lindebjerg J, Nielsen JN, Olsen DA, Bisgård C, Brandslund I, et al. Epidermal growth factor receptor analyses in colorectal cancer: A comparison of methods. Int J Oncol 2006;29:1159-65. |
|25.||Zhang W, Stoehlmacher J, Park DJ, Yang D, Borchard E, Gil J, et al. Gene polymorphisms of epidermal growth factor receptor and its downstream effector, interleukin-8, predict oxaliplatin efficacy in patients with advanced colorectal cancer. Clin Colorectal Cancer 2005;5:124-31. |
|26.||Brandt B, Meyer-Staeckling S, Schmidt H, Agelopoulos K, Buerger H. Mechanisms of egfr gene transcription modulation: Relationship to cancer risk and therapy response. Clin Cancer Res 2006;12:7252-60. |
|27.||Dahan L, Norguet E, Etienne-Grimaldi MC, Formento JL, Gasmi M, Nanni I, et al. Pharmacogenetic profiling and cetuximab outcome in patients with advanced colorectal cancer. BMC Cancer 2011;11:496. |
|28.||Moriai T, Kobrin MS, Hope C, Speck L, Korc M. A variant epidermal growth factor receptor exhibits altered type alpha transforming growth factor binding and transmembrane signaling. Proc Natl Acad Sci U S A 1994;91:10217-21. |
|29.||Bandrés E, Barricarte R, Cantero C, Honorato B, Malumbres R, Zárate R, et al. Epidermal growth factor receptor (EGFR) polymorphisms and survival in head and neck cancer patients. Oral Oncol 2007;43:713-9. |
|30.||Rebaï M, Kallel I, Hamza F, Charfeddine S, Kaffel R, Guermazi F, et al. Association of EGFR and HER2 polymorphisms with risk and clinical features of thyroid cancer. Genet Test Mol Biomarkers 2009;13:779-84. |
|31.||Sasaki H, Okuda K, Shimizu S, Takada M, Kawahara M, Kitahara N, et al. EGFR R497K polymorphism is a favorable prognostic factor for advanced lung cancer. J Cancer Res Clin Oncol 2009;135:313-8. |
|32.||Wang WS, Chen PM, Chiou TJ, Liu JH, Lin JK, Lin TC, et al. Epidermal growth factor receptor R497K polymorphism is a favorable prognostic factor for patients with colorectal carcinoma. Clin Cancer Res 2007;13:3597-604. |
|33.||Zhang W, Azuma M, Lurje G, Gordon MA, Yang D, Pohl A, et al. Molecular predictors of combination targeted therapies (cetuximab, bevacizumab) in irinotecan-refractory colorectal cancer (BOND-2 study). Anticancer Res 2010;30:4209-17. |
|34.||Zhang W, Park DJ, Lu B, Yang DY, Gordon M, Groshen S, et al. Epidermal growth factor receptor gene polymorphisms predict pelvic recurrence in patients with rectal cancer treated with chemoradiation. Clin Cancer Res 2005;11:600-5. |
|35.||Press OA, Zhang W, Gordon MA, Yang D, Lurje G, Iqbal S, et al. Gender-related survival differences associated with EGFR polymorphisms in metastatic colon cancer. Cancer Res 2008;68:3037-42. |
|36.||Hsieh YY, Tzeng CH, Chen MH, Chen PM, Wang WS. Epidermal growth factor receptor R521K polymorphism shows favorable outcomes in KRAS wild-type colorectal cancer patients treated with cetuximab-based chemotherapy. Cancer Sci 2012;103:791-6. |
|37.||Martinelli M, Ugolini G, Scapoli L, Rivetti S, Lauriola M, Mattei G, et al. The EGFR R521K polymorphism influences the risk to develop colorectal cancer. Cancer Biomark 2010;8:61-5. |
|38.||O'Donnell PH, Dolan ME. Cancer pharmacoethnicity: Ethnic differences in susceptibility to the effects of chemotherapy. Clin Cancer Res 2009;15:4806-14. |
|39.||Zeigler-Johnson CM, Walker AH, Mancke B, Spangler E, Jalloh M, McBride S, et al. Ethnic differences in the frequency of prostate cancer susceptibility alleles at SRD5A2 and CYP3A4. Hum Hered 2002;54:13-21. |
|40.||Gilliland FD. Ethnic differences in cancer incidence: A marker for inherited susceptibility? Environ Health Perspect 1997;105:897-900. |
|41.||Khrunin A, Ivanova F, Moisseev A, Khokhrin D, Sleptsova Y, Gorbunova V, et al. Pharmacogenomics of cisplatin-based chemotherapy in ovarian cancer patients of different ethnic origins. Pharmacogenomics 2012;13:171-8. |
[Table 1], [Table 2]