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Year : 2012  |  Volume : 49  |  Issue : 1  |  Page : 163--168

The frequency and spectrum of K-ras mutations among Iraqi patients with sporadic colorectal carcinoma

NA Al-Allawi1, AT Ismaeel2, NY Ahmed3, NS Merza4,  
1 Department of Pathology, College of Medicine, University of Dohuk, Dohuk, Iraq
2 Department of Pathology, College of Pharmacy, Hawler Medical University, Erbil, Iraq
3 Department of Pathology, College of Medicine, Hawler Medical University, Erbil, Iraq
4 Scientific Research Center, University of Dohuk, Dohuk, Iraq

Correspondence Address:
N A Al-Allawi
Department of Pathology, College of Medicine, University of Dohuk, Dohuk
Iraq

Abstract

Background: The epidemiology of colorectal cancers (CRC) is well known to differ in different geographical regions. K-ras mutations have been implicated in CRC carcinogenesis and they were extensively studied in developed countries; however, such studies are scarce from developing countries, like Iraq. Aim: To determine the frequency and spectrum of K-ras mutations among CRC Iraqi patients, and their clinico-pathological associations, if any. Materials and Methods: Fifty consecutive surgically resected sporadic CRC were evaluated. The evaluation included screening for ten K-ras mutations in codon 12 and 13 by mutant enriched polymerase chain reaction followed by reverse hybridization to oligospecific probes. Results: Out of the 50 enrolled patients, 24 (48%) had K-ras mutations. A total of 29 mutations were identified in the tumors of the latter 24 patients (20/24 tumors had single mutations, 3/24 had double mutations and 1/24 had triple mutations). The most frequently encountered mutations were the G>T transversions and G>A transitions (41.4% each). Codon 12 mutations constituted 89.7%, while codon 13 the remaining 10.3%. The most frequent mutation was GGT>GTT (Gly>Val) of codon 12 documented in 31%. No significant clinico-pathological correlations with K-ras mutational status were identified. Conclusion : The K-ras mutations are frequently encountered among Iraqi sporadic CRC patients, with relative higher frequencies of G>T transversions and Gly>Val codon 12 substitutions than encountered in their counterparts in developed countries. The latter is most likely to be related to differences in local carcinogens exposure, an aspect which requires further scrutiny.



How to cite this article:
Al-Allawi N A, Ismaeel A T, Ahmed N Y, Merza N S. The frequency and spectrum of K-ras mutations among Iraqi patients with sporadic colorectal carcinoma.Indian J Cancer 2012;49:163-168


How to cite this URL:
Al-Allawi N A, Ismaeel A T, Ahmed N Y, Merza N S. The frequency and spectrum of K-ras mutations among Iraqi patients with sporadic colorectal carcinoma. Indian J Cancer [serial online] 2012 [cited 2019 Oct 21 ];49:163-168
Available from: http://www.indianjcancer.com/text.asp?2012/49/1/163/98943


Full Text

 Introduction



Colorectal cancer (CRC) is a major cause of morbidity and mortality in Western developed countries, where age standardized rates are the highest in the world. The latter rates are much lower in developing countries in Asia and Africa. In Iraq, a developing Asian country in Eastern Mediterranean region, these rates are about fourfolds less than those in developed countries in Europe and North America. [1] However, CRC is still the seventh most common cancer among Iraqis. [2] Colorectal carcinogenesis is characterized by numerous genetic and epigenetic changes, including activating mutations in oncogenes, inactivating mutations in tumor suppressor genes, and mismatch repair genes, microsatellite instability, and methylation changes in gene promoters. [3]

According to the multi-step genetic model of CRC carcinogenesis, APC (adenomatous polyposis coli) gene is involved in adenoma formation and K-ras oncogene in the transition of adenoma to carcinoma in sporadic CRC. Thus, the latter is an early event in CRC carcinogenesis. [4] The K-ras gene encodes for a plasma membrane-bound GTP binding protein with a molecular weight of 21 kd. This protein controls multiple pathways affecting cell growth, differentiation, and apoptosis. [5] Certain mutations in the ras genes leads to amino acid substitutions in resultant protein, which would alter the signal transduction pathway leading to stimulation of clonal expansion of the cells, even in the absence of extracellular stimuli. [6] The K-ras mutations have been reported in up to half of sporadic CRC in developed countries. [7],[8],[9] The vast majority of these mutations (>90%) involved codons 12 and 13, with G>A transitions being the most frequent. [7],[10],[11]

Studies on K-ras mutations in CRC are scarce from developing countries, and there is a great need for such studies to learn more about the molecular signature of these tumors and ethnic differences if any, particularly with varying age standardized incidence and environmental influences between such countries and the Western ones. Therefore, the current study was undertaken to determine the frequency and spectrum of K-ras mutations among patients with sporadic CRC in Iraq, a country that was not previously investigated.

 Materials and Methods



In the period between September 2007 and August 2009, a total of 61 consecutive colectomy specimens from patients with colorectal carcinoma were referred to two major histopathology laboratories in Erbil city-Iraq. These two laboratories have special interest in gastro-intestinal malignancies.

The enrollment criteria for the study included electively resected colorectal adenocarcinoma based on endoscopic sampled tumor specimen and no history of chemotherapy or radiotherapy prior to surgery. The exclusion criteria included history of or pathological evidence for familial adenomatous polyposis (six patients) or hereditary non-polyposis colorectal cancer syndrome (one patient), history of or pathological evidence of idiopathic inflammatory bowel disease (one patient) and availability of DNA from tumor tissue (three patients). This left a total of 50 patients for inclusion in the study.

The research was approved by council of college of medicine in Hawler Medical University and informed consent was obtained from all patients. Demographic data (age at diagnosis, sex) and the topography of the tumor (tumor location, size, depth of invasion, and nodal status) were obtained from all enrolled patients.

Sections stained with Hematoxylin and Eosin were reviewed by two histopathologists (ATI and NYA). All the blocks were examined and the one which represented the tumor best (with no necrosis, no much mesenchymal tissue) was selected for the study.

The tumors were divided according to anatomical location based on embryonic location of the colorectum into three categories namely: right colon, left colon, and rectum. [12] The histological grading based on reviewing H and E stained representative slides was labeled as grade G1 for well differentiated, G2 for moderately differentiated, and G3 for poorly differentiated tumors. [13] Staging was performed according to American Joint Committee on cancer (AJCC) and the Union Internationale Contre Le Cancer (UICC), by grouping the various TNM components. [14]

DNA was extracted from formalin fixed paraffin blocks by QIAamp DNA FFPE Tissue Kit (QIAGEN, Austria). K-ras gene point mutation detection was performed with the K-ras strip assay kit (Viennalab Diagnostic GmbH, Austria) which utilizes a mutant enriched PCR technique and reverse hybridization. [15],[16] The DNA extracted was amplified with specific biotinylated primers using a thermocycler (ABI-USA) and the amplification program was Pre-PCR 94°C, following by 35 cycles of 94° C 1 min, 70° C 50 sec, 56° C 50 sec, 60°C 1 min, then by final extension at 60° C of 3 min. Following amplification the amplicons were reverse hybridized according to manufacturer's instructions, to readymade strips containing allele-specific oligonucleotide probes immobilized as an array of parallel lines to detect 10 different K-ras mutations, eight in codons 12 (GGT>GCT [Gly>Ala]; GGT>CGT [Gly>Arg]; GGT>GAT [Gly>Asp]; GGT>TGT [Gly>Cys]; GGT>ATT [Gly>Ile]; GGT>CTT [Gly>Leu]; GGT>AGT [Gly>Ser] and GGT>GTT [Gly>Val]), and two in codon 13 (GGC>GAC [Gly>Asp] and GGC>TGC [Gly>Cys]).

Statistical analysis utilized the Chi squared test and Mann Whitney U test when required. P<0.05 was considered significant.

 Results



Patient's characteristics

The 50 patients enrolled in the current study had a mean age (+ SD) of 55.4 + 15.25 years (median 59 years). They included 27 males and 23 females (M:F ratio of 1.2:1). The main clinico-pathological characters of the enrolled patients are outlined in [Table 1].{Table 1}

K-ras mutational status

Out of the 50 enrolled patients, 24 (48%) had K-ras mutations. A total of 29 mutations were identified in the tumors of the latter 24 patients (20/24 tumors had single mutations, 3/24 had double mutations and 1/24 had triple mutations). The most frequently encountered mutations were the G>T transversions and G>A transitions (41.4% each) followed by the G>C transversions at 17.2%. Codon 12 mutations constituted 89.7%, while codon 13 the remaining 10.3%. The most frequent mutation constituting 31% was GGT>GTT (Gly>Val), followed by GGT>GAT (Gly>Asp) at 24.1%, both of codon 12. Other less frequent codon 12 mutations were GGT>GCT (Gly>Ala), GGT>TGT (Gly>Cys) and GGT>AGT (Gly>Ser) at 17.2, 10.3 and 6.9% respectively. All three codon 13 mutations identified in this study were GGC>GAC (Gly>Asp). While the other codon 13 mutation which was screened for, namely GGC>TGC (Gly>Cys) was not detected in any of the 50 CRC tumors. [Table 2] details the mutations identified and their respective amino acid substitutions. The double mutations seen in three patients were all in codon 12. The triple mutations detected concomitantly in one patient were two in codon 12 and one in codon 13. All those with multiple mutations were reconfirmed by repeating the test, and in all four cases the results were unequivocal and clear cut.{Table 2}

There were no significant associations of age, gender, tumor location or histology, grading, staging, or lympho-vascular invasion with K-ras mutation status [Table 1].

 Discussion



The overall observed frequency of K-ras oncogene mutations in the 50 sporadic colorectal carcinoma Iraqi patients enrolled in the current study was 48%. The latter figure lies within the rates reported worldwide, which varies from 18.4% to 60%. [9],[8],[17],[18],[19],[20] The variations in the observed frequencies of K-ras mutations in different studies could be attributed to several factors, including the specificity and sensitivity of the method used for the detection, the number, and the type of the specific mutations tested, numbers and bias in patients' selection, and environmental factors. The latter has a major impact in different populations with different life styles and dietary habits and variable exposures to carcinogens. [17],[18],[19],[20]

The current study revealed that the G>T transversions and the G>A transitions were equal in frequency and constituted 41.4% each. The latter finding is similar to that reported from neighboring Iran in sporadic CRC, but is in contrast to the bulk of studies from developed countries [7],[8],[21],[22],[23],[24],[25],[26] and some developing countries, [18] where G>A transitions far exceeded G>T transversions. The variation in the pattern of specific alteration observed, i.e. G>A transitions versus G>T transversions, could be due to the difference in diet and/or other life style factors. It is believed that G>A transitions may either be induced by N-nitroso compound [27] or due to misreplication of unrepaired endogenously produced O 6 -Methylguanine from faulty S-adenosylmethionine methylation. [28] G>T transversions, however are likely to be induced by carcinogenic agents like the polycyclic aromatic hydrocarbons. [29] The similarities in the mutational spectrum observed among our patients and those from Iran may be related to similar dietary habits and probably local carcinogens in these two neighboring countries.

GGT>GTT (Gly>Val) in codon 12 was found in the current study to be the most frequent mutation accounting for around a third of all mutations detected. This has been shared by few other studies particularly those from Yugoslavia and Iran. [19],[30] However, most other studies and in contrast to ours showed predominance of the GGT>GAT (Gly>Asp) in codon 12, accounting of up to half the mutations in some. [7],[18],[31] [Table 3] The Gly>Val mutation is actually the most frequent K-ras mutation in pancreatic cancer. [10] In colorectal carcinoma, it has been associated with poor prognosis and metastasis. [26],[32] {Table 3}

Consistent with other studies worldwide, our study showed that K-ras codon 13 mutations were less frequent than those of codon 12 (although to variable degrees). [7],[8],[10],[11],[18],[19],[25],[30],[31] Furthermore, all three codon 13 mutations identified were GGC>GAC (Gly>Asp) mutations, which is consistent with the bulk of the literature showing that this mutation is the predominant in codon 13 in CRC [Table 3]. This amino acid substitution in codon 13 has been linked to reduced survival rate, less stable cancer and disease relapse and death. [20],[21]

Multiple K-ras mutations were detected in four cases (8%) in the current study. Multiple mutations are not unusual in CRC and a recent review by Macedo and coworkers (2011) reported that its frequency among CRC varies between 0.2% and 16.4%. [33] Multiple mutations may reflect increased genetic instability in cells that progressively acquire mutations or the presence of a heterogeneous group of neoplastic cells inside the tumor. [33] Analyzing several DNA samples taken from different parts of tumor might have provided useful information relevant to the latter observation. Although we confirmed all cases with multiple mutations by repeating the testing, and mutant enriched PCR used in the current study is validated as highly sensitive and specific, [15],[16] it may be argued that reaffirming results by another method may have been of added value. It is important to note here that the true clinical impact of multiple K-ras mutations is unknown, mainly due to the scarcity of such data.

The absence of significant correlation of K-ras status with age, gender, and tumor site of CRC patients is shared by most other researchers. [7],[26],[31] The current study did not find a significant association with TNM staging, which is contrary to some studies. [18],[20],[34] but is consistent with many others. [7],[24],[26],[31],[35],[36] This observation is consistent with the hypothesis that K-ras mutations are preferentially early events in the process of CRC tumorigenesis, but could appear at any stage depending on exposure to appropriate carcinogenic agent(s). [31],[37] An interesting though insignificant observation of the current study is that more differentiated tumors were more frequently harboring K-ras mutations than poorly differentiated ones. These results are in agreement with several previous studies [34],[35],[36],[38] and is consistent with the notion that the differentiation program of tumor tissue is not under the control of K-ras oncogene. [37]

The significance of knowing the K-ras mutational status in CRC patients has recently been augmented by the finding that it is the one of the most important predictors of resistance to targeted therapy using Epidermal Growth factor receptor (EGFR1) tyrosine kinase inhibitors. [10] The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, expressed constitutively throughout the body and found on many epithelial tissues, plays an important role in tumorigenesis and tumor progression of colorectal cancer. As a result, the EGFR has evolved as a relevant target in the treatment of metastatic CRC. K-ras serves as a mediator between extracellular ligand binding and intracellular transduction of signals from the EGFR to the nucleus. [39] Thus, mutations in K-ras would interrupt this pathway rendering EGFR inhibitors ineffective. [40]

In conclusion, this study has shown that K-ras mutations are common among Iraqi sporadic CRC patients with frequencies comparable to those reported in developed countries, however the spectrum of these mutations was relatively different, with higher relative frequencies of G>T transversions and thus codon 12 Gly>Val amino acid substitutions. [41] These observations are most likely related to differences in diet and local carcinogen exposures. Further studies on K-ras mutation associations among Iraqi CRC patients are needed, including focusing on any potential carcinogens that maybe present in diet or environment, with larger (and thus more representatives) numbers of patients and including survival data.

 Acknowledgment



We acknowledge with gratitude the technical support offered by Dalal Yousif BSc and Dilan Jassim BSc of Scientific Research center of University of Dohuk.

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