KRAS Gene Mutation in Patients with Primary Colorectal Cancer

Objective: KRAS mutation occurs in 30% to 50% of colorectal cancers. The aim of our study was to determine the frequency of KRAS mutations among patients with colorectal cancer; and the relationship with clinicopathologic features. Materials and Methods: 79 colorectal cancer cases at a hospital in Hai Phong of Vietnam were collected, including 45 colon cancer and 34 rectal cancer during January 2010 and July 2012. PCR amplification and DNA sequencing were used to detect mutations in exon 2 of KRAS gene. The study was based on informed consent and approval by the Ethics Committee of Viet Tiep Hospital. Results: KRAS mutation was found in 40.4% (225/557) colorectal cancer. All mutation locations were in codon 12. There was significant association (p < 0.05) between KRAS mutations and tumor size, tumor stage or metastatic stage. No significant association was observed between KRAS mutations and gender, tumor location, tumor grade or histologic presence of mucin (p>0.05). Conclusion: Determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers.


INTRODUCTION
Colorectal cancer (CC) is one of the most prevalent diseases in the world and the third leading cause of death among cancer types.According to the Union for ınternational Cancer Control (UıCC), there are approximately 1 million new cases of CC each year in the world and more than 500,000 people die from it each year [1].ıncidence rates across various regions and continents are different.ın Vietnam, CC is ranked fifth after stomach, lung, breast and nasopharynx cancers for mortality rates.Among gastrointestinal (gı) cancers, CC is ranked as the second most prevalent cancer, after stomach cancer [2].The mechanisms of initiation, development and progression of CC are attributed to gene modifications and accumulation of mutated genes in the mucosal cells of the colon.The gradual accumulation of genetic modifications often persists for many years (from 10 to 20 years), and this concurs with genetic and epidemiological studies that tumorigenesis progresses through many stages during that time [3].With the rapid development of the biomedical industry, it is now possible to accurately, thoroughly and quickly identify almost all major types of mutations in the most common tumor cells, such as in lung cancer, malignant lymphomas, and colorectal cancer.Some studies on gene-modified mouse models have confirmed the importance of targeting genes in many various cancers, including KRAS oncogene in colorectal cancer [4].From the above strategies, a new generation of anti-cancer drugs that accurately targets colon cancer cells can be developed-in a platform known as targeted therapy.ın Vietnam, there have been some studies on CC, but almost all of them have focused on clinical characteristics, endoscopic imaging, and histopathology.There have only been a few studies investigating mutations in cancer cells, in general, and in CC, in particular.ın this present study, we aim to determine the frequency of KRAS mutations among patients with CC; and the relationship with clinicopathologic features.

DNA Extraction
DNA was extracted from the patient's paraffin sample following the phenol/chloroform procedure after the precise selection of the cancerous tissue.Before extraction with phenol/chloroform, the paraffin was removed by xylene treatment.
DNA concentration and purity were measured on a Nano-Drop machine, and the DNA sample concentrations were quantified at OD 280/ OD260 (samples with purity ≥ 1.8 were used for further experiments).

Gene Sequencing
After gene amplification of the codon region 12, 13 of the KRAS gene using nested Polymerase Chain Reaction (PCR), the PCR products were purified from the agarose gel using Promega Wizard SV gel clean-up system (Promega, Madison, Wı, USA).The purified PCR products were sequenced using the BigDye terminator sequencing method (Applied Biosystems, Foster City, CA, USA).gene sequences were compared to wild-type KRAS gene sequences on geneBank (National Center for Biotechnology ınformation -NCBı).

RESULTS
Primary samples from 79 colorectal carcinoma patients were analyzed for KRAS gene mutations.Mutations of KRAS occurred in 58.2% (46/79) of CC.Mutated carcinomas were compared with non mutated carcinomas for sex, age, histological features and molecular characteristics (Table 1).Between subjects with KRAS mutation and KRAS wild type, there was no significant difference in age, sex, tumor location, histologic differentiation, and histologic presence of mucin.An association between KRAS mutation and tumor size (p=0.0072),tumor stage (T stage) (p=0.0179) or metastatic stage (M stage) (p=0.0442) was observed significantly.Figure 1 show the gene sequencing of a male 71-year-old patient with the mutation at codon 12.

DISSCUSION Frequency of KRAS Mutations Among Patients with CRC
The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancers, and KRAS mutational status testing has been highlighted in recent years.The most frequent mutations in this gene, point substitutions in codons 12 and 13, were validated as negative predictors of response to anti-epidermal growth factor receptor antibodies.Therefore, determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers [5].ın our study population, the overall mutational rate for KRAS was 58.2%, which seemed to be higher to those reported for other populations.Fu X et al [6], found KRAS exon 2 mutation rates were 37.7% in 5546 CRC patients diagnosed from 2010 to 2017.guo F et al [7], KRAS exon 2 mutations were identified in 42.2% in 353 CRC patients from two Chinese clinical centers.Hamzehzadeh l et [8], detected mutations in exon 2 (codons 12 and 13) of the KRAS and NRAS genes using high resolution melting analysis, ıntplex design and Sanger sequencing in 87 ıranian CRC patients.genomic DNA was isolated from fresh tissue samples of CRC patients.From 87 eligible cases, 51 were male and 36 were females.KRAS mutations in codons 12 and 13 were present in 28.7% of all analyzed CRCs.KRAS mutations were found in 37 (37.4%) of 99 patients in the report of ınoue Y et al [9].The genetic analyses of KRAS gene found mutations in 22 cases (45.3%) according to Dinu D et al [10].
As reported in 2011, the KRAS mutation frequencies in Asia, Europe, latin American were 24%, 36% and 40%, respectively (P < 0.0001) [11].ıt is unclear why a lower incidence is observed in Asian patients.
ın China, KRAS mutations were detected in 33.3% (30/90) of the CRC tumor samples using the nucleotide sequence analysis method [12].These results significantly correlated with the response rate and survival time of cetuximab-treated patients.The difference of mutation status may result from many aspects, such as the tissue, the percent of tumor cells, the extracted DNA quality, the testing methods and the testing target.

Relationship Between KRAS Gene Mutation and Clinicopathologic Features
The gene mutation and sites in the colon (p>0.05)[15].Monstein et al. (2004) showed that the percentage of cancer patients with KRAS mutation in the duodenum was 30% and in the colon was 44% [16].
According to a study by Zulhabri O. et al. (2012), the rates of KRAS mutations in the colon tumor sub-localizations were 37% in the right colon and 14% in the left colon (p>0.05)[17].Hence, our results are consistent with these studies that found no correlation between the KRAS mutagenic rate in cancer patients and colon tumor sub-localization.The rates of KRAS mutations in patients with tumor appearances were also evaluated.The mutations were found in 43.5% (20/46 cases) of ulcerated verrucous lesions, 26.1% (12/46 cases) of verrucous lesions, 13% (6/46 cases) of infiltrative lesions, and 8.7% (4/46 cases) of ulcerating lesions and infiltrative lesions.However, the differences in the rates were not significant (p>0.05).The results of our study are consistent with the results of Beranek M. et al. (1999) in that the rate of KRAS mutation in cancer patients were not related to tumor forms [15].Thus, there was no correlation between KRAS gene mutation with the tumor forms in the colon cancer patients.
Several studies have shown that the tumor size in combination with colon perimeter reflects tumor growth over time and is an independent prognostic factor for the patient's postoperative survival.
The results showed that the rate of KRAS mutagenic mutation in CC patients with different tumor sizes are different.The rates of KRAS mutation were found in 63.1% (29 cases) of tumors with size > 10 cm, in 32.6% (15 cases) of tumors with size ranging from 5-10 cm, and in 4.3% (2 cases) of tumors with size <5 cm.Thus, the rate of KRAS mutation in patients with CC patients gradually increased with tumor size; the differences were not statistically significant (p>0.05).
When studying the relationship between KRAS gene mutations in colon cancer patients with tumor invasion level (compared to the colon perimeter), the results showed that the rates of KRAS mutation in cancer patients were as follows: 63% The rate of KRAS mutation was also different among different malignancy grades but were also found to be non-significant (p>0.05).The KRAS mutations were detected in 73.9% of patients with colon tumors of low malignancy grade and 26.1% in patients with colon tumors of high malignancy grade (p>0.05).Thus, there was not any relation between KRAS mutation and histological types of tumors nor malignancy grades of the colon cancer.We also evaluated the relation of KRAS mutation with the invasion levels of tumors into the bowel wall.The mutations were seen in 35.0% of cases of muscle layer invasion, 60.0% of cases of serosa invasion, and 70.6% of cases of subserosa invasion (p<0.05).From these results, we can discern that there was a relation between KRAS gene mutation and tumor invasions in the colectoral carcinoma.The relationship between the rate of patients with KRAS mutation and their status of lymph node metastasis were also investigated.The results as shown in Table 1 showed that there were 50.0% of colectoral carcinoma patients with lymph node metastasis exhibiting KRAS mutation, while there were 60.6% colectoral carcinoma patients with no lymph node metastasis exhibiting KRAS mutation (p>0.05).Chang, M.Y. (2009) studied the KRAS mutation at codon 12 and 13 in colorectal cancer patients.Univariate analysis revealed a significant association between KRAS mutation at codon 12 and lymph node metastasis (p=0.048).Multivariate analysis was adjusted for tumor size, histologic grade, and lymph node metastasis; the analysis showed that KRAS mutations at codon 12 and 13 correlate We enrolled 79 CC patients at Viet Tiep Hospital of Hai Phong city.All patients were histologically diagnosed with CC during January 2010 and July 2012.

Table 1 .
Relationship of KRAS status with clinicopathological features Figure 1. gene sequencing of a male 71-year-old patient with the mutation at codon 12

Table 2 :
The rate of KRAS gene mutation in some studies