|Year : 2016 | Volume
| Issue : 4 | Page : 1272-1277
Modified mismatch polymerase chain reaction-restriction fragment length polymorphism detected mutations in codon 12 and 13 of exon 2 of K-ras gene in colorectal cancer patients and its association with liver metastases: Data from a South Asian country
Fathima Dhilhani Mohamed Faleel1, M. I. M. De Zoysa2, M. D. S. Lokuhetti3, Y. I. N. S. Gunawardena4, Vishvanath Naduviladath Chandrasekharan1, Ranil Samantha Dassanayake1
1 Department of Chemistry, Faculty of Science, University of Colombo, Colombo, Sri Lanka
2 Department of Surgery, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka
3 Department of Pathology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka
4 Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka
|Date of Web Publication||7-Feb-2017|
Ranil Samantha Dassanayake
Department of Chemistry, Faculty of Science, University of Colombo, Colombo
Source of Support: None, Conflict of Interest: None
Aim: Mutations in K-ras codon 12 and 13 of exon 2 are known to affect prognosis and impart resistance to anti-epidermal growth factor monoclonal antibody therapy in colorectal carcinoma (CRC). Our aim was to investigate the utility value of modified mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to detect mutation in K-ras codons of CRC patients and to relate the mutational status to liver metastasis.
Methodology: Mismatch PCR-RFLP was developed to detect K-ras mutations in DNA isolated from paraffinized tumor tissue of thirty CRC patients. All patients had 5 year follow-up data to detect liver metastasis. Cross-tabulations were generated between K-ras mutations and the metastatic status. The Chi-square test was used to indicate statistical significance of the association.
Results: Of the 30 CRC patients investigated, K-ras mutations of codons 12 and/or 13 of exon 2 were detected in 14 (46.6%). Meanwhile, 13 patients (43.3%) were observed to have developed liver metastases. There was a significant association between the presence of the K-ras mutation in codon 12 and the occurrence of liver metastasis (χ2 = 4.693, P = 0.030) on the contrary to the mutation in codon 13 to which such occurrence of liver metastases was not seen (χ2 = 1.884, P = 0.169).
Conclusion: Codon 12 of exon 2 of K--ras gene detected by modified mismatch PCR-RFLP assay is significantly associated with liver metastasis in CRC patients during the first 5 years after surgery. Thus, modified mismatch PCR-RFLP protocol is a suitable method in this setting to detect K-ras gene mutations predicting liver metastasis in CRC patients.
Keywords: Colorectal cancer, epidermal growth factor, K-ras, mismatch polymerase chain reaction-restriction fragment length polymorphism, mutations
|How to cite this article:|
Faleel FD, Zoysa MD, Lokuhetti M, Gunawardena Y, Chandrasekharan VN, Dassanayake RS. Modified mismatch polymerase chain reaction-restriction fragment length polymorphism detected mutations in codon 12 and 13 of exon 2 of K-ras gene in colorectal cancer patients and its association with liver metastases: Data from a South Asian country. J Can Res Ther 2016;12:1272-7
|How to cite this URL:|
Faleel FD, Zoysa MD, Lokuhetti M, Gunawardena Y, Chandrasekharan VN, Dassanayake RS. Modified mismatch polymerase chain reaction-restriction fragment length polymorphism detected mutations in codon 12 and 13 of exon 2 of K-ras gene in colorectal cancer patients and its association with liver metastases: Data from a South Asian country. J Can Res Ther [serial online] 2016 [cited 2021 May 7];12:1272-7. Available from: https://www.cancerjournal.net/text.asp?2016/12/4/1272/187294
| > Introduction|| |
Epidermal growth factor (EGFR) is a transmembrane cell surface receptor which is found in most epithelial tissues. It is activated by different ligands that initiate various signaling pathways including RAS-MAPK (mitogen-activated protein pathway) which influences gene transcription, cell cycle progression, cell proliferation and survival, adhesion, angiogenesis, migration, and invasion., EGFR is overexpressed in approximately 85% of colorectal carcinomas (CRC); hence, EGFR has been identified as a therapeutic target in CRC. Currently, two classes of EGFR monoclonal antibody (mAb) inhibitors, cetuximab and panitumumab, are used for the treatment of metastatic CRC (mCRC).,, Although EGFR is overexpressed in a large majority of CRC patients, only a subset of patients (8–23%) show the expected result for mAb therapy. Therefore, it is important to identify reliable biomarkers to select appropriate patients for anti-EGFR therapy. K-ras (Kirsten rat sarcoma viral oncogene homolog) is a downstream signaling molecule of the EGFR signaling pathway, which helps to personalize anti-EGFR therapy in CRC.,
K-ras is a RAS family of proto-oncogene, encoding a protein that possesses intrinsic GTPase activity. K-ras mutations are observed in approximately 35–40% of CRC. The K-ras mutations arise during early stage CRC and mostly occur in codons 12 (80%) and 13 (20%) of exon 2 of the K-ras gene. In addition, approximately 1% of mutations have been reported in codons 61 and 146. Clinical evidence shows that only patients who have the wild-type K-ras gene benefit from anti-EGFR mAb treatment. As this therapy is extremely expensive (costing approximately $80,000–100,000/year) and has potentially significant side effects, the national comprehensive cancer network and American society of clinical oncology guidelines recommended that all tumors with metastases should be tested for mutations in codon 12 and 13 of exon 2 to provide rational and beneficial use of EGFR-targeted drugs and also to prevent unnecessary toxicities, treatment delays, and to reduce health-care costs.,,,,
A variety of molecular methods such as real-time polymerase chain reaction (Q-PCR), allele-specific PCR assays, pyrosequencing, single-strand conformational polymorphism technique, and amplification refractory mutation system (ARMS) have been developed to screen mutations in K-ras. However, these methods are either costly or less reliable and are not practiced in resource-limited settings. The use of a molecular method to identify mutations in K-ras is recommended to personalized therapy in CRC. Therefore, the development of a screening method which is affordable is required. The main objective of this study is to develop an affordable screening method which is also simple, sensitive, reliable, and effective to detect the presence or absence of mutations in codon 12 and 13 of exon 2 of K-ras in CRC patients using limited resources. Further, this is the first study to ascertain the association between the mutations in codons 12 and 13 of exon 2 of K-ras gene with liver metastases in a cohort of Sri Lankan colorectal cancer patients.
| > Methodology|| |
Tissue sample collection
Thirty patients with CRC, who had undergone segmental resection followed with adjuvant chemotherapy were selected from the Faculty of Medicine, University of Colombo, Sri Lanka. These were CRC patients followed up for 5 years after surgery and adjuvant chemotherapy without receiving anti-EGFR therapy at the Department of Surgery, University of Colombo. The follow-up visits included clinical assessment, assessment of CEA levels, abdominal ultrasonography, and colonoscopy. Formalin-fixed paraffin-embedded (FFPE) tissue blocks of these patients were obtained from the Department of Pathology, University of Colombo, Sri Lanka. The study was approved by the Ethical Review Committee of the Faculty of Medicine, University of Kelaniya, Sri Lanka.
The paraffin-embedded sections were stained with hematoxylin and eosin for histopathological identification of the tumor-bearing area in tissue blocks. Twelve slices of 10 μm sections cut from each selected FFPE tissue block were subjected to paraffin removal process. DNA extraction was carried out using CEYGENGenoSpin GTM Genomic DNA extraction kit according to the manufacturer's instruction.
Evaluation of extracted genomic DNA
DNA concentration and purity were determined by measuring absorbance at 260 nm, 320 nm, and 280 nm via spectrophotometer. The quality of DNA was analyzed by DNA electrophoresis in 1% agarose gel stained with 0.5 μg/ml EtBr.
Primer designing for mismatch polymerase chain reaction-restriction fragment length polymorphism
Primer designing was carried out using full-length sequence of K-ras (GenBank acc. No. NG_007524.1) using OLIGOANALYSER web-based tool (http://sg.idtdna.com/analyzer/applications/oligoanalyzer) setting parameters to standard concentrations (0.2 μM Oligo, 50 mM Na +, 1.5 mM Mg 2+, and 0.2 mM dNTP). Restriction enzymes were selected subsequently using web-based tool, Enzyme Finder of New England Biolabs (https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder). In primer synthesis, mismatched nucleotides were introduced to forward primers to generate restriction site in PCR, whereas reverse primers were designed to have the same restriction site [Table 1].
|Table 1: Polymerase chain reaction-restriction fragment length polymorphism primers|
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Polymerase chain reaction-restriction fragment length polymorphism
PCR reaction was performed in 25 µl containing 80–100 ng of genomic DNA, 0.2 mM of dNTP, 1.5 mM MgCl2,1X colorless Go Taq ® DNA polymerase PCR buffer, 2.0 units of colorless Go Taq ® DNA polymerase (Promega), and 0.8 µM of forward and reverse primers [Table 1]. PCR cyclic parameters for codon 12 were initial denaturation for at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30s, primer annealing at 55°C for 30s, primer extension at 72°C for 30s, and final extension at 72°C for 6 min. For codon 13, the PCR conditions were initial denaturation at 95°C for 11 min, denaturation at 95°C for 30s, primer annealing at 56°C for 30s, primer extension at 72°C for 30s, followed by 35 cycles, and final extension at 72°C for 5 min. Custom synthesized DNA (GenScript, USA) containing mutations in codons 12 and 13 separately cloned into pUC19 vector were used as positive controls. The DNA obtained from clinically identified healthy person (wild-type DNA) was used as negative control, and the PCR mix without DNA was used as contamination controls. The PCR products were run in 2% agarose gel and visualized under UV-light before restriction fragment length polymorphism (RFLP) analysis. A fraction of PCR product (~6 µl) was digested with 5 unit of BstN l at 60°C and Bgl l at 37°C in 20 µl volume for 1 h to identify codon 12 and codon 13 mutations, respectively. Resultant mixture was separated by 12% native PAGE and then stained by EtBr (50 µl/ml) before visualization.
Cross-tabulations were generated between the K-ras mutations and the metastatic status. The Chi-square test was used to indicate statistical significance of the association.
| > Results|| |
In this study, mismatch primers were designed in a way that fabricates restriction sites for the wild type but not for the mutant type DNA in PCR. The banding profiles obtained following cleavage of PCR products resulting from mismatch primers were used to interpret the presence or absence of mutations in codons 12 and 13 of the K-ras gene. In PCR, the presence of mutation in the codon 12 of the K-ras gene eliminates the restriction site BstN l, as this requires the recognition sequence 3'….CCWGG….5' in the template DNA to detect the codon 12 mutations (GGT > XGT or GGT > GXT, and rarely GXX; X being the mutated nucleotide). Therefore, the mutant type for codon 12 results in a profile of two bands (140 bp and 20 bp), whereas the wild-type results in a profile of three bands (26 bp, 114 bp, and 20 bp). Similarly, in the presence of mutation in the codon 13 (has three reported mutations – GGT > XGT or GGT > GXT, rarely GXX; X is mutated nucleotide) of the K-ras gene, the restriction site for Bgl l (5'…GCCNNNNNGGC…3') is eliminated resulting in a profile of two bands for mutants (100 bp and 18 bp) and three bands (29 bp, 71 bp, and 18 bp) for wild type. However, because tumors generally contain heterogeneous cell populations, in terms of mutational status (wild type and mutant type K-ras), the presence of mutation is indicated by four bands (140 bp, 114 bp, 26 bp, and 20 bp for codon 12; 100 bp, 71 bp, 29 bp, and 18 bp for codon 13; [Figure 1]).
|Figure 1: Amplification of BstNl and Bgll primers create 160bp polymerase chain reaction product with BstNl restriction site to detect codon 12 mutations (a) and 118 bp polymerase chain reaction products with Bgll restriction site for codon 13 mutations (b), respectively. For wild type, KRAS gene restriction digestion generates three bands, but mutated gene remains intact at the first restriction site position resulting two bands. Amplification of reverse primer will create second restriction site at mutant and wild-type allele where both types can be cleaved and create extra fragments of 18 bp and 20 bp for codons 13 and codon 12, respectively|
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BstN I restriction digestion of the PCR products of KRAS 8 and KRAS 13 gave a banding profile of 114 bp, 26 bp, and 20 bp that corresponds to wild type samples having no mutations in K-ras [Figure 2]. However, samples KRAS P (positive control), 1, 4, and 5 elicited a profile having four bands (140 bp, 114 bp, 26 bp, and 20 bp) indicating that these samples are heterogeneous for codon 12, containing K-ras mutations. The sample KRASx obtained from a healthy person was used as a negative control and resulted in three bands as expected. Similarly, upon Bgl l enzyme digestion [Figure 3] of PCR products of KRAS4 and KRASx resulted a banding profile of 71 bp, 29 bp, and 18 bp indicating the absence of K-ras mutations. However, the samples KRASp and KRAS4 yielded four banding profile (100 bp, 71 bp, 29 bp, and 18 bp) in similar analysis indicating these samples are heterogeneous for codon 13. The sample KRASp [Figure 1], Lane 8 and [Figure 2], Lane 2], confirmed to have mutation in codon 13, was used as a positive control in this study to prove the validity of the assay. KRAS1 elicited very faint bands corresponding to mutant type (140 bp) compared with the wild-type bands (114 bp) demonstrating the presence of a low percentage of former compared to the latter [Figure 2], Lane 2]. Two samples (KRAS4 and KRAS5) were identified to have mutations in both codon 12 and 13, which is a rare incident seen in the development of CRC [Table 2]. A total of 14 (46.6%) CRC patients revealed the presence of mutations. Of the thirty patients, 13 (43.3%) developed liver metastases. There was a significant association between the presence of a K-ras mutation in codon 12 and the occurrence of liver metastasis (χ2 = 4.434, P = 0.035). However, mutations in codon 13 were not significantly associated with liver metastasis (χ2 = 1.884, P = 0.169).
|Figure 2: Restriction digested product of sample DNA by BstNl restriction enzyme (Lane 1: 9-uncut product (160 bp), Lane 2: KRAS 1, Lane 3: Sample KRAS 8, Lane 4: KRAS x (wild-type DNA), Lane 5: KRAS 13, Lane 6: KRAS 4, Lane 7: KRAS 5, Lane 8: KRAS p (positive sample), Lane 10: Molecular weight marker 100 bp, 200 bp)|
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|Figure 3: Restriction digested product of sample DNA by Bgll (Lane 1: Uncut product (118 bp), Lane 2: KRAS p (positive sample), Lane 3: KRAS 4, Lane 4: KRAS-6, Lane 5: KRAS x [wild type DNA])|
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|Table 2: The name of samples collected from colorectal carcinoma patients and the presence or absence of mutations in codons 12 and 13 of exon 2 of K-ras gene of these samples|
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| > Discussion|| |
K-ras mutation is one of the primary mutations during the early stage of colorectal cancer. While various methods for screening K-ras mutations, including sequencing, Q-PCR, ARMS, and allele-specific-PCR, have been introduced, each method has strengths and drawbacks. Although sequencing is the gold standard method for genotype analysis, at least 20% of selected cell population should have mutations for the detection by this method. With the allele-specific method, false positives may result due to degradation of bases at the 3′ end of the primer, and only the known mutations can be detected using this method. Meanwhile, the cost incurred with Q-PCR is relatively high,,,, and this method is not available in many laboratories in limited-resource settings.
Therefore, in this study, a modified version of the method based on mismatch PCR-RFLP followed by PAGE analyses was developed to screen K-ras mutation. This method has been reported to be sufficiently sensitive and capable of accurately detecting mutations in the presence of a large amount of wild-type alleles ,, and amenable to the limited-resource setting to detect all known and unknown mutations in codons 12 and 13 of the K-ras gene. Generally, plasmid containing the insert of target restriction site has been used as exterior internal control. The plasmid insert normally competes with the target gene (K-ras) in co-amplification and affect the sensitivity of the samples having a small number of tumor cells., This effect was minimized in the modified method using a reverse primer designed with an appropriate restriction site as an internal control to serve in amplification of both the mutated and wild alleles unbiasedly in PCR. A mismatched PCR approach has been used previously to detect K-ras mutations in different populations., However, to the best of our knowledge, this is the first study that has been carried out on paraffin-embedded CRC tissue samples of South Asian patients using a modified version of mismatched PCR followed up prospectively for over 5 years.
Colorectal cancer is a major problem in developed countries. Even though Sri Lanka is a developing country, CRC is the fourth and seventh most prevalent cancer in men and women, respectively. In this study, we have identified 43% of total mutations in the K-ras gene in the Sri Lankan population, of which 75% of mutations were at codon 12 and 25% were at codon 13, which are comparable with previous studies from other settings.,
Most studies on the K-ras mutation have been done in Western countries. As such, there is a paucity of data from Asian countries. A recent study from Saudi Arabia showed K-ras mutations in 56% of the patients; 48.7% of the mutations were in codon 12. Conversely, a study from Pakistan using denaturing gradient gel electrophoresis, RFLP analysis, and nucleotide sequencing showed a K-ras mutation frequency of only 13%, with most of the mutations occurring at codons 12 and 13. However, these studies did not examine the long-term prognosis of their patients.
Of the 30 patients included in this study, 13 (43.3%) developed liver metastases during the 5-year follow-up period. There was a significant association between K-ras in codon 12 mutation and the occurrence of liver metastasis (P = 0.035) [Table 3]. A recent meta-analysis shows a significant difference between the survival of patients with normal and mutated K-ras gene; however, no statistic differences were found between either codon 12 or codon 13 mutations and prognosis.”
|Table 3: Chi-square tables K-ras mutations of codon 12, 13 and 12 and 13 combined|
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It has been shown that the presence of a K-ras mutation is indicative of high tolerance for chemotherapy and poor prognosis in CRC patients. All our CRC patients received first-line chemotherapy following surgical resection of their tumor. Those patients who had mutations in codon 12 or 13 showed a significant association with the development of liver metastasis. As all our patients received chemotherapy following surgical resection, it is possible that patients with mutations in codon 12 of the k-ras gene were resistant to chemotherapy.
The mismatch PCR-RFLP assay developed in this study is a reliable and sensitive method to screen for K-ras mutations in CRC patients. The occurrence of mutations in codons 12 and 13 revealed by this assay was comparable to previous studies reported., Finally, as the mismatch PCR-RFLP assay developed in this study is cost–effective, it can be implemented for detection of K-ras mutations in CRC patients at a lower cost. This technique is ideally suited for use in developing countries, especially in the South Asian region. Knowledge of the presence or absence of K-ras mutations will be helpful in making therapeutic decisions regarding anti-EGFR mAb therapy, in these resource-limited settings to achieve better therapeutic efficacy.
Financial support and sponsorship
Grant (AP/3/2011/CG/03) awarded to Prof. R.S. Dassanayake by the University of Colombo, Sri Lanka.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]