Application and evaluation of KRAS gene mutation method for colorectal cancer

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Targeting drugs such as cetuximab and panitumumab have been widely used in clinic as effective therapeutic drugs for colorectal cancer. Clinical data show that patients with KRAS mutations have no significant effect on this monoclonal antibody drug, and only wild-type patients can benefit from it. Therefore, the KRAS gene mutation status is clinically regarded as an important therapeutic marker, which has a strong correlation with the prognosis and treatment effect of colorectal cancer. The 2009 National Cancer Comprehensive Network (NCCN) Colorectal Cancer Clinical Practice Guidelines stipulates that all patients with metastatic colorectal cancer must detect KRAS gene mutation status, and only KRAS wild type is recommended to receive EGFR targeted therapy. In the same year, the American Society of Clinical Oncology (ASCO) also issued the same clinical treatment  recommendations as a molecular marker for tumor targeted therapy, which shows its important guiding significance. At present, KRAS genetic testing has been widely carried out clinically. We mainly evaluate the domestic KRAS gene mutation detection methods for reference in clinical selection.

1. The positive rate of KRAS gene mutation in colorectal cancer

In colorectal cancer, the mutation rate of the KRAS gene is as high as 35% to 45%, and the high-risk mutation site is codons 12 and 13 on exon 2, and there are still rare ones such as 61 and 146. Mutation site. There are many detection methods for KRAS gene mutations, including direct sequencing, high resolution melting curve analysis (HRM), pyrosequencing, quantitative PCR, mutation amplification block system (amplinc atio)nrefractorymutation system (ARMS), restriction fragment length polymorphism (RFLP), polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-singlestrand confomation polymorphism (PCR-SSCP), co-amplification at lower denaturation temperatur PCR (COLD-PCR) and high-performance liquid chromatography analysis, etc.

2. Evaluation of KRAS mutation detection methods

1. Direct sequencing method: It is the most classic method for detecting KRAS gene mutations, and it is also the gold standard for detecting gene mutations. The direct sequencing method based on the principle of dideoxy sequencing can most intuitively show the change of gene sequence in the form of base peak map. The detection type is more comprehensive, and it is also the earliest applied mutation detection method. Despite the emergence of new generation sequencing platforms, scholars at home and abroad still use the results of direct sequencing as a scale to measure and determine the reliability of the new method. Gao Jing et al. Applied direct sequencing to the detection of KRAS and BRAF gene mutations in 966 patients with colorectal cancer. This is also the analysis of the KRAS gene mutation with the largest domestic sample reported in the literature. Ling Yun and others believe that the direct sequencing method is the most direct and effective detection method for understanding the mutation status of each gene, which can clarify the type of mutation, especially for the detection of unknown mutations. Although the sensitivity of this method is relatively low, it can be improved by methods such as microdissection to enrich tumor cells. The direct sequencing method has also been applied to the KRAS detection of larger sample sizes in other domestic research groups. However, lower sensitivity is the biggest disadvantage of direct sequencing. Judging from the results reported in China, the mutation detection rate by direct sequencing is not low. Liu Xiaojing et al. Compared direct sequencing and peptide nucleic acid clamp PCR (PNA-PCR) and found that 43 cases of KRAS gene mutations were detected by direct sequencing. In addition to these mutations, PNA-PCR was also detected by direct sequencing. Ten mutations were found in the wild type, and suggestions were made to determine the wild type patients by PCR and the direct sequencing method to determine the mutant patients. Qiu Tian et al. Detected 131 colorectal cancer specimens by fluorescent PCR-optimized oligonucleotide probe method and direct sequencing method, and the positive rates of KRAS gene mutations were 41.2% (54/131) and 40.5% (53/131) ). Bai Dongyu also discussed the detection sensitivity of different methods. Of the 200 colorectal cancer patients, 63 were detected by RT-qPCR mutations, and the mutation detection rate was 31.5%; 169 samples were successfully sequenced by direct sequencing 50 cases of mutation, mutation detection rate 29.6%. Although the direct sequencing method can accurately, objectively and specifically detect the KRAS gene mutation status, its shortcomings such as high technical requirements, complicated operation procedures, easy to cause cross-contamination, and time-consuming and laborious interpretation of the results are also very obvious. Often there is no sequencing equipment, and the specimen needs to be sent to the corresponding company for testing, which takes a long time and has a high cost, so it has great limitations. When extracting DNA, you must strictly control the specimen, scrape the HE tumor section of the tumor cell rich area, try to avoid the non-tumor area, necrosis area, make the tumor component account for a higher proportion, so as to minimize the occurrence of gene mutation detection by direct sequencing The possibility of false negatives.

Pyrosequencing method:

Pyrosequencing method is also a more convenient method for KRAS gene mutation detection in terms of sequencing sensitivity, detection cost and time to report. The repeatability of this method is better. According to the obtained peak map The quantitative study of the mutation frequency of a certain site and the comparison between the mutation frequencies of different sites are clear at a glance. In recent years, Ogino et al., Hutchins et al. Have used pyrosequencing technology to test KRAS mutations in patients with large samples of colorectal cancer. The results indicate that pyrosequencing technology is a powerful tool for screening patients for targeted therapy. Tumor molecular diagnosis has broad application prospects. Domestic scholars have also used pyrosequencing technology to clinically detect KRAS mutations in colorectal cancer, with good accuracy and reliability. This method has better specificity and higher sensitivity. SundstrÖm et al. Compared allelic-specific PCR and pyrosequencing in clinical applications and found that in 314 cases of KRAS mutations in colorectal cancer patients, the specificity of pyrosequencing was superior to that of alleles. PCR, and has good sensitivity to tissues with low tumor cell content. Dilute the proportion of tumor cells to 1.25% to 2.5%. Pyrosequencing can still detect mutation signals. When the minimum content of mutant alleles in the sample needs to reach 20% to be detected by Sanger sequencing, it can be detected by HRM method when it reaches 10%, and for pyrosequencing only mutations can be detected by 5%. Alleles. We used pyrosequencing to detect KRAS mutations in 717 patients with colorectal cancer and found that the frequency of KRAS mutations was 40.9%. The mutation rate of codon 12 was 30.1%, the mutation rate of codon 13 was 9.8%, and the mutation rate of codon 61 was 1.0%. We enriched the tissues with higher tumor content by manual microdissection before testing, making the results more reliable. The method has good sensitivity and specificity, and is easy to develop in clinical practice. The disadvantage of pyrosequencing is the high cost of detection, and the process of preparing single-stranded DNA for sequencing samples is cumbersome. In the future, pyrosequencing can be devoted to the development of technology for direct detection of double-stranded PCR products, which will greatly simplify the operation. And effectively reduce the cost of sequencing to achieve comprehensive promotion of clinical testing.

3. ARMS method:

This technology uses primers to distinguish between wild-type and mutant genes, wh
ich has been reported as early as the 1980s. The biggest advantage of this method is that it has a sensitivity of up to 1.0% and can detect mutant genes in samples as low as 1.0%. In design, the length of the target product can be shortened to the greatest extent, and the problem that accurate detection results cannot be obtained because most of the DNA extracted from the paraffin-embedded tissue specimen is fragmented. This technology combines the real-time PCR platform to achieve closed-tube operation during amplification. The operation is simple and does not require post-processing of the product, which can avoid the contamination of the amplified product to the greatest extent. At present, the scorpion-ARMS method combining scorpion probe and amplification block mutation system is more commonly used in the world. The combination of the two technologies can maximize the sensitivity and specificity of both sides. Gao Jie et al. Used this method to detect the KRAS gene mutation status in 167 patients with colorectal cancer, suggesting that this method is reliable and accurate. Wang Hui et al. Also used ARMS to detect KRAS mutations in 151 cases of formaldehyde-fixed and paraffin-embedded tissues. In the United States, the COBAS kit (Roche) approved by the FDA for clinical testing of KRAS and the Therascreen RGQ kit (Qiagen) certified by the European Union In Vitro Diagnostics (CE-IVD) all use the ARMS principle. Among the common methods, the ARMS method is the most sensitive and the cost is relatively reasonable. Therefore, a large part of the clinical detection of KRAS genes at home and abroad are using the ARMS method, but because the method is based on PCR technology, its shortcoming is that it can only be detected Known site mutations.

4. Real-time fluorescence quantitative PCR method:

It is a PCR-based detection method to determine the mutation by Ct value. It has the advantages of strong specificity, high sensitivity, accurate quantification, easy operation, and fully closed reaction. Many experimental groups have adopted this method for the detection of KRAS mutations in colorectal cancer. Compared with the direct sequencing method, quantitative PCR occupies a greater advantage in sensitivity. Most scholars comparing the two methods believe that quantitative PCR is more sensitive. Liu Wei et al. Used two methods to make a detailed analysis of the detection results of 280 cases of colorectal cancer KRAS gene mutations, 94 cases of KRAS gene sequencing mutations, the positive rate was 33.57% (94/280), of which, real-time fluorescence quantitative PCR was positive 91 cases had a sensitivity of 96.8% (91/94). Of the 186 gene sequencing wild-type cases, 184 were negative by real-time quantitative PCR, with a specificity of 98.9% (184/186). The coincidence rate between real-time fluorescence quantitative PCR method and direct gene sequencing method was 98.2%. In the two detection methods, the positive and negative coincidence rates of each mutation site were above 90%, and the coincidence rate of four sites reached 100%. The detection results of the two methods were highly consistent, indicating fluorescent quantitative PCR It is a more reliable method for mutation detection. However, PCR-based methods need to design primers and probes based on known mutation types, so all possible mutations cannot be detected, and only specific sites can be detected. If a certain site is not included in the detection range of the kit, even if there is actually a mutation, the kit result is still negative. In addition, although the sensitivity of quantitative PCR is high, whether there are false positives still needs to be verified by DNA sequencing technology, or retrospective and prospective clinical experiments with large sample sizes to confirm the correlation between KRAS mutation status and the efficacy of targeted drugs . Therefore, the high sensitivity of mutation detection should not be pursued blindly, while the specificity and accuracy of detection should be ignored. Under different laboratory conditions, the optimal method for mutation detection in specimens may also be different. For specimens with a higher proportion of mutations, Sanger sequencing method has a higher accuracy in detecting gene mutations, while for specimens with a lower proportion of mutations, Sanger sequencing method False negatives may occur, and the detection method using fluorescent PCR as the technical platform can be characterized by high sensitivity.

5. HRM method:

It is one of the more commonly used gene detection methods in recent years. It has the advantages of simple, fast, sensitive, and single tube to avoid pollution. In order to explore the feasibility of its use in clinical testing, Liu Liqin and others used the HRM method to detect KRAS gene mutations in 64 patients with colorectal cancer, and then used direct sequencing to verify the results. The results of HRM and direct sequencing are found to be consistent. Compared with direct sequencing, the detection of KRAS gene mutations by HRM is simple and accurate, suggesting that it is a reliable method suitable for clinical testing. Chen Zhihong et al. Used the HRM method to test a series of mixed samples containing different proportions of KRAS mutant plasmids to evaluate their sensitivity. It was found that the proportion of plasmid mutations in the mixed samples was 10%, and the sensitivity reached 10%. Subsequently, the method was used to detect KRAS gene mutations in 60 colorectal cancer tissue samples. Compared with the direct sequencing method, the sensitivity of the HRM method was 100%, and the specificity was 96% (43/45). The disadvantage of the HRM method is that it is impossible to accurately provide the specific mutation type and which codon is mutated. If an abnormality is found on the melting curve, sequencing method is needed to determine the mutation type. The Harlé research group used 156 cases of colorectal cancer tissue to compare fluorescent PCR, ARMS and HRM methods. The results suggest that although the three methods are suitable for clinical testing, the reliability of HRM is not as good as the other two methods.

6. Other methods:

In addition to the above mentioned methods, other detection methods have their own advantages and disadvantages in application, such as PCR-SSCP, high performance liquid chromatography, fluorescent PCR-optimized oligonucleotide probe method , Method of nested PCR and ARMS combination, COLD-PCR method, etc. High performance liquid chromatography has strong specificity, but the demand for samples is large; PCR-SSCP is low in cost and economical, but the operation is complicated; the mutation detection technology based on fluorescent PCR has strong specificity, high sensitivity, and accurate quantitative , Easy operation, fully blocked reaction and other advantages, but all need to design primers and probes according to the known mutation type, so only specific sites can be detected, and all possible mutations cannot be detected.

3. Summary

In summary, because the mutation sites and detection methods in different laboratories are not uniform, the size of the tumor specimens analyzed and the quality of DNA extraction are also uneven, resulting in the existence of large or small experimental results between laboratories Differences, the standardization of KRAS gene mutation detection has become a clinical detection issue of concern in various countries. At present, there are many methods to detect mutations in the KRAS gene. The sensitivity from high to low is ARMS, pyrosequencing, HRM, real-time quantitative PCR, and direct sequencing. From the clinical reality, low sensitivity is not conducive to clinical treatment, but too sensitive methods will cause the detection specificity to decline, and unnecessary false positive results may occur and affect the patient’s subsequent medication regimen. Considering the above aspects, combined with the method approved by FDA, the ARMS method is recommended. Of course, from a market perspective, molecular diagnostics should not emphasize me
thods, but focus on the final correct results. Different laboratories can adopt appropriate testing methods according to the actual situation, but only if they have good operator qualifications and internal quality control systems. Under the current domestic laboratory environmental conditions, it is necessary to carry out testing in a standardized PCR laboratory and participate in domestic and international inter-room quality control activities to ensure reliable laboratory testing quality. Standardized management is a necessary condition to ensure constant results. In China, there is an urgent need to unify and standardize the clinical testing of the KRAS gene, and to generate a standardized and standardized testing program according to different needs, and this program can be extended to the detection of BRAF, PIK23450_3CA, EGFR and other genes to promote clinical molecular pathology testing. 


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