Establishment Of High Resolution Melting Analysis For Detecting KRAS And BRAF Gene Mutations In Colorectal Cancer And Lung Cancer

BACKGROUNDThe KRAS gene is located on human chromosome12, is the downstream molecules of epidermal growth factor receptor (EGFR) functional signal, which plays an important role in signal transduction of membrane receptor to adenylate cyclase. About30%of the human tumor cells appear Ras gene mutations, which KRAS gene has the biggest influence on human cancers. KRAS gene is associated with tumor pathogenesis and prognosis, mainly in the relatively high mutation rate of colorectal cancer, lung cancer and pancreatic cancer. KRAS mutations occurr early in malignant tumors and keep highly consistent in primary and metastatic sites. Generally, the KRAS gene status does not change with treatment. The most common way of KRAS gene mutation is point mutation.Study shows that90%of KRAS mutations occur at codons12and13in exon2, and three common mutations are GGT>GAT(G12D), GGT>GTT(G12V) and GGC>GAC(G13D). The KRAS mutation rate is20%～30% in lung cancer and27%～43%in colorectal cancer.BRAF gene is located on human chromosome7q34, about190kb, is an important transduction factor of the Ras-Raf-MEK-ERK signaling transduction pathway, which performs function mainly through serine and threonine protein kinase. The enzyme connects cell surface receptors, Ras proteins and intranuclear transcription factor by MEK and ERK, starting a variety of factors involved in various biological events in cells, such as cell growth, differentiation and apoptosis, etc. The study found that BRAF gene mutation rate is about15%in colorectal cancer, mostly occur in exon15(T1799A). BRAF mutations in colorectal cancer high, medium and low differentiation are significant difference, the lower of tumor differentiation degree, the higher of the BRAF mutation rate. It provide a new basis for colorectal cancer diagnosis and treatment.At present, the treatment of colorectal cancer and lung cancer preferred to surgical treatment aided by chemotherapy and radiotherapy. However, with the development of molecular biology and genomics, targeted therapy predictors and drugs has attracted more and more attention and applied to clinical treatment. In recent years, the highlight of colorectal cancer and lung cancer treatment is that the correlation between KRAS gene and the BRAF gene status and anti-EGFR curative effect has been found. The latest research shows that the effectiveness of targeted therapy drugs cetuximab influenced by KRAS and BRAF gene status. KRAS and BRAF mutation type can automatically activate the EGFR pathway and start the downstream signal transduction without receiving signal, so only patients with wild-type KRAS and BRAF tumors can benefit from anti-EGFR therapy. Therefore, The National Comprehensive Cancer Network (NCCN) recommended that it is best to detect KRAS and BRAF gene mutation before use of anti-EGFR monoclonal antibody treatment and just wild-type use it.Currently, there are many ways to detect gene mutations, such as DNA sequencing, Taqman probe, single strand conformation polymorphism, restriction fragment length polymorphism, pyrosequencing, denaturing high performance liquid chromatography, and amplification refractory mutation system, etc. These methods are high cost, complicated operation or low sensitivity, which is unfavorable for the clinical test. Now there is lack of gene mutation testing standard, therefore, it is particularly important to look for a inexpensive, high throughput, simple and accurate method and apply to clinical.High resolution melting curve analysis (HRM) is a new molecular diagnostic technique based on real-time PCR, analysing the melting curve through saturated fluorescent dye monitoring nucleic acid melting curve. HRM is high sensitivity, high specificity, simple operation, low cost, high throughput, closed tube operation, and without the sequence-specific probe and limitation of mutation base sites and types, it can detect the paraffin-embedded tissues, blood, feces specimen and so on. After HRM analysis, the PCR products can be sequenced directly, because HRM does not damage DNA. It can be applied to mutation scanning, genotyping and other aspects. Therefore, if we can establish this method in clinical diagnosis of KRAS and BRAF gene mutations in colorectal cancer and lung cancer, it will contribute to individual therapy and reduce patient’s economic burden.ObjectiveThis study aims to use the current real-time PCR, combined with saturated fluorescent dyes and HRM analysis software, build HRM method on detecting KRAS and BRAF gene mutations in colorectal cancer and lung cancer, and apply to clinical test. Detecting KRAS and BRAF gene mutations by sensitive, specific, simple, rapid and accurate HRM analysis through optimizing test conditions, and to evaluate the advantages of HRM analysis on mutation scanning and the feasibility of clinical application through comparing with DNA sequencing and ARMS method. Analyze the correlations between specimen parameters and the results of KRAS gene mutation detected by HRM. Methods1. HRM analysis technology parameters optimizationTemperature gradient PCR and orthogonal design experiments were performed to optimize qPCR-HRM reaction system and reaction conditions with HT29, SW480and MDA-MB-231cell lines, which are known KRAS and BRAF gene mutation types. HT29cells DNA was serially diluted for PCR, and standard curve was drew with Ct value for vertical axis and different concentrations of starting DNA values for the abscissa. HRM analysis sensitivity testing was conducted by mixing a series of dilutions of100%,50%,25%,12.5%,6%,3%,1%of mutant KRAS DNA from SW480within wild-type KRAS DNA from HT29. In addition, these cell lines were also used as positive and negative controls. After HRM analysis, the PCR products were directly sequenced, and then compared the sensitivity of two methods.2. KRAS and BRAF mutation detection in colorectal cancer and lung cancer using HRMWe collected128paraffin-embedded tissue specimens of colorectal cancer and non-small cell lung cancer (NSCLC) from patients who planed to use the anti-EGFR monoclonal antibody drug postoperatively in Guangzhou General Hospital of Guangzhou Military Command, including64colorectal tumors and64NSCLCs respectively. Before DNA extraction, representative sections from tissues were stained with HE. DNA concentration was adjusted to30ng/μl and KRAS and BRAF mutations were detected by optimized qPCR-HRM reaction conditions and direct sequencing.4cases of colorectal cancer specimens KRAS mutations were detected by ARMS method, and compared with HRM and sequencing. For the intraassay variation, one wild-type and one mutant of KRAS were tested as15replicates for each sample at the same time. To test interassay variation, a series of10sampls (5mutant and5wild-type of KRAS) were tested every30days and three times totally. In order to analyze the minimum template quantity. Mutant KRAS DNA (C32) was diluted to30ng/μl,20ng/μl,10ng/μl,5ng/ul,2.5ng/ul and1.0ng/μl and detected by HRM. Finally, we wanted to determine whether the mutational status of KRAS gene was associated with any of the histological parameters evaluated (tumor cells, lymphocytes number and extracellular mucin) and tumor stages and metastasis factors. The statistical software SPSS13.0was used for data analysis, comparisons between groups were made using the chi-square test, correlation analysis between factors and mutation detection results tested by logistic regression analysis, the level of statistical significance was established at0.05.Results1. Temperature gradient PCR and orthogonal experiment results showed that:the optimal annealing temperature of KRAS gene was58℃and55℃for BRAF gene, the optimization of20μl qPCR-HRM reaction system for KRAS including DNA template40ng, MgCl22.5mmol/L and Primer0.5μmol/L. qPCR standard curve correlation coefficient R2is0.99928and amplification efficiency is1.05. The HRM detection limit was1%, namely it could detect mutation when1%mutant alleles. Sequencing could not detect mutant alleles below a level of25%and it was wild type when12.5%mutant alleles, indicating that HRM was more sensitive than direct sequencing.2. A total of23cases of colorectal cancer and9cases of NSCLC for KRAS mutations and four cases of colorectal cancer for BRAF mutations were detected by HRM. The others were wild type. All mutants were single mutation, KRAS and BRAF mutants were not found together at the same time. There were23colorectal cancer samples and7NSCLC samples were confirmed by sequencing, which occurred mutations in KRAS exon2, while2cases of NSCLC specimens could not confirmed by sequencing. The results showed that the mutation rate was no significant difference (P=0.500) between HRM and sequencing. The concordant rate between the two methods was high (κ=0.957). This study showed that HRM analysis sensitivity was100%and specificity was98%. ARMS method detected four cases of KRAS gene mutations, it was consistent with HRM and sequencing. The results of HRM reproducibility testing showed that15replicates of C16were wild-types and15replicates of C32were mutants, and5mutant samples and5wild-types on three different days were consistent. There was no significant difference between melting curve and wild standard melting curve when DNA template was1.0ng/μl, and there was significant difference between the melting curve of the rest of the template concentrations and wild standard melting curve, suggesting that the DNA template concentration of HRM as low as2.5ng. The KRAS mutation rate was significant difference (P=0.031) between groups of≤30%and>30%of tumor cells. It also was significant difference (P=0.025) between groups of≤10%and>10%of lymphocytes in the tumor. Logistic regression analysis results indicated that there was a significant relationship between the number of tumor cells, lymphocytes and extracellular mucin and KRAS gene mutation detection rate (P<0.05). The tumor stages and metastasis factors were no significant difference between groups.Conclusion1. HRM detection limit was1%. Sequencing could not detect mutant alleles below a level of20%. HRM is more sensitive than sequencing. The optimized qPCR-HRM reaction condition is suitable for gene mutation scanning because of its high efficiency and specificity.2. Direct sequencing, HRM analysis and ARMS test results are completely consistent, but ARMS is a costly method, so it is not suitable for conventional test. HRM method is reproducible, and does not change with the sample storage time. DNA template concentration of HRM as low as2.5ng, so detect the mutation just need a few DNA. Tumor cells, lymphocytes and extracellular mucin of tumor paraffin-embedded tissue specimen affect gene mutation detection rate, prompting the quality of specimen is very important. HRM is high sensitivity, high specificity, simple operation, low cost, high throughput, closed tube operation, and without the sequence-specific probe and limitation of mutation base sites and types. It can be applied to clinical gene mutation detection.