| Cancer poses a threat to the public, and the mortality caused by cancer in thedeveloped and developing countries are located in the first and second place respectively.Latest statistics show that United States is expected to appear nearly1.67million newcancer patients in2014, while the total death would reach to58million and more, with thelung cancer, colorectal cancer, prostate cancer (breast cancer) in the top three. The situationof tumor is so severe that the world could not afford to the serious economic and socialburden, and there are serious challenges for global prevention and control of cancer. Thecontrolling of tumor prevalence and mortality has become a major issue for healthpolicy-makers, clinicians and related researchers.It has been reported that the incidence, development, diagnosis, treatment andprognosis of cancer was significantly correlated with changes of gene loci in tumor. KRASmutation is one of the above changes. Now, the clinical studies have shown that KRASmutation was closely related to the failure of cancer therapy using monoclonal antibody.The Food and Drug Administration (FDA) in USA has recommended it as a detection whichmust be done before the treatment of cancer by monoclonal antibody drugs. However, smallnumber of patients with KRAS wild-type gene still faces the situation of failure with theabove treatment. The main reason is that the BRAF gene mutations, core of the downstreamof KRAS, prompt the mitogen-activated protein kinase (MAPK) pathway activation.Currently, the BRAF gene mutation has been detected in a variety of tumors. Theresearchers performed larger number of in-depth researches on the structure and propertiesof BRAF gene mutation and has achieved a breakthrough. At the same time, many studieson the relationship between BRAF mutation and clinicopathological features of commonmalignant tumors have been reported, but the conclusion is varied widely.Now, a number of methods are used to detect the BRAF gene mutation in clinical practice in order to provide some references for the therapy of cancer patients. However,the sensitivity of above methods is so low that could not detect rare mutation in BRAF gene.High resolution melting curve (HRMA) is a method with high sensitivity (about5%), andthe clinical studies about BRAF mutation detection by HRMA increased year by year.However, the number of samples collected in these studies is small and the accuracy ofdetection is difficult to assess. Besides, there have not been studies about the evaluation onthe accuracy by statistical methods. Therefore, it needs a further study and discussionbefore HRMA can be used to detect BRAF gene rare mutations.In recent years, it has made a rapid development on the techniques of molecularbiology, and new technologies on BRAF gene mutation detection have also been developed.Clamping-based PCR is one of the methods used to detect low abundance mutation. Itmainly used a locked nucleic acid probes for the identification of the nucleotide mismatchto improve the ability. However, this approach has many problems, such as need forspecific DNA polymerase and the high quality of amplified DNA, time-consuming toidentify BRAF mutations and so on.Based on the above background, we intended to resolve the dispute of relationshipbetween the BRAF gene mutation and clinicopathological features in common cancer usinga systematic review and meta-analysis. Therefore, we could have a good knowledge of thesignificance of BRAF mutations detection. On this basis, we performed a comprehensiveevaluation on the accuracy of BRAF mutation detection by HRMA in order to clear theneed of a new method with higher sensitivity. If the current sensitivity does not meet theclinical needs, we would build a high-sensitivity method using LNA/DNA probes on thebasis of Clamping-based PCR, and also detect BRAFV600E gene rare mutation incolorectal cancer using this new method.Objectives:1. To resolve the dispute of relationship between the BRAF gene mutation andclinicopathological features in common cancer and clear the significance of BRAFmutations detection.2. To evaluate the accuracy of BRAF mutation detection by HRMA with a largesample and clear the need of a new method with higher sensitivity.3. Construct a method with high sensitivity to detect BRAF rare mutation in colorectal cancer and verified by clinical samples.Methods:1. Searched PubMed, EmBase, SCI (Science Citation Index) databases, collectedrelated literature on the relationship between BRAF mutation and tumor pathology ordetection of BRAF mutation by HRMA; screened for literature accordance with theestablished inclusion criteria and evaluate the quality studies included; extracted data fromthe studies included and performed the data analysis and other relevant data analysis.2. Designed and synthesized the primers and LNA/DNA chimera; optimize thepreferred concentration of primers and annealing temperature, probe concentration,fluorescent dye concentration.3. Evaluate the sensitivity, specificity and reproducibility and accuracy of methodconducted based on LNA/DNA in our study and validated with50clinical specimens.Results:1. The systematic review on the relationship between BRAFV600E mutation andpathological characteristics of colorectal cancer included25literatures, involving11,955cases, and BRAFV600E mutation rate was about10.8%. BRAFV600E mutationsignificantly correlated with a number of characteristics of colorectal cancer patients: sex(OR=1.71;95%CI=1.42-2.07), age (OR=2.29;95%CI=1.13-4.61), TNM stage (OR=1.59;95%CI=1.16-2.17), differentiation (OR=3.89;95%CI=2.94-5.17), histologicaltype (OR=2.99;95%CI=2.20-4.07), tumor location (OR=4.85;95%CI=3.59-6.56),MSI status (OR=8.18;95%CI=5.08-13.17), CIMP status (OR=16.44;95%CI=6.72-40.21), MLH1status (OR=13.84;95%CI=1.75-109.24).2. The systematic review on the relationship between BRAFV600E mutation andpathological characteristics of colorectal cancer included10literatures, involving5,599cases, BRAF mutation rate was about3.0%. BRAF mutation was correlated withhistological type of lung cancer (OR=4.96;95%CI=2.29-10.75). BRAFV600E mutationwas correlated with other two features in patients with lung cancer: gender (OR=0.27;95%CI=0.12-0.59), smoking (OR=0.14;95%CI=0.05-0.42).3. The systematic review on the accuracy of BRAF mutation detection by HRMAincluded14studies, involving1324cases. The results showed that the sensitivity was0.99(95%CI=0.96-1.00), specificity was0.99(95%CI=0.99-1.00), positive likelihood ratiowas68.01(95%CI=25.33-182.64), negative likelihood ratio was0.06(95%CI= 0.03-0.11), diagnostic odds ratio was1263.76(95%CI=393.91-4064.39), AUC was0.995.4. Completed the design and synthesis of LNA/DNA chimera, Optimization wasdefined as follows: HQ-458/4610.2μM; Tm60℃; HQ-3560.5μM;2×EvaGreen,50cycles.5. Sensitivity was0.01%, and results of50clinical samples detection was16%(8/50) for positive, higher than conventional PCR and sequencing methods (10%,5/50).Conclusions:1. BRAFV600E mutation was significantly associated with gender, age, stage, grade,differential, type, location, MSI, CIMP and MLH1and so on. The results suggested thatBRAFV600E played an important role in the development, diagnosis, treatment andprognosis of the cancer; it should be used as a marker of prediction and prognosis ofcolorectal cancer and for screening of colorectal cancer patients before individualized drugtherapy.2. BRAF mutations in lung cancer patients were correlated with ADCs, so were theBRAFV600E and women or non-smokers. The results suggested that the ADCs and femalepatients were the focus of screening for BRAF mutation before personalized medicine.3. The accuracy of BRAF mutation detection by HRMA is very high, which is anideal method for clinical screening for BRAF mutation. However, the sensitivity is low andonly suitable for detection of advanced cancer specimens.4. The method called Real-Time WTB-PCR based on LNA/DNA in our study coulddetect BRAF mutation, and the sensitivity could reach to0.01%, which could be used to theanalysis of BRAFV600E mutation.5. Moreover, the accuracy of screening for clinical specimens is much higherconventional PCR and sequencing. This method has many advantages, such as highsensitivity, high accuracy, ease of operation, closed operation, low cost. If it could bewidespread used in the clinical practice in further, personalized treatment of cancer patientswill be benefited. |
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