The Primary Clinical Exploration Mutation Of EGFR, Kras And Braf In Early Non-small Cell Lung Cancer

Background:About 120 million of the global population were affected by lung cancer every year, lung cancer has become the first reason of death caused by cancer worldwide. 80% was non-small cell lung cancer (NSCLC), which included types of squamous carcinoma, adeno carcinoma and large cell carcinoma.Compared with small cell lung cancer, NSCLC had the slower proliferation and diffusion by lymphatic, hematogenous and infiltrating growth. Thanks to this, it has the possibility of further treatment.Follow the guidelines of National Comprehensive cancer Network (NCCN), NSCLC in stage Ⅰa to Ⅲa of TNM stage previously preferred to operation treatment. However, non-small cell lung cancer patients have a poor long-term survival situation,5 year survival rates of postoperative patients with NSCLS in stage Ⅰa, Ⅰb, Ⅱa, Ⅱb after were 80%,60%,55%,40%,respectively. Patients with lymph node metastasis to other side,5 years survival rate were 13%. In the last decades, NSCLC patients after stage Ⅲa and,the postoperation patients before stage Ⅲa but after stage Ib,their mainly systemic treatment were platinum-based chemotherapy,but about 20% of patients will appear side effects such as nausea, vomiting and other reaction. Despite the seriously effection of the quality of life of patients, only a small part of patients can benefit from the treatment. However,if untreat, the median survival period will be reduced to 4-5 months, and one year survival rate of only 10%.Therefore, surching the most effective way of treatment for patients in NSCLC,reduce the recurrence rate has become the hotspot for NSLC researching.In recent years, the discovery of epidermal growth factor receptor (EGFR), make the systematic treatment to non small cell lung cancer has ushered in a new stage. Molecule Targeting treatment based on Epidermal growthfactor receptor-tyrosine kinase inhibitors (EGFR-TKI) makes the systemic treatment of non small cell lung cancer gradually to the individual treatment transfor. The mechanism of EGFR-TKI is competitive inhibition with EGFR, thus inhibit tumor cell proliferation, metastasis and promote apoptosis of tumor cells. Molecule Targeting treatment has the advantages of high bioavailability, high selectivity, and low adverse reactions than chemotherapy. However, not all patients in NSCLC are suitable for EGFR-TKI treatment, a large number of clinical studies show that Asian female patients with adenocarcinoma and no smoking history were the advantage group of the TKI treatment,in which only exon 19 or 21 mutations in patients are sensitivity for TKI. and part of the patients could response such asrash, diarrhea after receiving the treatment. More worse, some patients will gain resistance to TKI, causing a huge impact on the patient’s follow-up treatment and survival. The resistance of TKI in current clinical application and basic research problem is in need of solution.Now a day, the researchers found that Phosphotylinosital kinase/protein kinase B/ the mammalian target of rapamyc in (PI3K/Akt/mTOR)was the signal transduction pathway playd an important role in thesurvival and apoptosis of tumor cells. Preliminary research indicates that, the activation of PI3K/Akt/mTOR signaling pathway is one of the reasons for EGFR-TKI resistance. In addition, recent resurch have found that activation of EGFR downstream pathways Ras-Raf-MEK-ERK is also play an important role in TKI resistance, but its mechanism has not been clarified, therefore, we design the clinical study in order to conduct a preliminary exploration of the Ras-Raf-MEK-ERK pathway.PurposeThis reserch collected the postoperative specimens of patients with NSCLC before stage Ⅲa after surgery, including tumor tissue, radical dissection lobar tissue and lymph nodes. Analyze mutation status of EGFR,KRAS and BRAF in non small cell lung cancer tumor tissue. Initially explore the relationship between Kras gene, Braf gene and EGFR gene combining with literature review, lay the foundation for further study of Ras-Raf-MEK-ERK pathways that leading to EGFR-TKI resistant.Material and method1.M aterial selection:74 cases of patients after operation for the treatment in the Navy General Hospitaland and 301 Hospital from 2011 to 2013,postoperative pathology were diagnosed with NSCLC, without chemotherapy or targeted therapy postoperative and preoperative, and no history of other tumors, TNM stage of Ⅲa previously NSCLC according to the guidelines of NCCN.Recorde their gender, age, smoking history, tumor type, tumor differentiation,tumor TNM stage, tumor size and degree of invasion, lymph node metastasis and other clinicalinformation.Obtain the fresh operation specimens, including tumor, lobectomy and lymph node dissection been excision, in the neutral formalin fixed 16h-24h. The sweeping lobectomy specimens and intraoperative lymph node specimens only as the basis of stages, no further gene detection and analysis.2.Detection Methods:2.1 Gene mutation detection use the DNA-liquid chip technology, detected the mutation status of each site of EGFR gene, Kras gene and Braf gene in operation resection tumor tissues, the specific steps are as follows:(1) Add the lysateinto FFPE samples fixed by Faure Marin,proceeding cracking reaction of 2 hours under the temperature of 55 ℃;(2) Pre hybridization:add sample liquid which had been completed cracking in the incubation platecracking,probe-microspheres, support extended probe and buffer are added to the sample solution, placed it in calorstat under the temperature of 55℃, concuss the liquid, incubating for 18-24 hours;(3)Learn supernate:the next day, place the supernatant liquid sample and incubated plate in the magnetic frame for 2 min, at this time the magnetic microspheres should be gathered at the bottom, magnetic microspheres is completely gathered at the bottom of the post, obtain supernatant and discard.(4) Washing:washing liquid is added to incubated plate discarded supernatant, placed in the concussion instrument washing 2 min, once again place incubation plate on the magnetic frame for 2 min, collecting supernatant and discard; Repeat steps above for 3 times.(5) Hybrid:Add amplification extension probes and labeled probe in the sample, then placement in calorstat under the temperature of 55 ℃, vibrate for 1 h;(6)Washing:Place incubation plate on the magnetic frame for 2 min, collecting supernatant and discard; Repeat steps above for 2 times;(7) Signal enhancement:Add streptavidin binding of phycoerythrin, placement in calorstat under the temperature of 55℃, concussion instrument vibrate 30 min;(8) Washing:Place incubation plate on the magnetic frame for 2 min, collecting supernatant and discard; Repeat steps above for 2 times;(9) Data collecting:Add the washing liquid into incubation plate, vibrate for 5 min, collect the data in the Luminex reading apparatus.(10) Analysis:data analysis, obtains test results.2.2 Detection of gene mutation (xTAG- liquid chip technology):(1) Extracted and purified Genomic DNA of FFPE using the Maxwell system;(2) PCR pre amplification:Purified DNA was used as the template for PCR amplification in advance, proceeding digestion reaction and ASPE reaction.(3) Hybridization:Add the product of PCR,microspheres mixture and hybrid liquid into the reaction tube, reacte under the temperature of 55 ℃ for 5min, and then proceed hybrid under the condition of constant temperature of 60 ℃ for 15min. Add streptavidin phycoerythrin inside, reaction at temperature of 60 ℃ for 5min.(4) Reading data results in the Luminex instrument and analysis, fluorescence value is less than or equal to 100 judge for the wild type, more than 200 judge for the mutation.3.Statistical methodsThe otherness between the mutation status of EGFR, Kras and Braf in patients and clinical information invoived gender, age, smoking history, tumor type, tumor differentiation, tumor TNM stage, tumor size and degree of invasion, lymph node metastasis using chi-square test.Correlation analysis of the mutation of EGFR,Kras and Braf using Spearman correlation analysis. Complete analysis with SPSS 13.0, p<0.01 that has statistical significance.Results:1. In 74 NSCLC patients, EGFR exon18 mutation rate was 1.35%(1/74), EGFR Exon19 mutation rate was 25.68%(19/74), EGFR exon20 mutation rate was 0% (0/74), EGFR Exon21 mutation rate was 20.27%(15/74), Kras gene mutation rate was 25.68%(19/74), Braf gene mutation rate was 6.76% (5/74). The EGFR, Kras, Braf gene mutation in different gender, age, smoking history, tumor differentiation, tumor TNM stage, tumor size and involvement, lymph node metastasis has no differences, but there are differences in different pathological types of patients, patients with adenocarcinoma had higher mutation rates, while the non adenocarcinoma one with low mutation rate.2. Through statistical analysis, E19, E21 locus mutation of the EGFR gene mutation and Kras gene has negative correlation (correlation coefficient=-0.345 and-0.309 respectively,p<0.01), but the relationship is not close (r<0.5), EGFR gene E18, E1 9 and E21 and Braf was not existing correlation between correlation(correlation coefficient r are respectively -0.032,-0.158,-0.011, p>0.1),nether was Kras and Braf (R=-0.158 p=0.178).Conclusion:1.The mutation of EGFR has differences in the patients with different pathological types.Patients with adenocarcinoma had higher mutation rate than the non-adenocarcinoma patients.But has no difference in the other clinical data。 The mutation rate of Kras and Braf in different clinical data has no difference history, tumor differentiation, tumor TNM stage, tumor size and involvement, lymph node metastasis patients has no difference, but2.Mutation of EGFR gene was negatively associated with the Kras mutation,but has less correlation.Mutation of EGFR and Braf gene is not related, neither was Kras gene and Braf gene.Our result suggest that the mutation of EGFR, Kras and Braf is independent course.3. For postoperative patients with non-small cell lung cancer in stage Ⅰa-Ⅲa, early gene detection has guiding significance for TKI targeted therapyhas.And further study Of the mechanism of resistance to TKI can be resurch. For postoperative patients with EGFR mutation positive after operation, if resistant to TKI, should be carried out to gene detection of Kras and Braf is necessary for surching other targeted therapeutic drugs.