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Research background and purpose of the epidermal growth factor receptor (epidermal growth factor receptor, EGFR) is a tyrosine kinase activity of the cell membrane receptor, an erbB receptor family belong to the cell surface. EGFR ligand binding, receptor phosphorylation caused by intracellular adapter molecules bind, causing the activation of a series of signaling pathways downstream. Many EGFR intracellular signal transduction pathway, RAS / RAF / MEK / ERK and PI3K/AKT pathway is one of the most important of the two pathways. Its principal involved in cell proliferation, growth, invasion, migration, angiogenesis and survival. View of EGFR and its downstream signaling pathways and tumorigenesis, development, invasion, the close relationship between the transfer and thus EGFR become an important target for targeted therapy. EGFR-targeting molecule targeted drugs are mainly two categories: a monoclonal antibody against EGFR extracellular domain, on behalf of the drug cetuximab, panitumumab; another EGFR intracellular The small molecule tyrosine kinase inhibitors, The representative drugs to gefitinib and erlotinib. The two types of drugs can effectively prevent downstream receptor-dependent signal transduction pathways, including the ERK / MAPK and P13K/AKT signal transduction pathway. To achieve a certain effect in gastric EGFR inhibitors cetuximab, multiple II clinical study of the efficacy and safety of cetuximab combined with different chemotherapy first-line treatment of advanced gastric cancer was observed. However, the total treatment efficiency is not high, and expensive, thus limiting its clinical reasonable application. How to accurately predict the efficacy of cetuximab to do \Cetuximab efficacy predictors in gastric less. Has confirmed that the K-ras mutant colorectal cancer patients can not benefit from cetuximab treatment, follow-up study reported absence of EGFR signal transduction pathways important regulating factor B-raf mutations of the PIK3CA and PTEN expression colorectal cancer patients do not benefit from cetuximab treatment. Whether like colorectal cancer, K-ras, B-raf, lack of the PIK3CA gene mutations and PTEN expression cetuximab treatment benefit of negative predictor? Therefore, it is necessary to understand the Chinese human gastric cancer K-ras , B-raf, PIK3CA gene mutation frequency. The mutation frequency of the gene is not only affected by the impact of the geography, race, but also with the quality of DNA, the relative content of the tumor cells in the sample and detection methods have a great relationship. Commonly used in clinical gene mutation detection method for direct sequencing, but the low sensitivity of conventional sequencing methods require at least 20% of the tumor specimens content. And clinical testing specimens majority of paraffin-embedded tissue, the integrity of the DNA is poor. And tumor genetic heterogeneity of tumor cells and normal cells mixed. Therefore, the application of the direct sequencing to detect gene mutations success rate is relatively low, and the detection process is complex and lengthy data analysis requirements, not easy to popularize. The ARMS assay of K-ras and B-raf, PIK3CA gene mutations, this study in order to understand the K-ras, B-raf and frequency of PIK3CA gene mutations in gastric cancer. ARMS (amplification refractory mutation system) that mutation specific amplification system, allele-specific PCR and fluorescent probe technology integrated. The method is sensitive, only 1% of the tumors content, and the detection process is simple and fast, low data analysis requirements. Gastric cancer is the most common malignant tumors of the gastrointestinal tract, and in cases of internal cause of cancer death worldwide, gastric cancer accounted seriously affect human health. And other malignancies, the pathogenesis of gastric cancer is very complex, multi-gene, the result of multiple factors. According to reports, the incidence of gastric cancer is associated with more than one hundred kinds of genes and their protein products, including oncogenes, tumor suppressor genes, cell cycle-related genes. The role of a variety of cancer-causing factors at different stages in different genes, leading to changes in gene structure and expression levels, including abnormal activation of oncogenes and inactivation of tumor suppressor genes. RAS is an oncogene that plays a key role in the cell signaling pathway. RAS gene family consists of three structurally similar genes that K-ras, H-ras and N-ras coding relative molecular weight of 21 kDa membrane-associated protein, namely p21 protein. The p21 protein is the hub of cell signaling, its activation can lead to unrestricted cell proliferation and immortalization, resulting in tumorigenesis. H-ras little studied. Study reported that the H-ras gene mRNA in normal parotid gland is not expressed or weakly expressed high levels of the salivary samples cystic carcinoma (SACC) in H-ras was abnormal expression, and the degree of malignancy of the SACC, infiltration and metastatic closely related. PTEN is a tumor suppressor gene found in recent years, studies have shown that PTEN is the only lipids dephosphorylated tumor suppressor gene, it can make PIP3 dephosphorylation into PIP2, it lost the role of the second messenger downregulate PI3K/AKT pathway. However, in many human malignant tumors, PTEN expression and function abnormalities. Based on the above research background and the problems, the purpose of this study are as follows: (1) In this study, ARMS method detection K-ras, B-raf, PIK3CA gene mutations, in order to understand the K-ras and B-raf and PIK3CA gene in gastric cancer in mutation frequency, further understanding of the impact of K-ras, B-raf, PIK3CA gene mutation status of the patient's survival; (2) using the method of immunohistochemical detection of H-ras, the positive expression of PTEN in gastric cancer rate, in order to understand H-ras, the relationship between the expression of PTEN impact on patient survival, and H-ras, PTEN expression and patient gender, age, degree of tumor differentiation, number of metastases. Research methods 1.K-ras, B-raf, PIK3CA gene mutation frequency detection from 1986 patients with gastric cancer screening Ⅳ gastric cancer patients complete the follow-up data of 143 patients met the inclusion criteria, extracted sample DNA. Application ARMS method for common types of mutations in K-ras G12D, G12A, G12V, G12S, G12R, G12C, G13D detection; common type of mutation V600E B-raf are reported to be detected; common outer reported PIK3CA exon 9 mutant type E542K/E545D, E545K, outer exon 20 common mutation type H1047R H1047L detected. Analysis of K-ras, B-raf, PIK3CA gene mutations in the relationship between the state and the survival period. 2.H-ras, PTEN expression in gastric cancer and clinical significance of 1986 patients with gastric cancer screening complete follow-up data of 143 patients met the inclusion criteria Ⅳ gastric cancer patients. H-ras, PTEN protein expression in gastric cancer were detected by immunohistochemistry; analysis of H-ras, PTEN protein expression in patients with clinicopathological parameters (gender, age, tumor differentiation, tumor metastasis number) The relationship between; analysis of the relationship between the H-ras, PTEN protein expression in gastric cancer patients survival time. 3 Statistical Methods SPSS 13.0 software for statistical analysis. The relationship between K-ras, B-raf, PIK3CA gene mutation status and survival in patients with the Kaplan-Meier method of survival analysis; χ2 test and Fisher's exact test, respectively, of the H-ras, PTEN protein expression was associated with each The statistical analysis of the relationship between clinicopathological parameters (gender, age, tumor differentiation, tumor metastasis number); relationship between the H-ras, PTEN protein expression in patients with gastric cancer survival of the survival of the Kaplan-Meier method Analysis. Results 1.K-ras, B-Raf, PIK3CA gene mutations in the frequency detected in the sample of K-ras, B-Raf, PIK3CA gene mutations in the number of cases were 2,1,4 cases, the mutation frequencies were 1.4 % (2/143), 0.7% (1/143), 2.8% (4/143). Two cases of K-ras mutations, the mutations were G12D; 1 case of B-raf V600E mutation; the four cases PIK3CA mutation type H1047L, E542K/E545D each case, E545K 2 Li. All samples were not detected in two kinds or more than two kinds of mutation type. 2. K-ras, B-raf, PIK3CA gene mutation status and survival in patients with the relationship between the Kaplan-Meier method survival analysis results: K-ras mutant survival of gastric cancer patients was significantly lower than in patients with wild-type K-ras (χ2 = 8.128, P = 0.004), while no significant difference between the survival period of the B-raf, PIK3CA gene mutation status. 3.H-ras protein expression and relationship between the patients with clinical pathological parameters exclude off-chip and can not determine the results of the sample, the sample of 123 cases, H-ras protein expression in 78 cases of positive and negative in 45 cases, The positive expression rate of 63.4% (78/123). The statistics showed that the H-ras protein expression and patient gender, age, degree of tumor differentiation and tumor metastases number no significant correlation, and there was no significant correlation between the H-ras protein expression and survival of patients. 4.PTEN protein expression and the relationship between patients with clinicopathological parameters exclude off piece and can not determine the sample, the sample of 100 cases, 89.0% positive in 89 cases, 11 cases of negative and positive expression rate of PTEN protein expression (89/100). PTEN protein expression-positive and-negative patients, the median survival time was 10.7 months, 5.6 months. PTEN protein expression difference between the positive and negative patient survival was statistically significant (χ 2 = 5.198, P = 0.023). Statistical results are shown PTEN protein expression and patient gender, age, tumor differentiation degree, and the number of tumor metastases is closely related to the number of metastases greater than three patients, low positive rate of PTEN protein expression, a statistically significance (P = 0.024). Research conclusions. The Ⅳ Opening treatment of gastric cancer patients detected, K-ras, B-raf, PIK3CA mutation frequency lower. 2.K-ras mutation status between patients survival significant difference, while the B-raf and PIK3CA mutation status between patients with no significant differences in survival, suggesting that the status of the K-ras gene can be somewhat predict patient prognosis. H-ras protein in gastric positive expression rate of 63.4%, but the statistical analysis showed that the H-ras protein expression and the patient's gender, age, degree of tumor differentiation, the number of tumor metastases and survival in patients with No significant correlation, but the H-ras protein in gastric cancer, development, worthy of further investigation. 4.PTEN protein in gastric positive expression rate of 89.0%, the statistical analysis showed significant correlation between PTEN protein expression and patient survival, and the expression of positive patients survive for long periods of time. Independent of PTEN protein expression and the patient's gender, age, tumor differentiation degree, is closely related to the number of tumor metastases. Prompted PTEN protein expression down or missing play an important role in gastric cancer invasion and metastasis and poor prognosis in patients.
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