Gene Mutations And Expressions In EGFR Signaling Network In Patients With Gastric Cancer Of Stage Ⅳ

Background and objectiveEpidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that belongs to erbB family. Ligand binding to the extracellular domain leads to EGFR activation,which then homodimerizes, resulting in the phosphorylation of the intra-cellular tyrosine kinase initiating a series of intracellular signals. The Ras/Raf/MEK /ERK and PI3K/PTEN/AKT signaling cascades are two main pathways which play critical roles in the transmission of signals from EGFR to regulate cell proliferation, growth, invasion, migration, angiogenesis, survival and so on.Because of EGFR and its downstream signaling pathways playing critical roles in tumorgenesis,it has been an important target for molecular targeted therapy.Two main anti-EGFR strategies are currently in clinical development:monoclonal anti-bodies that are directed at the ligand-binding extracellular domain. Cetuximab and panitum-umab are the typical drugs;Another is low-molecular-weight tyrosine kinase inhibit-ors such as Gefitinib and Erlotinib which compete with ATP for binding to the tyro-sine kinase portion of the receptor.Both the two main durgs could effectively prevent the downstream receptor-dependent signal transduction pathways, including ERK/ MAPK pathway and P13K/AKT pathway. At present, Cetuximab has been used to treat gastric cancer.The efficacy and safety of cetuximab combined with different chemotherapy regimens in first-line treatment of advanced gastric cancer were obs-erved in several phase II clinical studies. However, the low treatment efficiency and it's expensive price limited its clinical reasonable applications. How to predict the efficacy of cetuximab in treatment gastric cancer is especially important.Neverthel-ess, the research about predictors of cetuximab in treatment gastric cancer is very less. It has been confirmed that K-ras gene mutation could lead to failure of anti-EGFR monoclonal antibody therapy.The follow-up studys reported that B-raf, PIK3CA mutation and PTEN loss in patients with colorectal cancer could not get benefits from the treatment of cetuximab. Whether it is the same as colorectal cancer, K-ras,B-raf, PIK3CA mutation and PTEN loss is the negative indicator to predict the efficacy of anti-EGFR therapy. Therefore, it is necessary to understand K-ras, B-raf, PIK3CA mutant frequency in Chinese gastric cancer.Gene mutant frequency is not only influenced by geographical area, racial,but also by the DNA quality, content of tumor cells and detection methods. Currently, DNA direct sequencing is one of the most widely used methods for analysing gene mutation. But sequencing is not exquisitely sensitive which requires at least 20% tumor content in the samples. Nevertheless the samples for clinical testing are most of paraffin-embedded tissue in which DNA integrity is poor. In addition, tumour specimens are heterogeneous. They can contain surrounding and infiltrating normal cells, and not all tumour cells are identical.Thus,the success rate of DNA direct seq-uencing is relatively low in detecting the gene mutations. In addition, this assay is routinely a long testing process, resulting in difficult to spread. In this study, ARMS assays were used to detect K-ras,B-raf and PIK3CA mutations in order to under-stand the mutant frequency in gastric cancer. Amplification refractory mutation system(ARMS) is the combine of allele-specific PCR and fluorescent probe. This assay is sensitive, routinely being able to detect at least 1% mutant in a normal DNA background, and are quick and easy to use.Gastric cancer is the most common gastrointestinal malignancy and the second most common cause of cancer death worldwide with approximately one million cases diagnosed annually. Like other malignancies, the pathogenesis of gastric cancer is very complex, which is the results of multiple genes and factors. It has reported that the incidence of gastric cancer was related with over one hundred kinds of genes and their protein products including oncogenes, tumor suppressor genes, cell cycle-related genes. Carcinogenesis is a multistep process featuring the accumulation of several ge-netic alterations, including the abnormal activation of oncogenes and the inactivation of tumour suppressor genes. Ras constitutes a family of proto-oncogenes which plays a key role in the cell signaling pathways encoding small G-proteins with a molecular weight of 21 kDa (p21). Three different members contribute to the RAS gene family known as K-ras, H-ras, and N -ras. P21 protein plays a key role in the cell signal-ing and its activation can lead to unlimitedproliferation and immortalization of cells, resulting in tumorigenesis. It has reported that H -ras mRNA in normal parotid gland tissue is weak expression or absence of expression. But in salivary adenoid cystic car-cinoma (SACC), the H -ras shows abnormal expression of a high level which is clo-sely correlated with the differentiation, infiltration and metastasis of SACC.PTEN has been identified as the the only tumor suppressor gene that can make PIP3 dephospho-rylate into PIP2 resulting in losting the role of second messenger.Thus, it can down deregulate PI3K/AKT signaling pathways. However, there is a PTEN expression and function abnormalities in a variety of human malignant tumors.Based on the above background and problems, the purpose of this study are as follows:(1) Apply ARMS assay to detect K-ras,B-raf, PIK3CA gene mutations in order to understand the mutant frequency in gastric cancer, and further understand the influences of K-ras,B-raf, PIK3CA mutant status on the survival of patients with gastric cancer; (2) Apply immunohistochemical method to detect H-ras, PTEN exp-ression in gastric cancer in order to understand the relationship between H-ras,PTEN expression and patients' survival,gender, age, tumor differentiation and the number of metastasis.Research Methods1.The detection of K-ras,B-raf, PIK3CA mutant frequency143 cases were screened which met the inclusion criteria from 1986 patients with gastric cancer of stage IV.DNA was extracted from paraffin-embedded tissue of gast-ric cancer. The commonly reported mutant types G12D,G12A,G12V,G12S,G12R,G12C. G13D of K-ras, V600E of B-raf, mutations in exon 9 (E542K/E545D and E545K) and mutations in exon 20 (H1047R and H1047L) of PIK3CA were dete-cted respectively by ARMS assay in real-time PCR reactions.Analyze the relationship between K-ras, B-raf, PIK3CA mutant status and the survival of patients.2.The expression H-ras, PTEN protein in gastric cancer and its clinical significance143 cases were screened which met the inclusion criteria from 1986 patients with gastric cancer of stageⅣ. H-ras, PTEN protein expression in gastric cancer were detected by immunohistochemical methods. Analyze the relationship between H-ras, PTEN protein expression with clinicopathological parameters including patients'sur-vival,gender, age, tumor differentiation and numbers of metastasis.3.Statistical MethodsSPSS 13.0 software was used for statistical analysis. Kaplan-Meier survival analysis was used to analyze the relationship between K-ras, B-raf, PIK3CA mutant status and patients' survival. And the relationships between the expression of H-ras, PTEN protein and their clinical-pathologic features including patients' survival,gender, age, tumor differentiation and numbers of metastasis were analyzed using Chi-square test and Fisher's exact test.Meanwhile,the relationships between the expression of H-ras, PTEN protein and survival of patients with gastric cancer were analyzed by Kaplan-Meier survival analysis.Results1.Frequencies of K-ras, B-raf and PIK3CA mutationsIn the samples collected from 143 GC patients, the mutant frequency of K-ras, B-raf and PIK3CA was 1.4%(2/143),0.7%(1/143),2.8%(4/143) respectively. No samples bear 2 or more mutations. All the two mutatd cases of K-ras carried G12 D mutation. One cases of B-raf carried V600E mutation.There were one cases carri-ed E542K/E545D mutation,while two cases E545K mutations of PIK3CA. 2.The relationship between K-ras,B-raf, PIK3CA mutant status and patients'surv-ivalThe results of Kaplan-Meier survival analysis showed that survival of patients with K-RAS mutant-type was significantly lower than that of K-ras wild-type (χ2= 8.128, P=0.004). But, there were no significant differences in cumulated survival between patients with mutations of B-raf, PIK3CA gene and those with wild-type ones.3.The relationships between the expression of H-ras protein and their clinical-patho logic featuresEliminating the off-chip and undeterminable samples, there were 123 samples left. Positive expression of H-ras protein was detected in 78 cases,while negative expression in 45 cases. The positive rate of H-ras protein was 63.4%(78/123). There were no significant associations between the expression of H-ras protein and patient' gender, age, tumor differentiation and numbers of metastasis. 4.The relationships between the expression of PTEN protein and their clinical-pathol-ogic featuresPositive expression of PTEN protein was detected in 89 cases,while negative expression in 11 cases. The positive rate of PTEN protein was 89.0%(89/100). The median survival time of patients who detected positive expression and negative expr-ession of PTEN protein was 10.7months and 5.6 months respectively. There were significant differences in cumulated survival between patients with positive expressi-on of PTEN protein and those with negative ones (χ2=5.198, P=0.023).The statistic results show that there were no significant associations between the expression of PTEN protein and patient'gender,age,tumor differentiation.But there was significant association between the expression of PTEN protein and numbers of metastasis. The positive rate of PTEN protein in patients whose metastatic lesions numbers more than 3 was significant lower than that of less than 3 (P=0.024)Conclusions1.The mutant frequency of K-ras,B-raf, PIK3CA detected in the initial treatment of gastric cancer patients with stageⅣwas low.2.There was significant differences in cumulated survival between patients with mut-ations of K-ras gene and those with wild-type ones,but not for B-raf,PIK3CA genes genes.This results suggest that the status of K-RAS may predict the prognosis of patients in a certain way.3.The positive rate of H-ras protein was 63.4%.The statistic analysis showed that th-ere were no significant associations between the expression of H-ras protein and patient' gender, age, tumor differentiation and numbers of metastasis. However, it is worth further studying the role of H-ras protein playing in pathogenesis and develo-pment of gastric cancer.4.The positive rate of PTEN protein was 89.0%. There were significant differences in cumulated survival between patients with positive expression of PTEN protein and those with negative ones.The survival of patients with positive expression of PTEN protein was longer than that of negative ones.The statistic analysis showed that there were no significant associations between the expression of PTEN protein and patient' gender, age, tumor differentiation. While, the expression of PTEN protein correlated closely with numbers of metastasis. The results suggest that PTEN loss or its'low level expression play an important role in metastasis and poor prognosis of patients with gastric cancer.