Non-small Cell Lung Cancer Clinical Pathology Are Associated With GNAS1Gene Polymorphisms And EGFR Mutations

Background and objectiveLung cancer is the most frequent cause of cancer-related deaths in worldwide. The main reason is high tumor aggressivity and high metastasis potential. According to the World Health Organization estimates that if no intervention, during2005-2015, there will be have84million people died of cancer, new cancer cases worldwide each year up to900million people, the death of about500million people. Male lung cancer incidence has topped the list in2008, the first breakthrough in the incidence of70thousand, far ahead of other organ malignancies. In recent years, due to multiple factors such as the environment, the incidence of lung cancer in women also showed an upward trend year after year. Lung cancer has become the top killer disease in humans, resulting in a heavy social burden. Therefore, it is a primary task of mankind to overcome the causes of cancer and find a solution as the primary task of mankind. It is generally agreed that the incidence and the development of lung cancer involving many pathophysiological responses, multi-factor, multi-mechanism. According to epidemiological studies have found that the main risk factors for lung cancer are closely related personal habits,environment, drug usage and around individual genetic factors, but the exact mechanism of lung cancer is still so far fully understood. Since nearly85percent of lung cancers are non-small cell lung cancer (Non-small cell lung cancer, NSCLC), so research the development of mechanisms for non-small cell lung cancer will have a significant impact on the prevention and treatment of lung cancer. With the era of biomedical research into the molecular and clinical cancer treatment is also advancing into the molecular era. For the cause of human disease, pathogenesis, prognosis study and understanding, also from the traditional concept down to the molecular biological level, currently, molecular studies in tumor genes, oncogenes and tumor suppressor genes related mainly become a hot research field. Some molecular markers have entered the clinical application.Single nucleotide polymorphisms (SNPs) are a new generation of genetic markers. As the single nucleotide polymorphism information content-rich, the feasibility of detection, and the exact relationship between the diseases, they were used in more and more researches to explain the genetic susceptibility to cancer, and the prognosis of the effectiveness of chemotherapy drugs and many other aspects.The GNAS1gene encoding the G subunit (Gs) of the heterodimeric G protein, is located on chromosome20q13.2-13.3, containing13exons and12introns. GNAS1is a complex locus encoding multiple overlapping transcripts. The ubiquitously expressed Gs is essential for coupling of multiple receptors to adenylyl cyclase and stimulation of proapoptotic processes within a cell. In previous studies, our group found the different genotypes of the T393C SNP in the gene GNAS1being significantly associated with the clinical outcome of patients with carcinomas of the urinary bladder, kidney, colorectum, and oro/hypopharynx. Patients carrying the TT genotype showed a prolonged survival compared with patients carrying either TC or CC genotypes. This occurrence was suggested to be mediated by an increase in the apoptotic rate in TT genotypes. Different in vitro experiments suggest that increased expression of G protein is associated with enhanced apoptosis. The second messenger cyclic AMP (cAMP) that is generated subsequently to the activation of G a s, seems to play a major role in this proapoptotic process. cAMP can augment or suppress extracellular regulated kinase activity, depending on the cell type. As an example, Raf-1is inhibited by protein kinase A, which is one of the downstream effectors of cAMP. A recent report has shown that cAMP blocks Raf-1activity in different cell types resulting in suppression of the oncogenic activity of Raf-1. Subsequently, Raf-1inhibition by antisense nucleotide treatment induced potent anti-proliferative effects in tumor cell lines. In line with these results, Frey UH recently found that the GNAS1 TT genotype is associated with increased Gαs mRNA expression in different tissues which led them to hypothesize that the T393C exchange itself could have an effect on mRNA stability. Indeed, Frey UH found that the T393C polymorphism influences GNAS mRNA folding which could have a major impact on mRNA stability and Gs expression. Determinants of mRNA stability have been described in the coding region of some other genes. These results prompted us to further investigate whether the T393C polymorphism of the GNAS1gene may predict the clinical course of NSCLC as well.Epidermal growth factor receptor (epidermal growth factor receptor, EGFR) is widely distributed in human cell membranes, protein tyrosine kinase receptors. After EGFR ligand binding, it can form homo or heterodimers, activating of the tyrosine kinase signal transduction pathways downstream, and promoting the proliferation and differentiation of cells. Current study found:EGFR gene mutations had a correlation with malignant tumors clinic-pathological features、treatment of drug sensitivity and disease prognosis.In recent years, studies of the relationship between G protein gene family SNPs with a variety of tumors are increasing, particularly with regard to the relationship between GNAS1gene T393C with clinical pathological features and prognosis of disease susceptibility. Therefore, in this experiment, DNA samples in57patients with non-small cell lung cancer patients in the GNAS1gene T393C SNP genotypes were detected; simultaneous detection of DNA in EGFR exon18-21mutations. The purpose of the experiment was to investigate the correlation between GNAS1gene T393C genotype and allele frequencies of non-small cell lung cancer; and correlation with EGFR mutation rate of non-small cell lung cancer.Materials and methods1. The paraffin tissue samples were collected from57unrelated patients with NSCLC from February2012to March2013.All patients with NSCLC of paraffin tissue samples were collected from patients before surgery in the first affiliated hospital of Zhengzhou University.2. Commercially available kit (QIAAmp, Qiagen, Hilden, Germany) was used to extract DNA from fifty-seven paraffin tissue samples from patients with NSCLC.3. Using the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to detect GNAS1T393C genotype.4. Using the method of amplification refractory mutation system (AEMS) to detect EGFR gene mutations.5. The methods of statistical analysis:using the SPSS17.0statistical software package to analyse collected data. Analysing ordered categorical datas by rank sum test, the mean ranks were used to express risk degree. Analysing unordered categorical datas by χ2test, Odds ratio(OR) and95%confidence interval(95%CI) were used to express risk degree(OR and95%CI were computed by Logistic regression model). Using two-sides probability test when there were two kinds of side tests. Method of Bonferroni was used to mutiple comparision. Significant level was0.05.Results1. The genotype frequencies of GNAS1exon5T393C T/T、T/C、C/C were38.60%(22/57)、49.12%(28/57)、12.28%(7/57) respectively. The allele frequencies of GNASl exon5T393C T and C were63.16%、36.84%respectively.2. The genotype frequencies of GNAS1T393C site had no correlation with the NSCLC patients age、gender、smoking history、TNM stage、tumor size、tumor differentiation and the histological type(P>0.05); The genotype frequencies of GNAS1T393C site had significant correlation with NSCLC lymph node metastasis (P<0.01). The allele frequencies of GNAS1T393C site had no obviously correlation with the NSCLC patients age, gender, smoking history, tumor TNM stage, tumor differentiation, and the histological type (P>0.05); The allele frequencies of GNAS1T393C site had significant correlation with NSCLC lymph node metastasis (P<0.01).3. NSCLC patients who carring T/C、C/C genetype can raise the NSCLC patients lymph node metastasis risk (OR of genotype C/C, genotype T/C to genotype T/T were8.500、6.120respectively; The results of mutiple comparision by Bonferroni (a’=0.017):P<0.01when genotype T/T was compared with genotype T/C, P<0.01 when genotype T/T was compared with genotype C/C; P>0.05when genotype T/C was compared with genotype C/C). GNAS1T393C site allele C raised the risk of NSCLC patients’ lymph node metastasis (OR of allele C to allele T was3.143, P<0.01).4. EGFR mutation were found in61.54%(35/57) of our NSCLC samples,of which,15cases located at the exon21,all the mutant types are L858R;20cases located at exon19,all mutant types are delE.5. There were no significant differences between the EGFR mutation and NSCLC patients’ age, Lymph node metastasis or tumor TNM stage (P>0.05).6. The EGFR mutant rate was higher in females71.43%(25/35) than in males45.57%(10/22).The EGFR mutant rate was higher in adenocarcinoma67.74%(21/31)than non-adenocarcinoma53.85%(14/26). The mutation rate was higher in smoker patients72.73%(16/22) than non-smokers54.29%(19/35) Moreover, the mutant rate was higher in patients who’s tumor well-differentiated and tumor smaller (<3cm).%2test all the results proving meaningful (P<0.01).Conclusions1. The genotype frequencies and the allele frequencies of GNAS1T393C site had significant correlation with NSCLC lymph node metastasis. NSCLC patients who earring allele C can raise the risk of lymph node metastasis.2. The EGFR mutant rate had correlation with NSCLC patients gender、smoking history、TNM stage、tumor differentiation and the histological type and tumor size.