| Objective:1.To investigate the common sites of KRAS,PIK3 CA and TP53 gene mutation in non-small cell lung cancer(NSCLC)patients2.To explore the relationship between KRAS,PIK3 CA and TP53 gene mutation and the clinical features in non-small cell lung cancer(NSCLC)patients3.To explore the relationship between KRAS,PIK3 CA and TP53 gene mutation and the survival of the prognosis in order to provide the basis for the individualized treatment.Methods:1.Patient selectionOne hundred and twenty-two patients who meeted the inclusion criteria and accepted NGS for advanced NSCLC at the Department of Respirtory and the Department of Chest surgery in Shanghai Changhai Hospital were collected from January 2015 to December 2016.Thirty-there patients were excluded due to lack of information and loss of follow-up.Blood samples and clinical data of 89 patients with identified genes were collected from all patients,including gender,age,smoking,symptom,tumor mark,chest CT,tumor location,pathological type,TNM stage and metastasis site.All the samples were identified.Ethics Committee of Shanghai Changhai Hospital approved this study and a written informed consent was obtained from each participant.2.Gene detectioncSMART was used to detect KRAS,PIK3 CA and TP53 mutation in all NSCLC patients which was based on tumor gene mutation high throughput sequencing platforms to identifiy the type,sites and abundances of KRAS,PIK3 CA and TP53 genes mutations in patients with non-small cell lung cancer.First,genomic DNA was extracted from patients’ 10 ml plasma.The libraries was prepared from plasma DNA by ligation of universal sequencing adaptors.Then,cSMART was used to label,circularize,amplify and high-throughput sequencing the free DNA fragments from tumor cells in tumor tissue DNA or blood,and finally bioinformatics analysis was applied.3.Survival analysisAll patients were given a first-line chemotherapy regimen of pemetrexed+carboplatin(non-squamous non-small cell lung cancer patients)and paclitaxel+carboplatin(Lung squamous cell carcinoma patients).All patients have signed informed consent.Tumors were evaluated during chemotherapy treatment every 2 cycles,or were evaluated early when significant signs of progression appeared.Progression-free survival(PFS)were determined according to the Response Evaluation Criteria in Solid Tumors(RECIST1.1).The PFS was defined as the time from the beginning of chemotherapy to the presence of objective evidence of progression or dead.The last follow-up was June 30,2017.4.Statistical analysisSurvival curves were calculated using the Kaplan-Meier method from chemotherapy to documented progression or death from any cause.Statistical analysis was performed with the SPSS 21 software.The Chi-square test was used to compare the categorical variables.P<0.05 was considered statistically significant.Results:1.Baseline demographic characteristics122 NSCLC patients who meeted the inclusion criteria had received Circulating Single-Molecule Amplification and Resequencing Technology(cSMART)sequencing and 33 patients were excluded because of missing information.Then 89 patients had been enrolled in this study.The Baseline demographic characteristics of the 89 patients are shown in Table 1.There were 52 male and 37 female with a median age of 61.0 years.Seventy-five patients presented with a histology of adenocarcinoma,two typical of adenosquamous carcinoma and twelve typical of squamous carcinoma.Forty-one patients are smokers and forty-eight never smoke.2.Mutation status of KRAS,PIK3 CA and TP53 in NSCLC patientsFifty patients with KRAS/TP53/PIK3 CA mutations that harbored at least one driver gene were identified.The most common concurrent gene was TP53 mutation(40/89,44.9%),followed by KRAS mutation(21/89,23.6%)and PIK3 CA mutation(8/89,9.0%).Coexisting mutations were identified in 17 patients(17/89,19.1%)including KRAS+TP53 co-mutations(10/89,11.2%),PIK3CA+TP53 co-mutations(4/89,4.5%),KRAS+PIK3CA co-mutations(1/89,1.1%)and KRAS+PIK3CA+TP53 co-mutations(2/89,2.2%).There were 32 cases with EGFR mutations(32/89,36.0%),3 cases with EML4-ALK fusion oncogene(3/89,3.4%),and 3 cases of c-Met exon 14 skipping(3/89,3.4%).3.Relationship between mutation status of KRAS,PIK3 CA and TP53 in patientswith NSCLC and their clinical featuresCompared with those with wild type gene,the tumors of patients with KRAS mutations,TP53 gene mutation and KRAS + TP53 co-mutations showed invasive growth(76.2% vs 48.7%,P=0.04;72.5% vs 48.7%,P=0.03;90.0% vs 48.7%,P=0.046).The mutation of KRAS,PIK3 CA and TP53 in NSCLC was not directly related to its clinical characteristics beside margin lobulation sign in chest CT imaging.The incidence of distant metastasis(bone and thoracic lymph nodes)of KRAS mutations,TP53 gene mutation and KRAS + TP53 co-mutations in NSCLC patients is more than that of wild type gene group.However,there was no significant difference in the incidence of local invasion compared with wild type gene group.4.Relationship between mutation status of KRAS,PIK3 CA and TP53 in patients with NSCLC and their prognosisThe PFS of patients with wild-type gene(15.3±1.6 months)was significantly longer than that of those with single KRAS mutation(8.9±2.3 months,P= 0.045),single TP53 mutation(7.8±1.5months,P < 0.001),KRAS/TP53 coexisting mutation(6.6±1.6 months,P < 0.001)and PIK3CA/TP53 coexisting mutation(7.1±2.1 months,P= 0.012)had a significantly shorter progression-free survival.Compared with single KRAS gene mutation group and single TP53 gene mutation group,the PFS of KRAS/TP53 gene coexisting mutation group had a shorter trend.A similar phenomenon occurred in single TP53 gene mutation group and PIK3CA/TP53 gene coexisting mutation group.The PFS of two patients with KRAS/PIK3CA/TP53 gene coexisting mutation was average 6.15 months,that was a significantly shorter than those with wild-type gene.Due to the limited sample size,further research will be needed.Conclusion:1.Patients with NSCLC who have two genes co-mutations in KRAS,PIK3 CA,and TP53 have a significantly shorter PFS than those with wild-type gene,that suggests the existence of two genes co-mutations is one of the predictors of poor prognosis in NSCLC patients;2.Patients with two genes co-mutations and three genes co-mutations have a shorter tendency in PFS than those with single gene mutation.There may be "gene superposition" effect,which indicates that the type and number of gene mutations are also one of the important factors affecting patients’ prognosis;3.NSCLC with KRAS,PIK3 CA,and TP53 gene mutations are more aggressive and more likely to have distant metastases,but there is no direct relationship between gene mutations and clinical characteristics4.We believe that it is necessary for NSCLC patients to carry out routine KRAS,PIK3 CA and TP53 gene sequencing to clarify single gene mutation or multiple gene mutations,which has important clinical significance in the analysis of clinical characteristics,invasion and prognosis. |
PDF Full Text Request