Rarisk Variables Analysis And Molecular Chacteristics Of Postoperative Survival And Recurrence In Resected Stage-Ⅰ Non-small Cell Lung Cancer

Objective:Early diagnosis and treatment is key to improve the five-year survival rate of lung cancer,but there are still about 20% of patients with resected stage Ⅰ lung cancer developing recurrence within 5 years,which has become the main reason affecting the long-term post-operative survival.At present,there still require analyses on the risk factors for survival and predictors of postoperative recurrence in stage Ⅰ lung cancer the existing results of different studies are inconsistent.Compared with advanced lung cancer patients,whose metastatic characteristics have been investigated by a large number of previous omics studies,early lung cancer cases were less explored and their characteristics accounting for recurrence remain elusive.Therefore,here,I investigated the the risk factors affecting postoperative survival and recurrence based on a a large scale of stage Ⅰ non-small cell lung cancer(NSCLC)clinical dataset.Reliable risk factors were selected to establish an easy and practical risk prediction model for postoperative recurrence.Further,I explored the molecular landscape of resected stage Ⅰ lung cancer with the WES techniques,characterizing the recurrent and non-recurrent groups in both tumor tissues and somatic cells.Fundamental differences in the genetic mutations were discovered between patients with and without recurrence.I also sought to identify the pivotal signaling pathway and influential germline mutation characteristics of postoperative recurrence.Materials and methods:Chapter 1: Risk factors for postoperative survival and recurrence of stage Ⅰ non-small cell lung cancerIn this study,6148 patients with stage Ⅰ non-small cell lung cancer who were pathologically diagnosed in West China Hospital of Sichuan University from January2009 to December 2018 were included.The pathological staging(IA1,IA2,IA3,IB)was conducted according to the Eighth Edition TNM staging system published by the Eighth Edition International Association for the Study of Lung Cancer.The clinical,pathological,and imaging information of the patients were collected.Patients enrolled from January 2009 to December 2015 were followed up for 5 consecutive years or more.Genetic testing was performed by amplification refractory mutation system or multi-gene panel.Kaplan-Meier(K-M)method was used to draw the survival curve and carry out survival analysis.Cox proportional risk model was used to explore the risk factors affecting survival,and Logistic regression model was used to explore the risk factors for postoperative recurrence.Lasso method selected predictors to establish XGBoost model,SPSS 22.0 for data analysis,P<0.05 showed statistical difference.Chapter 2: Comparison of molecular characteristics between stage Ⅰ NSCLC groups with and without recurrenceIn this study,61 patients with stage Ⅰ NSCLC were included in West China Hospital of Sichuan University after standard radical resection of lung cancer and pathologically confirmed to be stage Ⅰ NSCLC without recurrence or recurrence specimens within 5 years.The samples were excluded due to insufficient paraffin specimens,unmatched cancer and adjacent tissues,tumor proportion less than 60%,and substandard DNA quality control.Total exome sequencing was performed on tumor tissues and matched normal tissues from 61 patients.Somatic SNV mutation analysis,somatic copy number mutation analysis and susceptibility gene analysis were performed,respectively.Clinicopathological and follow-up data were collected.SPSS 22.0 was used for data analysis,Kaplan-Meier method was used to draw survival curves,P<0.05 showed statistical difference.Results:Chapter 1: Risk factors for postoperative survival and recurrence in stage Ⅰ resected non-small cell lung cancer1.From 2009 to 2015,the proportion of stage Ⅰ NSCLC in primary lung cancer grew from 12.83% to 37.32%.The proportion of male patients decreased from59.17% to 42.40%,while the proportion of women increased correspondingly from40.83% to 57.60%.The proportion of young and middle-aged group rose from8.28%,36.69% to 12.59% and 45.14% respectively,while the proportion of the elderly group diminished from 55.03% to 42.26%.And the proportion of current smokers dropped from 45.56% down to 29.67%,while the proportion of never smokers climbed from 53.85% to 70.19%,the proportion of people who smoke without knowing it is very small.The proportion of adenocarcinoma jumped from69.23% to 91.43%,while the proportion of squamous cell carcinoma fell from23.08% to 7.10%.The proportion of stage ⅠA1 and IA2 increased from 9.47%,20.12% to 29.87%,40.05% respectively,while the proportion of stage ⅠA3 and IB decreased from 39.64% and 30.77% to 20.83% and 9.24%,respectively.2.The EGFR mutations were frequent in our stage Ⅰ NSCLC sample with an incidence of 58.77%,mainly L858 R point mutation in exon 21 and deletions in exon19.The prevalence of EGFR mutations were correlated with gender,age,smoking status,pathological type,and TNM stage.Non-smoking,female,older age,and relatively advanced stage(IA2 and IA3)were positively related with the occrrence of EGFR mutations in the LUAD group.In the young group,the dominant subtype of EGFR mutation was 19 DEL while in the old group L858 R was most common.Among the common genes of stage Ⅰ NSCLC,EGFR had the highest mutation rate,followed by TP53 and KRAS.3.The 5-year overall survival rate of stage Ⅰ NSCLC was 78.7%,and that of stage ⅠA1,IA2,IA3 and IB was 90.3%,81.9%,78.3%,and 72.5%,respectively.Univariate analysis showed that gender,age,smoking history,tumor size,TNM stage,pathological type,subclassification within lung adenocarcinoma,pleural invasion,and vascular invasion(P<0.05)were statistically significant correlated with survival.Further,in the multivariate analysis,TNM stage,and subclassification within lung adenocarcinoma(P<0.05)were the independent prognostic factors.4.During follow-ups,420 patients in a total of 2004 patients with resected stage Ⅰ NSCLC had experienced recurrence,with a recurrence rate of 20.96%.211 cases harbored local recurrence(50.24%),and 185 cases had distant metastasis(44.05%),while 24 cases developed both local recurrence and distant metastasis(5.71%).5.Univariate analysis results showed tumor size,TNM stage,pathological type,subclassification within lung adenocarcinoma,vascular invasion,and pleural invasion(P<0.05)was the risk factors for postoperative recurrence.Then,multivariate analysis validated that TNM stage and vascular invasion(P<0.05)was the independent risk factors for postoperative recurrence.Univariate and multivariate analysis showed the pathological type and EGFR mutation(P<0.05)and distant metastasis was statistically significant.In the recurrent group,adenocarcinoma and EGFR mutated patients mainly developed distant metastasis,while squamous cell carcinoma and EGFR wild-type patients mainly carried local recurrence.6.The area under the curve of the training set of Model1,a prediction model for postoperative recurrence of stage Ⅰ NSCLC established based on clinicopathological information,was 0.889,specificity 0.976 and accuracy 0.861.The area under the curve of the test set was 0.759,the specificity was 0.945 and the accuracy was0.800.Based on clinicopathological information and gene mutation characteristics,the area under the curve of the training set of Model2 was 0.915,the specificity was0.948,and the accuracy was 0.842.The area under the curve of the test set was 0.852,the specificity was 0.913 and the accuracy was 0.791.Chapter 2: Comparison of molecular characteristics between stage Ⅰ NSCLC groups with and without recurrence1.A total of 41 high-frequency mutations were found in 61 patients with stage Ⅰ NSCLC,including TP53(38%),EGFR(31%),TTN(21%),Ry R2(16%),MUC16(15%),CDKN2A(8%)and so on.There was a significant increase in TP53 mutation frequency in the recurrence group(6/29 vs.17/32,P<0.005).2.Regarding the differentially mutated genes in the somatic cells,mutations in ANK3,DNAH5,FSIP2,LRP1 B,MUC16 and so on were more prevalent in the recurrent adenocarcinoma group.In 23.8% of the patients,lung cancer or other cancer-related signaling pathways were activated,in which the RTK/RAS,Wnt and TP53 pathways were significantly activated in the recurrence group.3.There were several known CNV genes associated with lung cancer in the relapsed stage Ⅰ lung adenocarcinoma group,in which CDKN2 A and TP53 copy number deletion were enriched in the recurrence stage Ⅰ lung adenocarcinoma group.4.In stage Ⅰ NSCLC,germline mutations in BNC2,BRD3 and RECQL4 were merely specific to the recurrent group.Conclusion:1.The 5-year survival rate gradually decreased according to the disease progression in stage Ⅰ NSCLC patients,from stage ⅠA1,next to IA2,then to IA3,and finally to IB.TNM staging and subclassification within lung adenocarcinoma(P<0.05)are the independent risk factors for survival.2.The recurrence rate of stage Ⅰ NSCLC during postoperative follow-up period was 20.96%.TNM staging,vascular invasion(P<0.05)was an independent risk factor for postoperative recurrence.3.Based on clinicopathological information and gene mutation characteristics,the prediction efficiency of stage Ⅰ NSCLC recurrence can be significantly improved.4.Stage I NSCLC has common EGFR,TP53,CDKN2 A and other high-frequency mutations.TP53 mutation frequency and somatic mutation load were significantly increased in the relapse group.The recurrence of stage Ⅰ lung adenocarcinoma may be related to smoking.5.The differentially mutated genes in the somatic cells between groups with and without recurrence were ANK3,DNAH5,FSIP2,LRP1 B,MUC16 and so on which were more frequent in the recurrent group.ANK3 was suspected to be the key gene promoting the recurrence of stage Ⅰ lung adenocarcinoma.And germline mutations in BNC2,BRD3 and RECQL4 might also be responsible for stage Ⅰ NSCLC recurrence.