| Background and objective:Globally,lung cancer is one of the most common cancers and the leading cause of cancer-related deaths.Recently,many signaling pathways have been reported to be related with lung cancer development and progression.Among of them,Notch pathway,which involves gene regulation mechanisms that control cell proliferation,differentiation and apoptosis processes,has been shown to have biological significance and therapeutic application in non-small cell lung cancer(NSCLC).Therefore,we hypothesize that genetic variants in the Notch signaling pathway genes are associated with OS of NSCLC patients.Objective:(1)Explored the risk genes which were associated with NSCLC prognosis in the Notch pathway;(2)Explored the risk SNPs which were associated with NSCLC prognosis in the Notch pathway;(3)Tried to find out the functional evidences to support our findings,to understand the biological mechanism that how the SNPs affected the prognosis of NSCLC.Materials and Methods:We investigated associations of 19 571 single nucleotide polymorphisms(SNPs)in 132 Notch pathway genes with survival of 1 185 NSCLC patients available from the Prostate,Lung,Colorectal and Ovarian(PLCO)Cancer Screening Trial.We performed univariate and multivariate Cox proportional hazards regression analysis to evaluate the associations.False discovery rate(FDR)was used for multiple testing corrections.All statistical analyses were performed with SAS software(version 9.4),if not specified otherwise.The details about other softwares and websites like Haploview,R(Gen ABEL),Stata,SNPinfo、Regulome DB and UCSC will be described in the paper.Results:(1)The associations between clinical variables of 1 185 NSCLC patients in PLCO database and the overall survival(OS)of NSCLCIn our current project,we performed univariate and multivariate Cox proportional hazards regression analysis to evaluate the associations of clinical variables and prognosis of NSCLC.We found clinical variables like age,sex,smoking status,histology,tumor stage,chemotherapy,radiotherapy,and surgery were related with NSCLC OS(P ? 0.05).Therefore,these clinical variables were selected out for further analyses.(2)The associations between SNPs of Notch pathway genes and NSCLC OSWe first performed multivariate Cox proportional hazards regression analysis to evaluate the associations between 19 571 SNPs(i.e.,2 167 genotyped and 17 404 imputed SNPs)of Notch signaling pathway genes and NSCLC OS with adjustment of age,sex,smoking status,histology,tumor stage,chemotherapy,radiotherapy and surgery.Among of these SNPs,2 103 SNPs were individually significantly associated with OS at P < 0.05 in an additive genetic model.After the multiple test corrections,144 SNPs in 10 genes(ADAM12,CNTN1,CNTN6,DTX1,HDAC9,LNX1,NCOR2,RAB6 A,TLE1,and E2F3)with FDR < 0.05 were selected for further analyses.We then used the expression quantitative trait loci(e QTL)analyses to identify SNPs that were associated with m RNA expression levels of the corresponding genes.Of the 144 SNPs,24 SNPs in ADAM12,8SNPs in DTX1,4 SNPs in TLE1,and 1 SNP in E2F3 with positive e QTL results were selected(Padditive < 0.05).Finally,the five tag SNPs like ADAM12 rs10794069,DTX1 rs1732793,TLE1 rs199731120,TLE1 rs35970494 and E2F3 rs3806116 were selected after linkage disequilibrium(LD)analyses.(3)The associations between the five tag SNPs of Notch pathway and NSCLC OSIn our study,we found ADAM12 rs10794069 A>G,DTX1 rs1732793 G>A,TLE1 rs199731120 C>CA,TLE1 rs35970494 T>TC,E2F3 rs3806116 G>T were associated with a poor OS,with a variant-allele attributed hazard ratio(HR)of 1.27 [95% confidence interval(95% CI)= 1.13-1.42,P = 3.62×10-5],1.30(1.14-1.49,8.16×10-5),1.40(1.16-1.68,3.47×10-4),1.27(1.11-1.44,3.38×10-4),and 1.21(1.09-1.33,2.56×10-4),respectively.(4)Haplotype analysisWe performed univariate and multivariate Cox proportional hazards regression analysis to assess the prognosis of different haplotypes existing between the two SNPs(rs199731120 and rs35970494)locating in the same gene(TLE1).We found that the most favorable haplotype with rs199731120 C and rs35970494 T account for 82.2%,while,the most unfavorable haplotype with rs199731120 CA and rs35970494 TC account for only 7.6%.Compared with those with the most favorable haplotype,individuals with the most unfavorable haplotype had an adjusted HR of 1.41(P = 2.18×10-4).(5)Combined analyses of five tag SNPsTo evaluate the joint effect of the five tag SNPs on OS of NSCLC patients,we combined the genotypes of rs10794069 AG+GG,rs1732793 GA+AA,rs199731120 C/CA+CA/CA,rs35970494 T/TC+TC/TC and rs3806116 GT+TT(under a dominant genetic model)into a genetic score to define the combined risk genotypes.We firstly categorized all the patients into six groups: 0 to 5 risk genotypes.As a result,we found that there was a dose-response in the effect on OS associated with the genetic score(Trend test: P=3.44×10-13)after adjustment for other host and clinical covariates.Then,we dichotomized all the patients into a low-risk group(0-1 risk genotype)and a high-risk group(2-5 risk genotypes).A similar result was observed that the high-risk group notably had 1.56 fold increased risk of death(95% CI = 1.34-1.81,P = 1.46×10-8),compared with the low-risk group.(6)Stratified analyses for the effect of five tag SNPs and the combined risk genotypes on NSCLC OSWe first performed stratified analyses to evaluate the effect of each tag SNPs on NSCLC OS.We found that individuals with ADAM12 rs10794069 AG or GG genotype have a worse prognosis in the subgroup of radiotherapy(yes),compared with non-radiotherapy subgroup(Pheterogeneity = 0.024);individuals with TLE1 rs35970494 T/TC or TC/TC genotype have a worse prognosis in the subgroup of age > 71,compared with that of age ≤71(Pheterogeneity = 0.047);and individuals with E2F3 rs3806116 T/TC or TC/TC genotype have a worse prognosis in the subgroup of late tumor stage(IIIB-IV),compared with individuals with early stage(I-IIIA)(Pheterogeneity = 0.042).We then performed stratified analyses to evaluate whether the combined effect of risk genotypes as defined by the genetic score on NSCLC OS was affected by host and clinical characteristics.In the multivariate analyses,we found that patients with the high score(2-5)risk genotypes showed significantly worse prognosis in most subgroups,except for never smoking group,however,we found no difference between the subgroups of each clinical characteristic(Pheterogeneity > 0.05)as assessed by the heterogeneity test.(7)Receiver operating characteristic(ROC)curveIn our present study,we found that tumor stage combining with the five risk genotypes could increase the prognostic predictive effect of NSCLC,compared with the effect of tumor stage only [Area under curve(AUC)= 0.781 & 0.762,P = 0.018].While,we did not find the same effect from other models including clinical variables of age,sex,smoking status,histology,stage,chemotherapy,radiotherapy,and surgery(P > 0.05).(8)SNP-gene expressionWe performed e QTL analyses to select the potential functional SNPs.About the five tag SNPs,we found that rs10794069 GG,rs199731120 CA/CA,and rs35970494 TC/TC genotypes were associated with increased levels of the corresponding m RNA expression(Padditive = 0.003,0.001 and 0.012,respectively),whereas,rs1732793 AA and rs3806116 TT genotypes were found to be associated with decreased m RNA expression levels(Padditive = 0.014 and 0.046,respectively).Conclusions:(1)Clinical variables including age,sex,smoking status,histology,tumor stage,chemotherapy,radiotherapy,and surgery were associated with NSCLC prognosis in the PLCO database.(2)ADAM12 rs10794069 G,DTX1 rs1732793 A,TLE1 rs199731120 CA,TLE1 rs35970494 TC,E2F3 rs3806116 T were associated with a poor NSCLC prognosis.(3)The most favorable haplotype of rs199731120 C and rs35970494 T had the highest incidence,and the haplotype of rs199731120 CA and rs35970494 TC with the lowest incidence was related with the worst prognosis of NSCLC.(4)There was a dose-response in the effect on NSCLC OS associated with the number of risk genotypes.More risk genotypes increased the risk of death.(5)Individuals who accepted radiotherapy with ADAM12 rs10794069 AG or GG risk genotype,and individuals whose age were greater than 71 with TLE1 rs35970494 T/TC or TC/TC risk genotype,and individuals who were diagnosed at late tumor stage with E2F3 rs3806116 T/TC or TC/TC risk genotype had a worse prognosis,compared with the individuals of non-radiotherapy,age ≤ 71,and early stage,respectively.(6)Tumor stage combining with the five risk genotypes might increase the prognostic predictive effect of NSCLC,compared with the effect of tumor stage only.(7)The five SNPs played an important role in NSCLC OS by affecting the corresponding gene m RNA expression: rs10794069 GG,rs199731120 CA/CA,and rs35970494 TC/TC genotypes increased the levels of the corresponding gene m RNA expression,and rs1732793 AA and rs3806116 TT genotypes were associated with decreased m RNA expression levels of the corresponding genes. |
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