Mutational Spectrum Of Promoter And Its Correlation With Tumor Mutational Burden In Lung Squamous Cell Carcinoma Concomitant TP53 Mutations With Response To Crizotinib Treatment In Patients With ALK-rearranged Non-small Cell Lung Cancer

Background:Lung squamous cell carcinoma is one of the most common pathological subtypes of lung cancer,causing approximately 400,000 deaths per year worldwide.Previous studies have reported the mutational profile of the gene coding region in lung squamous cell carcinoma,but little attention has been paid to the mutational characteristics of gene regulatory region because of the lack of bioinformatics analysis algorithm and sequencing accuracy.Therefore,we designed a sequencing scheme based on capturing promoter and exon region to sequence both promoters and exons in lung squamous cell carcinoma,in order to systematically analyze the mutation profile of promoter and exon in lung squamous cell carcinoma and its relationship with tumor mutation load.This study aims to explore the potential molecular therapeutic targets in the promoter region of lung squamous cell carcinoma and the immunotherapy markers that can replace the tumor mutation burden.Methods:This study included 109 patients with lung squamous cell carcinoma who underwent surgery in our hospital from December 2017 to May 2019 and the samples of lung squamous cell carcinoma tissues,paired normal lung tissue and periph eral blood were collected.Promoter sequencing,whole-exome sequencing,and transcriptome sequencing were performed on Illumina HiSeq 2500 sequencing platform following the Illumina protocols.The mutational characterization of lung squamous cell carcinoma was analyzed by bioinformatics,and the tumor mutational burden was calculated to explore the relationship between tumor mutational characterization and tumor mutation burden.Results:In the whole-exome sequencing results of 109 patients with lung squamous cell carcinoma,we found that 96 patients(88.07%)had at least one mutation site in their genomes.Of all the mutations detected in whole exons,the vast majority were missense mutations.Among all missense mutations,the main mutation was C>A(adenine mutation to cytosine).After analysis by MutSigCV algorithm,it was found that the top 10 genes with high mutation frequency were TP53(82%),KMT2D(23%),NFE2L2(20%),NOTCH1(17%),ANKS1A(13%),CDKN2A(10%),SIX3(8%),RB1(7%),KRT76(7%)and PTH2(6%).We found for the first time that ANKS1A gene had high frequency mutation in Chinese patients with lung squamous cell carcinoma and significantly low expression in tumor tissues.In copy number variations,we found that high frequent fusion of AMDHD1 and GTF2IRD2 occurred in lung squamous cell carcinoma(12.8%).Among all the mutations detected,there were 13879 mutations in the promoter region,most of which were single nucleotide variants(SNV).Among all the single nucleotide variants,the main mutation was C>A.After analysis byMutSigCV algorithm,it was found that only promoter mutations of 40 genes were significant.Among the 40 promoter mutated genes,only the promoter mutation of WDR74 gene had a significant effect on the expression of WDR74,and the transcriptional expression of WDR74 was significantly increased(P=0.048).Among the 109 patients with lung squamous cell carcinoma,16 patients(14.7%)had promoter mutations of WDR74 gene:14 cases were SNV and the other 2 cases were insertion mutation and deletion mutation.The TMB value was calculated based on the sum of nonsynonymous single nucleotide and indel variants.It was found that the highest TMB value was 42.19 mu/Mb,the lowest TMB value was 0.05 mu/Mb,the average TMB value was 9.67 mu/Mb,and the median TMB value was 8.30 mu/Mb.We analyzed the relationship between TMB and clinicopathological features of patients with lung squamous cell carcinoma and the results showed that only advanced age was significantly correlated with high TMB(P=0.006).We used 11 genes with promoter mutations,ZNF595,DUSP22,GLYAT,MYCT1,OR1C1,OR4C3,OR4D11,OR4M2,OR4N4,TMEM132C,WDR74 to construct the TMB prediction model,and found that the 11 genes were highly correlated with TMB value(R2=0.8017,P<10-16).We identified the PNAEL of the 11 genes as Promoter-TMB PANEL(PT-PANEL).We used 10 mu/Mb as the cutoff value of TMB,and found that the AUC value of TMB predicted by PT-PANEL is 0.899±0.065.Conclusions:In this study,whole exon,whole promoter and RNA sequencing were performed simultaneously in a large sample of Chinese patients with lung squamous cell carcinoma for the first time.Among exon mutations,we found that the ANKS1A gene frequently mutated in lung squamous cell carcinoma and ANKS1A expression was significantly decreased in tumor tissues.In copy number variations,we found that high frequent fusion of AMDHD1 and GTF2IRD2 occurred in lung squamous cell carcinoma,and the role of AMDHD1-GTF2IRD2 fusion in lung squamous cell carcinoma needs to be further studied.Among the promoter mutations,we found that 11 genes with promoter mutations,ZNF595,DUSP22,GLYAT,MYCT1,OR1C1,OR4C3,OR4D11,OR4M2,OR4N4,TMEM132C,WDR74,were highly correlated with TMB.It is expected to confirm the feasibility and accuracy of promoter mutations in predicting immunotherapy through further clinical validation.Background:Non-small cell lung cancer(NSCLC)is one of most common malignancies and the leading cause of cancer deaths worldwide.TP53 mutations are the most prevalent mutations detected in NSCLC and have been reported to be associated with outcome.ALK-rearranged NSCLC patients are sensitive to crizotinib,but not all ALK-rearranged patients benefit equally from crizotinib.The aim of study was to investigate the impact of concomitant TP53 mutations in efficiency of crizotinib treatment in ALK-rearranged NSCLC.Methods:Tumor samples from 64 ALK-rearranged NSCLC patients receiving crizotinib treatment were subjected to next-generation sequencing(NGS)to identify TP53 mutational status.The clinicopathologic features of the TP53 mutations and its impact on the effect of crizotinib treatment were analyzed.Results:Among the 64 ALK-rearranged patients,15(23.4%)patients showed a TP53 mutation.Of these,6 cases had disruptive mutations and 9 with nondisruptive mutations.The objective response rate(ORR)and disease control rate(DCR)for TP53 mutated patients were both significantly lower compared with those for TP53 wild-type patients(P=0.003 and P=0.023,respectively).A significantly shorter progression-free survival(PFS)was found in TP53 mutated patients compared with TP53 wild-type patients(P=0.045).Nondisruptive TP53 mutations were associated with a shorter PFS in comparison with disruptive TP53 mutations in ALK-rearranged patients(P=0.069).When nondisruptive TP53 mutated patients were in comparison with TP53 wild-type patients,nondisruptive TP53 mutations were associated with a significant reduced PFS(P=0.003).Conclusions:TP53 mutations,especially nondisruptive mutations,negatively affected the response to crizotinib and correlated with shorter PFS in ALK-rearranged NSCLC patients.