| Objective:Genome-wide and tumor mutation burden(TMB)detection of cancerous tissues of patients with lung adenosquamous carcinoma(ASC)by next-generation gene sequencing(NGS)technology,and the determination of programmed cell death in cancerous tissues by immunohistochemistry(IHC)-Ligand 1(PD-L1),to clarify the mutation status of each gene in ASC,to screen potential target genes,and to focus on the analysis and discussion of the main mutant genotypes of ASC,as well as the relationship between TMB,PD-L1 and the clinical pathology of ASC.Methods:A retrospective study was conducted to investigate 21 ASC patients confirmed by surgical resection in the Second Affiliated Hospital of Nanchang University,the First Affiliated Hospital of Nanchang University,and the People's Hospital of Jiangxi Province through an electronic medical record query system from April 2014 to May2019.Case data,collecting relevant clinical information,including gender,age,smoking history,family history,TNM stage,clinical stage,treatment history,and pathology report results.Follow-up process: Determine the condition by investigating case data and telephone consultation,and record the date of recurrence or death of each patient in detail.If information is missing,it is recorded as lost follow-up.The follow-up date is December 2019.Cancer tissue specimens: Paraffin-embedded cancer tissue sections and adjacent tissues or normal lymph node tissue sections provided by the pathology departments of three medical institutions.The specimens were confirmed by two pathologists under microscope before obtaining the specimens.In the later stage,the samples were tested for genome,TMB and PD-L1 by NGS detection technology and immunohistochemistry.The data were analyzed and processed by SPSS24.0 statistical software.Result:1.Among 21 ASC patients,11 were male(52.4%),10 were female(47.6%);15(71.4),6(28.6%);7(33.3%)were smokers,14(66.7%)were nonsmokers;clinical stage: 12(57.1%)in stage I-II,9(42.9%)in stage III;N stage: 12(57.1%)in stage N0,9(42.9%)in stage n1-n2.2.Genome-wide mutation detection of 21 ASC specimens via NGS revealed that 29 mutant genotypes were screened: 13 cases of TP53(61.9%);10 cases of EGFR(47.6%);TP53 and EGFR double mutations were found in 7 cases(33.3%);3cases of PIK3 CA,PTPRD,and TSHZ3(14.3%);2 cases each of ALK,BRAF,CDKN2 A,DDR2,EP300,FANCA,GATA3,GNAS,JAK3,KMT2 C,LRRK2,NF1,NF2,PBRM1,PTEN,RBM10,ROS1,SDHA,STAT3,etc.(9.5%);AKT3,ARID2,ERBB2,MET,etc.1 case each(4.8%).There are three types of appealing gene mutations: single nucleotide variation(SNV),insertion or deletion mutation(INDEL),single nucleotide variation + insertion or deletion mutation(SNV + INDEL);SNV is the most common,except for MET,The ARID2 and NF2 genes were shown outside of the three genes.There were 11 cases(84.6%)of TP53 mutation type as SNV;7cases(70%)of EGFR mutation type were mainly INDEL;only one case of EGFR mutation was SNV + INDEL.3.EGFR mutation subtypes are mainly concentrated in exon 19 deletion mutations,and in E19 there are E746-A750,L747-A750,E746-S752,L747-P753,P753 S,and L747-S752 + E758 D double mutations;20 Exon N771-H773;Exon 21L858 R and so on.Analysis found no correlation between EGFR mutations and gender,age,smoking history,clinical stage,N stage,and ASC subtypes(P> 0.05);survival analysis revealed EGFR mutation status with overall survival(OS)and relapse-free survival(There was no correlation between RFS),and the difference was not statistically significant(P> 0.05).4.Copy number variation test(CNV)results: 21 samples were tested,and six gene copy number variations were found: 2 cases of CDK4 amplification(9.5%),1 of CCND1,EGFR,FGFR1,MDM2,and MYCN amplification(4.8% each).5.TMB test results: 21 samples were tested,and TMB?10Mut / Mb was high expression,and <10Mut / Mb was low expression.There were 4 cases(19.0%)with high TMB expression and 17 cases(81.0%)with low TMB expression;at the same time,there were 2 cases with high TMB and 9 cases with low TMB in adenocarcinoma-based ASC(gland component ?60%);1 case of gland squamous equivalent type(proportion of gland squamous ratio)has low TBM,squamous cellcarcinoma is the main type of high TMB in 2 cases,low TMB in 7 cases,analysis shows no correlation between TMB expression level and ASC subtype(P> 0.05).analysis found that there was a significant positive correlation between clinical stage and N stage and TMB expression level,and the difference was statistically significant(both P <0.05);but There was no correlation with age,gender,smoking or not,TP53 and EGFR gene mutation status(P> 0.05);survival analysis showed a significant negative correlation between OS and TMB expression levels,the difference was statistically significant(P <0.05));And there was no correlation between RFS and TMB expression levels(P> 0.05).6.PD-L1 test results: 18 cases of ASC samples were tested for PD-L1,and stipulated that> 50%(Tumor cells expressed PD-L1/total number of tumor cells)is high expression,and <50% is low expression,8 cases(44.4%)of high PD-L1 expression and 10 cases(55.6%)of low PD-L1 expression were found;mainly in adenocarcinoma There were 4 cases of high PD-L1 and 7 cases of low PD-L1 in ASC;4 cases of high PD-L1 and 3 cases of low PD-L1 in squamous cell carcinoma-based ASC;analysis showed that the expression level of PD-L1 was similar to that of ASC.There was no correlation between types(P> 0.05);analysis found that there was a significant positive correlation between smoking and PD-L1 expression levels,and the difference was statistically significant(all P <0.05);however,it was related to age,gender,clinical stage,There was no correlation between N stage,TP53 and EGFR gene mutation status(P> 0.05);survival analysis showed no correlation between OS and RFS and PD-L1 expression levels(all P> 0.05).Conclusions:1?Genes such as TP53 and EGFR have a high mutation rate in ASC,and there are multiple potential targeted driver gene mutations,which may bring a new approach to targeted treatment of ASC.2?The higher the expression level of TMB,the higher the clinicopathological stage and N stage,and the lower the total survival time.TMB can be used as an effective biomarker to predict the prognosis of ASC.3?PD-L1 was expressed in ASC,especially in smoking patients,suggesting that immunosuppressant might be effective in the treatment of ASC. |
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