1.Construction Of Lung Adenocarcinoma Prognostic Model Based On Transcriptome Data 2.The Function And Prognosis Of FABP5 In EMT And Sarcomatoid Transformation Of Non-small Cell Lung Cancer

Background:The prognosis of patients with lung adenocarcinoma(LUAD)is highly variable.The 5-year overall survival rates of stage IA LUAD are only 63-79%.Therefore,in order to identify high-risk patients and help clinical decision making,it is necessary to explore new prognostic markers to achieve more optimal risk stratification in combination with TNM staging system.Gene sequencing technologies have developed rapidly over this century,enabling us to identify an increasing number of cancer-related molecular prognostic markers with promising applications.Method:We used transcriptomic data from The Cancer Genome Atlas Lung Adenocarcinoma Cohort(TCGA-LUAD)as a training set to identify prognostic genes,and combined these genes with clinical data to develop new prognostic models for LUAD.We validated the model with an independent dataset(GSE50081)in the GEO database.Kaplan-Meier survival curves,Lasso regression and Cox multifactorial regression were used to identify prognostic genes.Area under the subject operating characteristic curve(AUC-ROC)was used to assess the discrimination of the prognostic model,and calibration curve(Calibration)was used to assess the accuracy of the model in predicting probabilities.An online analysis tool(TIMER 2.0)was applied to determine the relative abundance of immune infiltrating lymphocytes.Result:This study identified a prognostic model for four genes(CENPH,MYLIP,PITX3 and TRAF3IP3),with CENPH(HR=1.31,p<0.00 1)and PITX3(HR=1.24,p<0.001)as risk genes and MYLIP(HR=0.62,p<0.001)and TRAF3IP3(HR=0.75,p=0.017)as protective genes.A four-gene risk score was calculated based on the coefficients and expression levels.Using univariate COX regression analysis,we found that the four-gene risk score could serve as a prognostic predictor in the TCGA-LUAD cohort and the GSE50081 cohort.Compared to the low-risk group,HRs were 2.73(p<0.001)and 2.72(p<0.001)in the TCGA-LUAD and GSE50081 cohorts,respectively.Combining clinical factors,the multifactorial Cox regression analysis showed that the risk score was an independent prognostic factor in the TCGA-LUAD cohort and the GSE50081 cohort.Compared to the low-risk group,HRs were 2.34(p<0.001)and 2.10(p=0.017)in the TCGA-LUAD and GSE50081 cohorts,respectively.We combined risk score and clinical factors to develop a comprehensive prognostic model.The AUCs for 1-year,3-year,and 5-year OS in the training cohort were 0.750,0.737,and 0.719,respectively;and in the validation dataset were 0.645,0.766,and 0.725,respectively.Calibration curves showed a good match between predicted and actual probabilities.In addition,we determined the relative abundance of immune infiltrating lymphocytes by an online analysis tool(TIMER 2.0),revealing a more abundance of CD4+T cells in the low-risk group than the high-risk group in both cohorts(both p<0.001).Conclusions:We identified four prognostic genes(CENPH,MYLIP,PITX3 and TRAF3IP3)in this study.We have developed the four-gene risk score as an independent prognostic factor for LUAD and the score can be used to identify high-risk patients with different TNM stages.Compared to conventional TNM staging system,a comprehensive prognostic model combining prognostic genes with clinical features showed better predictive ability for OS.BACKGROUND:Lung cancer is the cancer with the highest mortality rate.Despite great advances in the treatment of lung cancer,treatment is often suboptimal because of the susceptibility to distant metastasis or drug resistance,resulting in a 5-year survival rate of only approximately 4-17%for non-small cell lung cancer(NSCLC).Epithelial mesenchymal transition(EMT)is a dynamic process associated with invasion,metastasis and drug resistance in a variety of tumors.The occurrence of EMT is often manifested by a decrease in the expression of its marker E-Cadherin and/or an increase in the expression of Vimentin.Pulmonary sarcomatoid carcinoma(PSC)is a non-small cell lung cancer with high malignancy,which contains epithelial and mesenchymal components.It metastasizes easily and has poor outcomes with chemotherapy.Studies have confirmed that PSCs often contains both classic cancer components and sarcomatoid components,and EMT is an important factor in the occurrence of sarcomatoid components.NSCLC is a tumor of epithelial origin,which acquires greater invasiveness,metastatic ability and drug resistance during the occurrence of EMT and sarcomatoid transformation,and this transformation process has important research value.METHODS:Transcriptomic data of PSCs(distinguishing different components)and TCGA lung adenocarcinoma were used to screen differentially expressed genes,and further screen genes negatively correlated with CDH1 and positively correlated with VIM(using Spearman correlation coefficient method)to obtain candidate gene markers.Then the GEPIA2 online analysis platform was used to find genes associated with prognosis of LUAD to further narrow down and identify target genes.The correlation between target gene and EMT were then validated at the transcriptome level in TIMER,LCR,GEPIA2,K-M Plotter and other databases.Finally,transcriptome and protein level(immunohistochemical staining)validation was performed in our independent LUAD and pulmonary sarcomatoid carcinoma cohorts,respectively,and then evaluate the prognostic impact of protein expression.R software(Version 4.0.3)was applied for differentially expressed gene screening,gene correlation validation(using Spearman correlation coefficient method)and mapping.SPSS(Version 25.0)software was used for data analysis,including independent sample t-test,KM survival analysis,and Cox multi-factor regression analysis.Survival time was measured in months.Survival analysis of prognostic factors was performed using Log-rank test.Fisher exact test and Wilcoxon test were used for statistical analysis of categorical and continuous variables,respectively.P<0.05 was considered statistically different.RESULTS:Transcriptome samples of pulmonary sarcomatoid carcinoma were obtained from 14 patients,including data from 7 adenocarcinoma components and 14 sarcomatoid component samples.1986 DEGs(differentially expressed genes)were obtained from the differential expression analysis of the two groups of sarcomatoid component and adenocarcinoma component,and a total of 575 genes were screened for upregulation in the sarcomatoid component.The TCGA-LUAD transcriptome data were downloaded,and genes negatively associated with CDH1 and positively associated with VIM were screened out from the 575 genes,and 22 candidate genes associated with EMT were obtained.The GEPIA2 database was used to analyze the effect of candidate gene expression on prognosis,and only the expression of FABP5(HR=1.6,p=0.001)and CTSL(HR=1.65 p=0.003)was found to be statistically different from prognosis,and finally FABP5 with a large correlation coefficient with EMT(correlation coefficient with CDH1 was-0.194,correlation coefficient with VIM 0.247)was selected as the key gene for study.Validation was performed in public databases,the analysis of the GSE72094 dataset came to similar results,and there were positive correlation trends for other important genes associated with EMT,SNAI1,SNAI2,and ZEB1 and ZEB2.Further studies in pan-cancer revealed that FABP5 showed similar trends of correlation with EMT-related indicators in breast,kidney,liver and pancreatic cancers.This was then re-validated in our independent adenocarcinoma cohort,where FABP5 was highly expressed in the VIM high expression group and low in the CDH1 high expression group,but neither reached statistical differences.Finally,immunohistochemical staining in an independent cohort of pulmonary sarcomatoid carcinomas at our institution revealed that FABP5 expression was negatively correlated with E-Cadherin(R=-0.17,p=0.173)and positively correlated with Vimentin(R=0.39,p=0.001).Multifactorial proportional risk model analysis showed that the expression of FABP5 was an independent prognostic factor for pulmonary sarcomatoid carcinoma(HR=2.17,CI:1.11-4.30,p=0.024).CONCLUSION:In this study,we mainly analyzed the transcriptomic data of different components of pulmonary sarcomatoid carcinoma and TCGA-LUAD to screen out FABP5 that was closely associated with EMT and sarcomatoid transformation.With multiple validations in multiple public databases and our independent cohort,we concluded that 1.FABP5 in NSCLC is closely associated with EMT and sarcomatoid transformation,but the exact molecular mechanism still needs further study.2.FABP5 is an independent prognostic factor in NSCLC,including pulmonary sarcomatoid carcinoma,and its high expression is associated with poor prognosis.