Identification Of Two Distinct Immune Subtypes In Lung Adenocarcinoma

ObjectiveImmune checkpoint blockade(ICB)therapies have dramatically improved the survival of lung adenocarcinoma(LUAD).However,in currently clinical practice,only a small subset of patients could benefit from ICB therapy.Although biomarkers that were broadly used to some extent could predict ICB therapy efficacy,the lower response rate driving us to explore novel and more effective indicators.MethodsWe retrospectively collected 2300 LUAD samples of 10 datasets from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO).All these samples contained genome-wide expression data and clinical information.TCGA dataset was used as the training cohort and datasets from GEO as validation cohorts.The 2995 immune-related genes were curated from the TCGA pan-cancer immune landscape and association of these gene expression with LUAD prognosis was estimated with univariate Cox proportional hazard model.Identification of molecular subtypes was performed with nonnegative matrix factorization(NMF)algorithm.Tumor Immune Dysfunction and Exclusion(TIDE)method was applied to evaluate the potential immunotherapy efficacy of distinct LUAD subtypes.Differences of PD-L1 and TMB between subtypes were calculated with Wilcoxon rank sum test.Gene set enrichment analysis(GSEA)was used to identify the dysregulated signaling pathways.Significantly mutated genes(SMGs)were identified via Mut Sig CV algorithm.ResultsIn TCGA cohort,433 immune-related genes were significantly correlated with LUAD prognosis(FDR < 0.05).NMF clustering analysis with these 433 genes indicated that model estimation parameters could obtain the maximum value when the clustering number was 2.Survival analysis of these 2 subtypes revealed that they are prognostically significantly different(HR: 1.99,95% CI: 1.43-2.76,Log rank P < 0.001).The result was still significant after taking clinical variables into account(HR: 1.80,95% CI: 1.28-2.53,P < 0.001).The identified 2 subtypes also had significantly distinct prognosis in 8 of 9 GEO cohorts(Log rank P < 0.01),another cohort did not reach statistical significance but exhibited the similar trend(Log rank P = 0.092).The subtype with worse prognosis was named as ‘high-risk’ subtype,another was ‘low-risk’ subtype.Results of TIDE algorithm for TCGA cohort indicated that the high-risk subtype had significantly lower TIDE score than low-risk subtype(Wilcoxon rank sum test,P < 0.001).The lower TIDE score,the weaker potential of immune escape,the better immune microenvironment.Multivariate logistic regression model with clinical variables was still significant(OR: 0.13,95% CI: 0.08-0.20,P < 0.001).These results were also validated in 9 GEO cohorts(Wilcoxon rank sum test,P < 0.001).The high-risk subtype harbored significantly higher PD-L1 expression than lowrisk patients in TCGA and 7 validation cohorts with PD-L1 probes(Wilcoxon rank sum test,P = 0.003).In TCGA cohort,high-risk patients had significantly higher TMB than low-risk patients(Wilcoxon rank sum test,P < 0.001).Multivariate logistic model was still significant with above signatures,clinical variables and mutations of DNA damage repair(DDR)genes taken into account(OR: 3.99,95% CI: 2.19-7.46,P < 0.001).GSEA analysis suggested that cell cycle relevant pathways were markedly found in high-risk patients(FDR < 0.05)and checkpoints in distinct stage were also significantly upregulated in high-risk patients(Wilcoxon rank sum test,P < 0.001).In TCGA cohort,TP53,NAV3,COL11A1,KEAP1 and SMARCA4 were frequently mutated in high-risk subtype using univariate and multivariate analysis(OR > 1,P < 0.01).Association of higher TP53 mutation rate with high-risk subtype was also found in 3 GEO cohorts contained TP53 mutation data(OR > 1,P < 0.01).ConclusionThis study identified 2 LUAD subtypes with distinct prognosis(i.e.,high-risk and low-risk subtypes)by using a total of 2300 LUAD samples and comprehensive immune-related signatures.The high-risk subtype was characterized by lower TIDE score,upregulation of PD-L1 expression,higher TMB,activated cell cycle and TP53 mutation.ICB immunotherapy may be efficacious for high-risk subtype of LUAD.