Characterization Of The Immune Microenvironment And Identification Of Prognosis-related Immune Genes In KRAS-mutant Colorectal Cancer

Objective: KRAS mutation is one of the most common mutation types in colorectal cancer(CRC),and its impact on CRC tumor immunity remains to be elucidated.In this study,we aimed to investigate the effect of KRAS mutation on the immune microenvironment of CRC,identify the immune genes associated with the prognosis of KRAS-mutated CRC,and establish an immune risk prognostic model.Methods: 535 cases of CRC transcriptome sequencing data from The Cancer Genome Atlas(TCGA)database were used as the training set,and 566 CRC cases of transcripts from the Gene Expression Omnibus(GEO)database were used as the validation set and transcriptome sequencing data from 8surgically resected CRC primary tumor samples from the Department of Gastrointestinal Surgery,Affiliated Tumor Hospital of Guangxi Medical University,was served as the internal validation set.The clinical data of 335 CRC patients in our hospital were used as the clinical data set.Patients were divided into KRAS-MT group and KRAS wild-type(KRAS-WT)group according to KRAS gene status.The abundance of Tumors infiltrate immune cells(TIICs)was assessed by TIMER and CIBERSORT.Univariate Cox regression was used to identify prognosis-related TIICs and Immune-related genes(IRGs).A multivariate Cox regression model was used to construct an immune risk model integrating TIICs and IRGs.And the serum inflammatory immune markers were compared between the two groups of patients.Results: Pathway enrichment analysis revealed significant inhibition of immune-related pathways in KRAS-MT CRC patients,such as the B-cell receptor signaling pathway,cytokine receptor interactions,and nuclear factor-κB signaling pathway(p<0.05 for all).Compared to KRAS-WT CRC patients,KRAS-MT CRC patients had significantly higher abundance of regulatory T cells(Tregs)(KRAS-MT: 0.048;KRAS-WT: 0.038;p=0.0012)in the tumor microenvironment,while M1 macrophage(Macrophage M1,M1 MΦ)(KRAS-MT: 0.058;KRAS-WT: 0.063;p=0.045)and activated CD4+ effective memory T cells(CD4+Tem)(KRAS-MT: 0.036;KRAS-WT: 0.045;p= 0.014)were significantly decreased.Among KRAS-MT patients,those with elevated M1 MΦ and CD4+Tem had significantly better overall survival than those with reduced(M1 MΦ: p=0.03;CD4+Tem: p=0.001),while increased Tregs suggested poor prognosis(p=0.006).These phenomena were not observed in KRAS-WT CRC patients.This result was validated in the validation set(M1MΦ: p<0.001;CD4+Tem: p<0.001;Treg: p=0.002).Multivariate COX regression analysis suggested that the abundance of the three cells was an independent prognostic factor for patients with KRAS-MT CRC.Univariate Cox proportional risk regression model analysis for immune-related genes in KRAS-MT CRC suggested that the expression levels of VGF,RLN3 and CT45A1 genes were significantly associated with patient prognosis(VGF:HR=1.81,p=0.025;RLN3: HR= 2.16,p=0.0030;CT45A1: HR=1.98 p=0.0074).Comparison of serum inflammatory immune markers for both groups suggested that C-reactive protein(KRAS-MT: 7.07;KRAS-WT: 10.61;p=0.020),ultrasensitive C-reactive protein(KRAS-MT: 1.60;KRAS-WT: 2.43;p=0.020)and Ig M(KRAS-MT: 0.91;KRAS-WT: 1.08;p=0.024)levels were statistically different in the two groups of patients.By integrating the above immune cells and immune genes,we constructed an immune risk prognostic model for KRAS-MT CRC patients,which had AUCs of 0.76 and 0.68 under the ROC curves of the predicted training and validation sets,respectively.Conclusion: This study systematically compared the characteristics of the immune microenvironment of KRAS-MT and KRAS-WT CRC,the differential IRGs,serum inflammatory immune-related indicators,and constructed an immune risk model for KRAS-MT CRC patients.The results of this study will deepen the understanding of the impact of KRAS mutations on the immune microenvironment of CRC and provide new targets for immunotherapy.The main conclusions are as follows.1.immune-related pathways such as B-cell receptor signaling pathway,cytokine receptor interaction,and nuclear factor κB signaling pathway were significantly down-regulated in KRAS-MT CRC patients,suggesting that KRAS mutations is associated with suppression of immune pathways in CRC patients.2.Immune cells in the KRAS-MT CRC tumor microenvironment were altered,characterized by a significant increase in Tregs abundance and a significant decrease in M1 MΦ and CD4+Tem abundance,suggesting that KRAS mutations is associated with abnormal immune infiltration in CRC.3.KRAS-MT CRC serum C-reactive protein,high-sensitivity C-reactive protein and Ig M levels were significantly decreased,suggesting that KRAS mutations is associated with reduced levels of systemic inflammation in CRC.4.The immune risk model developed by integrating TIICs and IRGs could predict the prognosis of patients with KRAS-mutated CRC.