Neutrophil in the suppressed immune microenvironment: Critical prognostic factor for lung adenocarcinoma patients with KEAP1 mutation

Abstract Purpose: In recent studies, mutation in the Kelch-like ECH-associated protein 1 (KEAP1) was found to be associated with resistance tochemotherapy and immunotherapy in lung adenocarcinoma (LUAD) patients. Nevertheless, some studies have shown that KEAP1 mutation correlates with better objective survival (OS) following immunotherapy. Although it is unclear whether KEAP1 mutation is detrimental to LUAD patients, increasing evidence suggests that KEAP1 mutation enables a suppressed tumour immune microenvironment (TME) in LUAD. The purpose of our study was to analyse the exact changes in the TME in LUAD patients with KEAP1 mutations and to identify key factors influencing prognosis. Experimental design: A total of 1011 patients with lung squamous carcinoma (LUSC) or LUAD with data obtained from The Cancer Genome Atlas (TCGA) were included in this study. The TME and OS of patients with LUAD and LUSC stratified by mutant versus wild-type KEAP1 status were comprehensively measured. Moreover, we classified LUAD patients with KEAP1 mutations into three subtypes, clusters 1, 2 and 3, by unsupervised consensus clustering. We further analysed the TME, OS, commutated genes and metabolic pathways of patients in cluster 2 and clusters 1 and 3 and LUAD patients with wild-type KEAP1. A total of 40 patients who were confirmed lung adenocarcinoma pathologically underwent immunotherapy were collected and classified into KEAP1-MT(mutant KEAP1) groups (n=10)and KEAP1-WT (wild-type KEAP1) groups(n=30). We also conducted immunohistochemical staining in KEAP1-MT groups. Result: The presence of KEAP1 mutations correlated with a significantly lower density of infiltrated immune cells,including CD8+ T cells, CD4+ T lymphocytes, B lymphocytes, macrophages and natural killer cells, and suppressed TME was observed not only in LUAD patients but also in LUSC patients. LUAD patients with mutant KEAP1 underwent immunotherapy had worse PFS than with wild-type KEAP1. Despite, the difference in OS between patients from TGCA with mutant and wild-type KEAP1 was not statistically significant in either LUAD or LUSC patients. Unsupervised consensus clustering analysis suggested that the three subtypes of patients exhibited different densities of neutrophil and type 2 T helper cell (Th2) infiltration and had different OS results: cluster 2 patients had significantly higher levels of neutrophils and Th2 cells and had significantlyworse prognoses than those of patients in clusters 1 and 3 and patients with wild-type KEAP1. Enrichment analysis of differentially expressed genes demonstrated that the nitrogen metabolism pathway may play an important role in thehigh density of infiltrated neutrophils observed in cluster 2 patients. Univariate and multivariate Cox analyses proved that a high density of neutrophils was significantly associated with worse OS and immunohistochemical staining proved that shorter PFS showed high density of neutrophils. Conclusion: KEAP1 mutation significantly suppresses the tumour immune microenvironment in both LUAD and LUSC patients. LUAD patients with mutant KEAP1 underwent immunotherapy had worse PFS than with wild-type KEAP1.Neutrophils and Th2 cells may play a more important role in the prognosis of LUAD patients with KEAP1 mutations and may provide a promising therapeutic target.