Objective To explore the role and potential molecular mechanism of CDKN2A homozygous deletion in mediating resistance to anti-programmed death 1/ programmed death-ligand 1 (PD-1/PD-L1) immunotherapy in non-small cell lung cancer (NSCLC).

Methods A total of 486 NSCLC patients who received immunotherapy were enrolled as the study cohort, and 348 patients from the MSK-IMPACT cohort were included as the external validation cohort. The genetic variation status of CDKN2A was detected by next-generation sequencing (NGS). Techniques including RNA sequencing (RNAseq), single-cell RNA sequencing (scRNA-seq), multiplex immunofluorescence immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay (ELISA), combined with bioinformatics analyses, including differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA), and survival analysis, were used to investigate the effects of CDKN2A homozygous deletion on the tumor microenvironment, antigen presentation pattern, and related signaling pathways.

Results Thirteen percent (63/486) of patients in the study cohort had CDKN2A homozygous deletion. These patients had significantly shorter overall survival 13.2 months vs. 24.9 months, hazard ratio (HR)=2.71, 95% confidence interval (CI) 1.67~4.36, P<0.001 and progression-free survival (8.4 months vs. 13.2 months, HR=2.73, 95%CI 1.85~4.01, P<0.001) than wild-type patients, and this result was validated in the external cohort (14.7 months vs. 26.2 months, HR=1.56, 95%CI 1.15~2.34, P=0.012). Multivariate Cox regression analysis confirmed that CDKN2A homozygous deletion was an independent poor prognostic predictor independent of PD-L1 expression and tumor mutational burden (TMB) (HR=0.43, P=0.003). Patients with CDKN2A homozygous deletion showed significantly reduced tumor microenvironment immune infiltration gene expression profile score (GEP score) and cytolytic activity score (CYT score). Gene set enrichment analysis (GSEA) revealed enrichment of the major histocompatibility complex class Ⅱ (MHC-Ⅱ) antigen presentation pathway and cell cycle pathway, along with downregulation of the T cell activation pathway in these patients. Additionally, MHC-Ⅱ was abnormally highly expressed on the surface of tumor cells. Single-cell analysis showed decreased CD8⁺ T cell infiltration and increased differentiation of CD4⁺ T cells into regulatory T cells (Treg) in the tumor microenvironment of patients with CDKN2A homozygous deletion. Further mechanistic validation demonstrated that CDKN2A homozygous deletion activated the PI3K/Akt pathway to promote transforming growth factor-β (TGF-β) secretion, synergistically inducing the formation of an immunosuppressive tumor microenvironment.

Conclusions CDKN2A homozygous deletion mediates NSCLC immunotherapy resistance by aberrantly regulating the MHC-Ⅱ antigen presentation pattern and activating the PI3K/Akt-TGF-β pathway, which induces Treg enrichment and the formation of an immunosuppressive tumor microenvironment. This study provides a basis for clinically precise screening of patients who may benefit from immunotherapy and formulating strategies to reverse resistance.