Abstract
BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with rising incidence globally, shows heterogeneous responses to immune checkpoint blockade (ICB). DNA methylation dysregulation impacts tumor immunity, necessitating predictive CpG methylation biomarkers for ICB efficacy. METHODS: Genome-wide methylation profiling (TCGA/GEO) identified prognostic promoter CpG islands linked to CD8⁺ T cell infiltration. Single-CpG resolution analysis guided TaqMan probe design, validated in two independent cohorts: non-ICB (n = 335) for prognosis and ICB-treated (n = 45) for immunotherapy prediction. RESULTS: High-throughput methylation analysis identified hypermethylation at the FBLN2 promoter (chr3:13590414-13591008), which correlated with advanced tumor stage, higher grade, and poorer overall survival (OS) and progression-free survival (PFS). This locus exhibited significant associations with CD8⁺ T cell infiltration and IFNγ pathway activation. In the non-ICB cohort, validation using a 5-CpG TaqMan assay confirmed FBLN2 hypermethylation as an independent prognostic marker for both OS (HR = 3.45, P < 0.001) and PFS (HR = 2.43, P = 0.023). Subsequent IHC analysis of 74 tumor specimens demonstrated strong concordance between FBLN2 percent methylation ratio (PMR) and CD8⁺ T cell density (r = 0.62, P < 0.0001). In the anti-PD-1-treated cohort, high FBLN2 methylation was associated with a favorable immunotherapy response (Cohen's weighted Kappa = 0.607, P < 0.001) and predicted superior PFS (median 11.2 vs 3.4 months, HR = 0.35, P = 0.006), suggesting its potential as a novel predictive biomarker for ICB efficacy. CONCLUSIONS: FBLN2 promoter hypermethylation is a promising prognostic biomarker in ccRCC and a potential predictor of immunotherapy response. These findings warrant further investigation to validate its potential for enhancing personalized patient management.