BTLA promoter hypomethylation correlates with enhanced immune cell infiltration, favorable prognosis, and immunotherapy response in melanoma

Background Immune checkpoint blockade (ICB)-based immunotherapy has significantly improved survival in advanced melanoma. However, many patients exhibit resistance to these therapies. This study examines the impact of BTLA promoter methylation on its expression, immune cell infiltration, and clinical outcomes, evaluating its potential as a prognostic and predictive biomarker for immunotherapy response.Methods We analyzed methylation and gene expression data from public datasets (The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO)) and an in-house cohort of melanoma patients treated with ICB therapy at the First Affiliated Hospital of Zhengzhou University. We developed a quantitative methylation-specific PCR (qMSP) assay to measure methylation levels of the cg24157392 and cg03995631 CpG sites, and a targeted bisulfite sequencing assay was used to validate the accuracy of qMSP. We measured BTLA protein expression using multiplex immunofluorescence and immunohistochemical staining methods. Pearson correlation, survival analysis, and immune cell infiltration estimation were conducted to explore the associations between BTLA promoter methylation, mRNA and protein expression, clinical outcomes, and immune characteristics.Results Hypomethylation at CpG sites cg24157392 and cg03995631 in the BTLA promoter were significantly associated with higher BTLA mRNA and protein expression. In the TCGA dataset, low methylation at these sites predicted longer overall survival and was validated in an independent cohort of 50 stage III/IV melanoma patients, with an area under the curve of 0.94 for predicting 5-year survival. Furthermore, BTLA promoter hypomethylation correlated with higher infiltration of immune cells, such as CD8+T cells, CD4+T cells, B cells, and macrophages. Additionally, low methylation at cg24157392 and cg03995631, as quantified by the qMSP assay, was significantly associated with better progression-free survival in patients treated with immune checkpoint inhibitors. These findings were further validated using GEO datasets.Conclusions BTLA promoter hypomethylation serves as a significant biomarker for favorable prognosis and enhanced response to ICB therapy in melanoma. The developed qMSP assays for cg24157392 and cg03995631 accurately quantified methylation levels and demonstrated their potential for clinical application in patient stratification and personalized immunotherapy.