Jie Dai

Increased immune cell infiltration and enriched immune-related pathways in PMME compared with NEMM. A, Comparison of infiltrated immune cells between PMME and NEMM. B, Significantly enriched immune-related pathways identified through GSEA when comparing PMME and NEMM.

Supplementary Table 3. The immune-related pathways that were enriched in TERT methylation high group versus TERT methylation low group using GSEA analysis.

Supplementary Table 1. The univariate and multivariate COX analyses for OS associated DMRs in PMME patients.

Supplementary Figure S3. Heatmap depicting the association between methylation status of TERT and genes within 7-DMR panel and the immune cell infiltration in NEMM.

Increased immune cell infiltration and enriched immune-related pathways in PMME compared with NEMM. A, Comparison of infiltrated immune cells between PMME and NEMM. B, Significantly enriched immune-related pathways identified through GSEA when comparing PMME and NEMM.

Supplementary Table 2. The immune-related pathways that were enriched in PMME when compared with NEMM using GSEA analysis.

Supplementary Table 2. The immune-related pathways that were enriched in PMME when compared with NEMM using GSEA analysis.

Mucosal melanoma exhibits limited responsiveness to anti-PD-1 therapy. However, a subgroup of mucosal melanomas, particularly those situated at specific anatomic sites like primary malignant melanoma of the esophagus (PMME), display remarkable sensitivity to anti-PD-1 treatment. The underlying mechanisms driving this superior response and the DNA methylation patterns in mucosal melanoma have not been thoroughly investigated. We collected tumor samples from 50 patients with mucosal melanoma, including 31 PMME and 19 non-esophageal mucosal melanoma (NEMM). Targeted bisulfite sequencing was conducted to characterize the DNA methylation landscape of mucosal melanoma and explore the epigenetic profiling differences between PMME and NEMM. Bulk RNA sequencing and multiplex immunofluorescence staining were performed to confirm the impact of methylation on gene expression and immune microenvironment. Our analysis revealed distinct epigenetic signatures that distinguish mucosal melanomas of different origins. Notably, PMME exhibited distinct epigenetic profiling characterized by a global hypermethylation alteration compared with NEMM. The prognostic model based on the methylation scores of a 7-DMR panel could effectively predict the overall survival of patients with PMME and potentially serve as a prognostic factor. PMME displayed a substantial enrichment of immune-activating cells in contrast to NEMM. Furthermore, we observed hypermethylation of the TERT promoter in PMME, which correlated with heightened CD8+ T-cell infiltration, and patients with hypermethylated TERT were likely to have improved responses to immunotherapy. Our results indicated that PMME shows a distinct methylation landscape compared with NEMM, and the epigenetic status of TERT might be used to estimate prognosis and direct anti-PD-1 treatment for mucosal melanoma.Significance:This study investigated the intricate epigenetic factor of mucosal melanomas contributed to the differential immune checkpoint inhibitor response, and found that PMME exhibited a global hypermethylation pattern and lower gene expression in comparison to NEMM. TERT hypermethylation may contribute to the favorable responses observed in patients with mucosal melanoma undergoing immunotherapy.

Demographic and clinical characteristics of the patients with PMME and NEMM included in the study

Supplementary Table 3. The immune-related pathways that were enriched in TERT methylation high group versus TERT methylation low group using GSEA analysis.