The role and mechanism of tertiary lymphoid structure and its associated B cells in the resistance of immune checkpoint inhibitor therapy in acral melanoma.

e14591 Background: Recently studies have suggested that TEX, TLS and B cells are associated with prognosis and sensitivity to ICI therapy in a variety of tumors. Based on these findings, we propose to utilize the previously collected AM and CM samples with different ICI therapy responses to explore the mechanism of ICI therapy resistance of AM by single-cell multi-omics methods. Methods: The subtypes, phenotypes, effector pathways, and regulatory factors of TEX in AM will be detected and investigated. The regional characteristics, cell lineages, maturation levels, and key molecules and signaling pathways of TLS of AM will be detected and analyzed. Classification, BCR clone variations, surface immune checkpoint molecule expression, interactions with other immune cells, and functional regulatory factors of B cells will be explored. Results: 1. TLS Architecture and Maturation: TLS in melanoma exhibit distinct structural organization with a central B-cell zone (CD20+) surrounded by T-cell zones (CD4+, CD8+). Three maturation stages were identified: immature aggregates (Agg), primary follicles (FL1), and secondary follicles (FL2), distinguished by differential expression of CD21 and CD23 markers, with mature TLS showing denser B-cell clusters.2. Prognostic Impact: In a cohort of 174 melanoma patients, the presence and abundance of TLS demonstrated significant prognostic value. Patients with TLS had substantially superior overall survival (OS) and progression-free survival (PFS) compared to those without TLS. Moreover, TLS-rich patients showed better outcomes than TLS-poor patients, with statistically significant differences (p < 0.0001 for OS, p = 0.00021 for PFS).3. Cellular Landscape: Single-cell transcriptomic analysis of 22 patients revealed that TLS-high (TLShi) tumors were enriched in T and B cells, while showing reduced proportions of melanoma cells and endothelial cells compared to TLS-low (TLSlow) tumors.4. Spatial Validation: Stereo-seq spatial transcriptomics confirmed the precise localization of TLS through integrative analysis of histopathology (HE/IHC) and computational deconvolution. TLS signature gene sets accurately colocalized with anatomically defined TLS structures, validating the molecular characterization. Conclusions: This study provides compelling evidence that TLS serve as a robust positive prognostic biomarker in melanoma. The correlation between TLS abundance and improved survival suggests that TLS actively contribute to anti-tumor immunity. The multi-omics approach successfully mapped the spatial architecture and cellular composition of TLS, establishing a foundation for understanding their functional role in the melanoma microenvironment and potential therapeutic targeting.