Single-cell analysis and histopathological evaluation of tertiary lymphoid structures and association with survival in melanoma.

e21509 Background: Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates composed of immune cells such as B cells, T cells, and dendritic cells (DCs), and have been observed in various solid tumors. TLS are associated with favorable clinical outcomes. While TLS are believed to contribute to adaptive antitumor cellular and humoral responses, the mechanisms driving TLS formation and their immune regulatory functions in tumor immune microenvironment (TIME) remain poorly understood. Methods: We collected formalin-fixed paraffin-embedded (FFPE) tissue sections from 86 MM patients and performed hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and multiplex IHC (mIHC) to assess TLS density, maturation, and distribution. Clinical data were integrated to investigate the relationship between TLS and patient prognosis in MM. Additionally, single-cell data from 20 patients were analyzed to explore the abundance of immune cell types and subtypes in the TIME at single-cell resolution, along with potential intercellular interactions. Results: Based on histopathological analysis of MM samples, patients were stratified into two groups: those poccessing TLS (TLS+, n = 44) and those lacking TLS (TLS-, n = 42). Across the entire cohort, the TLS+ group showed a trend toward improved overall survival (OS), though not statistically significant (p = 0.27). A higher proportion of advanced-stage (T3 and T4) patients in the TLS+ group (T1-T3a: TLS- = 12, TLS+ = 5; T3a-T4b: TLS- = 17, TLS+ = 21) may have influenced the prognostic value of TLS in the overall patient cohort. Further supporting this notion, subgroup analysis restricted to patients with AJCC stage IIB and IIC revealed significantly improved OS in the TLS+ group (p = 0.046). Single-cell data revealed significant differences in the abundance of immune-related cells between the TLS+ and TLS- groups. All B cell subtypes (naïve, memory, germinal center (GC), and plasma cells) were enriched in the TLS+ group compared to the TLS- group. CD4+ and CD8+ naïve T cells also exhibited similar trends, whereas the distribution of CD4+ and CD8+ memory T cells was comparable between the two groups. Furthermore, plasma cells in the TLS+ group were predominantly IgG-producing. Intercellular communication analysis with B cells identified a CAF subpopulation highly expressing TLS-associated genes. A CCL19-CCR7 and CCL21-CCR7 chemokine axis from this CAF subpopulation was observed exclusively in TLS+ tumors but was deficient in TLS- tumors. Multiple T cell subsets exhibited high expression of the CXCL13-CXCR5 axis when communicating with B cells. Conclusions: Our findings highlight the prognostic value of TLS in patients within MM. TLS formation may be associated with specific CAF and T cell subtypes and their interactions with B cells.