Distinct maturity and spatial distribution of tertiary lymphoid structures in head and neck squamous cell carcinoma: implications for tumor immunity and clinical outcomes

The influence of tertiary lymphoid structures (TLSs) on disease progression and the response to immunotherapy in head and neck squamous cell carcinoma (HNSCC) is well established, yet the heterogeneity among these structures remains largely unexplored. We utilized digital spatial profiling technology to perform in situ transcriptomic sequencing of TLSs across varying levels of maturation and distinct tumor regions within HNSCC. We assessed the prognostic significance of TLS maturation and spatial distribution in 260 patients with HNSCC through hematoxylin and eosin staining and multiplex immunohistochemistry. Furthermore, we established a TLS scoring system to predict survival in patients with HNSCC. Our study revealed that mature TLSs in the intratumor region (Intra-TLSs) of HNSCC, enriched with memory B cells, plasma cells, and CD4⁺ T cells, presented increased B-cell activity gene expression. Conversely, early TLSs (E-TLSs), abundant in endothelial cells, fibroblasts, and regulatory T cells, express epithelial‒mesenchymal transition (EMT)-related genes, potentially fostering tumor growth. Compared with mature TLSs within the peritumoral region (Peri-TLSs), mature Intra-TLSs have greater memory B-cell and macrophage densities and upregulate genes involved in B-cell receptor signaling and immune effector processes. Mature Peri-TLSs, characterized by endothelial cell enrichment and EMT receptor interaction genes, may contribute to tumor progression and immune evasion. Patients with mature Intra-TLSs or invasive margin TLSs (Invas-TLSs) have improved 5-year survival, whereas those with mature Peri-TLSs have poorer prognoses. By integrating TLS maturity and distribution in HNSCC, we developed a TLS scoring system to guide personalized treatments, which is crucial for predicting outcomes.