Clinical and immunological significance of tertiary lymphoid structure maturation heterogeneity in brain metastases of lung adenocarcinoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that arise within the tumor microenvironment and orchestrate local immune responses. While TLSs have been associated with favorable prognosis and enhanced immunotherapy efficacy in various solid tumors, their heterogeneity, spatial organization, and functional significance in brain metastases from lung adenocarcinoma (LUAD-BrM) remain poorly understood. METHODS: We retrospectively analyzed 283 surgically resected LUAD-BrM specimens. TLSs were identified on H&E and classified by serial IHC into immature (E-TLS) and mature subtypes (PFL-TLS/SFL-TLS). Multiplex IHC incorporating follicular network and vascular-stromal markers (including CD35, CD31, and COL1A1) was used for spatial validation. Survival was assessed using Kaplan-Meier and multivariable Cox models. RESULTS: Patients were stratified into TLS-negative (n = 200), immature TLS (n = 43), and mature TLS (n = 40) groups. Spatial mapping revealed that mature TLS subtypes were more frequently intratumoral, whereas early TLSs were relatively enriched at peritumoral regions. Clinically, mature TLSs were associated with significantly prolonged survival (median PFS: 14.8 vs. 9.9 vs. 6.3 months; median OS: not reached vs. 13.3 vs. 10.2 months; all P < 0.001) and remained an independent favorable prognostic factor. Multiplex IHC profiling demonstrated that mature TLSs were associated with an immune-activated niche characterized by higher CD8⁺ infiltration, reduced PD-1⁺CD8⁺ exhaustion-like features, and decreased immunosuppressive macrophage and PD-L1-associated signals. Single-slide spatial mIHC further validated mature TLS architecture, demonstrating stronger B/T compartmentalization, prominent follicular network features, and enriched vascular-stromal contextualization compared with immature TLSs. CONCLUSION: TLSs in LUAD brain metastases exhibit marked heterogeneity in maturation and spatial distribution, which stratifies immune contexture and patient outcomes. Mature, intratumoral TLSs define an immune-permissive niche and are associated with significantly improved survival. Collectively, these results highlight TLS maturity as a clinically actionable feature for risk stratification and may inform future TLS-guided precision immunotherapy approaches in LUAD-BrM.