Distribution and density of tertiary lymphoid structures as predictors of clinical outcomes and immunotherapy responses in gallbladder cancer.

Abstract

4127 Background: Gallbladder cancer (GBC) is an aggressive malignancy with limited treatments and is often immunologically “cold.” Tertiary lymphoid structures (TLS) are key regulators of antitumor immunity, but their spatial heterogeneity and clinical impact in GBC remain unknown. This study explores how TLS distribution and maturation influence outcomes and immunotherapy response in GBC. Methods: We analyzed 99 GBC patients who underwent curative resection (2015–2020). H&E and multiplex immunohistochemistry(mIHC) identified TLS number, maturation, and spatial distribution. TLS were categorized into intratumoral (T) and peritumoral (P) regions. Novel TLS scores (T score, P score) were developed to quantify TLS abundance and correlate with overall survival (OS). Pre-treatment samples from anti-PD-1-treated advanced GBC patients were assessed for TLS association with immunotherapy response. Patient-derived xenograft (PDX) models were used to compare tumor progression and anti-PD-1 efficacy between TLS-high and TLS-low groups. Results: T score correlated with improved OS (p < 0.001), while P score negatively correlated with OS (p < 0.001). Intratumoral TLS contained more Fol-I/Fol-II structures; peritumoral TLS were predominantly Agg (p < 0.001). Responders to immunotherapy had higher intratumoral TLS scores (median 2.5 vs. 0.5, p < 0.01), TLS numbers (8.0 vs. 3.0/ROI, p < 0.05), and TLS area (29.0% vs. 12.0%, p < 0.05). Intratumoral TLS showed higher CD8⁺ T cells, CD4⁺ T cells, and Tfh cells (all p < 0.05), whereas peritumoral TLS were enriched in Tregs (p < 0.01). Higher T scores linked with increased CD8⁺ T cells, Tfh cells, CD21⁺CD23⁺ FDC, and LAMP3⁺ DCs, but decreased Tregs and M2 macrophages (all p < 0.05). An immune classification based on T/P scores stratified patients into four prognostic subgroups with distinct OS (p < 0.001). In PDX models, TLS-high tumors progressed slower (p < 0.05) and responded better to anti-PD-1 therapy(p < 0.01). Conclusions: TLS spatial distribution in GBC exerts opposing prognostic effects: intratumoral TLS promote antitumor immunity, while peritumoral TLS may support tolerance. TLS-high tumors show greater sensitivity to immunotherapy. A TLS-based immune classification may guide personalized treatment.