Integrative characterization of the HER2-TLS maturation axis reveals immune heterogeneity and prognostic subtypes in gastric cancer

While HER2 is an established therapeutic target in gastric cancer, HER2-positive tumors remain heterogeneous. Tertiary lymphoid structures (TLS) are organized immune aggregates associated with antitumor immunity, but the relationship between HER2 activity and TLS maturation has not been systematically defined. Bulk transcriptomic, genomic, clinical, single-cell RNA-seq and immunohistochemical data from TCGA-STAD and GSE26253 were integrated. TLS maturation scores were calculated using TLS-related signatures, and HER2 activity was assessed by ERBB2 expression and HER2-axis genes. HER2-TLS phenotypes were defined by combined ERBB2 expression and TLS maturation status. A HER2-TLS Maturation-Related Score (HTMRS) was constructed using WGCNA and LASSO-Cox regression. Single-cell RNA-seq and an independent IHC cohort were used for cellular and tissue-level validation. ERBB2 expression inversely correlated with TLS maturation score. HER2-TLS stratification identified four phenotypes with distinct immune-clinicopathologic features. TLS-mature tumors had higher B-cell, plasma-cell, Tfh-like, cytotoxic T-cell, antigen-presentation and checkpoint-related activity, while HER2-high/TLS-low tumors were immune-cold. HTMRS significantly stratified overall survival in TCGA-STAD and recurrence-free survival in GSE26253. Time-dependent ROC analysis showed TCGA-STAD 1-, 3- and 5-year OS AUCs of 0.660 (95% CI: 0.594-0.720), 0.709 (95% CI: 0.622-0.788) and 0.673 (95% CI: 0.514-0.819), and GSE26253 1-, 3- and 5-year RFS AUCs of 0.540 (95% CI: 0.453-0.630), 0.594 (95% CI: 0.540-0.652) and 0.626 (95% CI: 0.572-0.684). Low-HTMRS tumors showed higher TMB, TCGA MSI subtype enrichment and stronger MSI-related immune programs, whereas high-HTMRS tumors showed increased EMT, TGF-β, cell-cycle and DNA damage response activity. Single-cell and IHC analyses confirmed ERBB2 localization in epithelial cells and TLS-associated activity in immune compartments; across the full IHC cohort, HER2 H-score correlated positively with CD8 density, TLS density and PD-L1 H-score. The HER2-TLS maturation axis captures immune heterogeneity and prognostic risk. HTMRS provides a biological stratification framework with moderate predictive performance for interpreting distinct tumor states. Findings support integrating HER2 activity with TLS maturation for improved biological stratification.