Neoadjuvant chemotherapy interferes with tertiary lymphoid structure formation in Epstein-Barr virus-associated gastric cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a molecularly distinct, highly immunogenic gastric cancer subtype, yet the role of tertiary lymphoid structure (TLS) during therapy remains unclear. Using a large locally advanced EBVaGC cohort (144 samples), combined with spatial transcriptomics (11 samples) and a single-cell atlas (77 samples), we show that EBVaGC exhibits enhanced TLS formation compared with EBV-negative gastric cancer, with invasive-margin TLS (IM-TLS) predicting favorable prognosis. Neoadjuvant chemotherapy disrupts TLS formation and shows limited efficacy in EBVaGC. Recurrent tumors display increased neutrophil infiltration around TLS, with aging neutrophils recruited via the CXCR4-CXCL12 axis. Mechanistically, chemotherapy induces EBV reactivation, activating neutrophil pattern recognition receptors and promoting neutrophil extracellular trap (NET) formation, which impairs lymphocyte migration and TLS maturation. These findings identify EBV reactivation-driven neutrophil aging and NET formation as a mechanism underlying poor prognosis in EBVaGC, positioning IM-TLS as a prognostic biomarker and neutrophil dysfunction as a therapeutic target.