AbstractBackgroundWe aimed to determine the molecular and immune microenvironment characteristics of HER2‐positive gastric cancer (GC) related to the patient's response to first‐line trastuzumab‐based treatment.MethodsEighty‐three cases of HER2‐positive advanced gastric adenocarcinoma patients treated with trastuzumab were enrolled. Targeted deep sequencing and transcriptome analysis were performed on selected 21 cases (exploration cohort) along with two post‐treatment samples. The results were compared between patients progressed before 6 months (Group 2) and others (Group 1), and were validated by FISH and immunohistochemistry in total cohort. Tumor‐infiltrating immune cells were evaluated using RNA sequencing data and multiplex immunohistochemistry. Progression‐free survival (PFS) analysis was performed.ResultsGroup 1 showed frequent amplification of G1/S cell cycle checkpoint‐related genes and upregulated KEGG pathways related to cell proliferation. In contrast, Group 2 had more frequent EGFR, HER3, and MET amplification and higher RNA expression in immune‐related KEGG pathways than Group 1. In total cohort, significant predictors of better PFS were cell cycle‐related including CCNE1 amplification, Cyclin A and PLK1 overexpression, and decreased Cyclin D3 and HER3 expression (p< 0.05), or immune‐related including high density of CD3−CD57+NK cells and PD‐L1 combined positive score ≥5 (p< 0.05). The best prognostic predictors were a combination of Cyclin A, Cyclin E, p21, and HER3 (p< 0.001).ConclusionHER2‐positive GC with favorable response to trastuzumab were characterized by cell cycle‐related gene alterations and increased CD3−CD57+NK cell infiltration. These findings would be helpful to the fine modulation of therapeutic strategies for patients with HER2‐positive GC.