AbstractIn this multi-center phase II trial, we evaluated the efficacy and safety of a quadruplet regimen (pembrolizumab, trastuzumab, and doublet chemotherapy) as first-line therapy for unresectable or metastatic human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (AGC) (NCT02901301). The primary endpoints were recommended phase 2 dose (RP2D) for phase Ib and objective response rate (ORR) for phase II. The secondary endpoints included progression-free survival (PFS), overall survival (OS), duration of response, time to response and safety. Without dose-limiting or unexpected toxicities, the starting dose in the phase Ib trial was selected as RP2D. In 43 patients, the primary endpoint was achieved: the objective response rate was 76.7% (95% confidence interval [CI]: 61.4–88.2), with complete and partial responses in 14% and 62.8% of patients, respectively. The median progression-free survival, overall survival, and duration of response were 8.6 months, 19.3 months, and 10.8 months, respectively. No patients discontinued pembrolizumab because of immune-related adverse events. Programmed death ligand-1 status was not related to survival. Post hoc analyses of pretreatment tumor specimens via targeted sequencing indicated that ERBB2 amplification, RTK/RAS pathway alterations, and high neoantigen load corrected by HLA-B were positively related to survival. The current quadruplet regimen shows durable efficacy and safety for patients with HER2-positive AGC.