AbstractBackgroundCommon treatments for metastatic/unresectable HER2‐negative gastric cancer include chemotherapy, immune checkpoint inhibitor monotherapy and chemotherapy plus immune checkpoint inhibitor. However, significant drug resistance exists regardless of the treatment regimen.MethodsPatients with metastatic/unresectable HER2‐negative gastric/gastroesophageal junction adenocarcinoma were enrolled. All patients were divided into three groups according to the treatment regimen and were further divided into responders and non‐responders according to efficacy evaluation. Metagenomics sequencing were performed to analyze gut microbiome signature of patients receiving different treatments at baseline and throughout treatment.ResultsOne hundred seventeen patients with HER2‐negative advanced gastric or gastroesophageal junction adenocarcinoma receiving chemotherapy alone, anti PD‐1/PD‐L1 immunotherapy alone or combined regimen were included in this study. Microbiome signatures related to clinical response are distinct among the three treatment groups. Among which, 14, 8 and 13 species were significantly different between responders and non‐responders in immunotherapy, immunotherapy plus chemotherapy and chemotherapy group, respectively. Patients with higher relative abundance ofLactobacilluspossessed higher microbiome diversity and significantly better response to anti‐PD‐1/PD‐L1 immunotherapy and had a trend to achieve better progression‐free survival. Another cohort of 101 patients has been used as an external validation set to confirm the stability and reliability of these findings.ConclusionsGut microbiome affects response of treatments in HER2‐negative advanced gastric cancer in a treatment‐specific way, immunotherapy plus chemotherapy did not equal to a simple superposition of immunotherapy and chemotherapy.Lactobacillusis expected to become a novel choice as an adjuvant agent in promoting the efficacy of immunotherapy in gastric cancer.