<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Gastric cancer (GC) is a heterogenous disease consisted of several subtypes with distinct molecular traits. The clinical implication of molecular classification has been limited especially in association with treatment efficacy of ramucirumab or various targeted agents.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We conducted a prospective non-randomized phase II single-arm trial of ramucirumab plus paclitaxel as second-line chemotherapy in 62 patients with metastatic GC who failed to respond to first-line fluoropyrimidine plus platinum treatment. For integrative molecular characterization, all patients underwent pre-ramucirumab treatment tissue biopsy for whole-exome/whole-transcriptome sequencing to categorize patients based on molecular subtypes. We also systematically performed integrative analysis, combining genomic, transcriptomic, and clinical features, to identify potential molecular predictors of sensitivity and resistance to ramucirumab treatment.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Sixty-two patients were enrolled in this study between May 2016 and October 2017. Survival follow-up in all patients was completed as of the date of cut-off on January 2, 2019. No patient attained complete response (CR), while 22 patients achieved confirmed partial response (PR), resulting in a response rate (RR) of 35.5% (95% CI, 23.6–47.4). According to TCGA molecular classification, there were 30 GS, 18 CIN, 3 EBV, and 0 MSI tumors. The RR was 33% in GS (10/30), 33% in CIN (6/18), and 100% in EBV-positive GC patients with significant statistical difference for EBV(+) against EBV(−) tumors (<jats:italic>P</jats:italic> = 0.016; chi-squared test). Moreover, responsive patients were marked by activation of angiogenesis, VEGF, and TCR-associated pathways, while non-responder patients demonstrated enrichments of sonic hedgehog signaling pathway and metabolism activity. Integrative multi-layer data analysis further identified molecular determinants, including EBV status, and somatic mutation in <jats:italic>GNAQ</jats:italic> to ramucirumab activity.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Prospective molecular characterization identified a subset of GC patients with distinct clinical response to ramucirumab therapy, and our results demonstrate the feasibility of personalized therapeutic opportunities in gastric cancer.</jats:p> </jats:sec><jats:sec> <jats:title>Trial registration</jats:title> <jats:p>The study was registered on ClinicalTrial.gov (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://clinicaltrials.gov/ct2/show/NCT02628951">NCT02628951</jats:ext-link>) on June 12, 2015.</jats:p> </jats:sec>