Background: Durvalumab/cis/gem improved overall survival (OS) in pts with advanced BTC versus placebo/cis/gem (Oh et al. NEJM Evid 2022). Disruption of the microbiota may impair tumour response to immunotherapy and chemotherapy and a better understanding of its role in the efficacy of these therapeutics in advanced BTC is required. Methods: This is a multi-centre, single arm trial exploring the microbiome in pts receiving durvalumab 1500 mg intravenously (IV) Q3w, in combination with cis 25 mg/m², gem 1000 mg/m² (Days 1 and 8, Q3w) up to 8 cycles, followed by durvalumab 1500 mg as monotherapy Q4w, until progression or intolerable toxicity. Pts with an ECOG performance status of ≤1 and histologically-proven BTC, including cholangiocarcinoma and gallbladder carcinoma, who have had no prior systemic chemotherapy for locally advanced or metastatic disease are eligible. Pts must provide a saliva and stool sample prior to commencement of durvalumab/cis/gem and at 18 weeks, or at progression (if earlier than 18 weeks). Taxonomic profiling via 16S Ribosomal ribonucleic acid gene sequencing will examine the differences in the diversity and composition of the pt gut microbiome. Pts must also have availability of a tumour biopsy. This study plans to recruit 70 pts from 10 UK centres (over 12 months). The primary objective is to determine the difference in baseline alpha diversity between “responders” (partial or complete response) and “non-responders” at 18 weeks (RECIST 1.1) in patients treated with durvalumab/cis/gem. Secondary objectives include investigation of the association between microbiome parameters and objective response rate, tumour control (partial + complete response + stable disease), progression-free and OS, and to investigate the interaction between treatment effect and microbiome parameters on clinical outcomes. The tumour biopsy will be used for research into the tumour microbiome and/or factors that may influence response to chemotherapy/immunotherapy, including, but not limited to tumour mutation burden, programmed cell death 1/programmed death-ligand 1 status, and microsatellite instability status. Clinical trial information: ISRCTN11210442.