IntroductionMetastatic biliary tract cancer (BTC) is a rare and aggressive entity associated with poor prognosis. It represents a major challenge for adequate treatment strategies. In recent years, BTC has become a model for precision medicine in gastrointestinal oncology. Therefore, the analysis of the individual molecular profile in BTC patients may lead to targeted therapies for the benefit of patients.MethodsIn this Austrian, tricentric, real-world, retrospective analysis, we investigated patients diagnosed with metastatic BTC who underwent molecular profiling between 2013 and 2022.ResultsIn total, 92 patients were identified in this tricentric analysis and 205 molecular aberrations, including 198 mutations affecting 89 different genes in 61 patients were found. The predominant mutations were inKRAS(n=17; 22.4%),TP53(n=17; 22.4%),PIK3CA(n=7; 9.2%),FGFR2(n=7; 9.2%),DNMT3A(n=7; 9.2%),IDH1(n=7; 9.2%),IDH2(n=6; 7.9%),CDKN2A(n=6; 7.9%),BAP1(n=4; 5.3%),NF1(n=4; 5.3%), andNF2(n=4; 5.3%). Three patients hadHER2amplification. MSI-H status andFGFR2fusion genes were each observed in two different patients. One patient had aBRAF V600Emutation. Eventually, 10 patients received targeted therapy, of whom one-half derived clinical benefit.ConclusionsMolecular profiling of BTC patients is implementable in routine clinical practice and should be regularly employed to detect and exploit molecular vulnerabilities.