Consensus molecular subtypes and divergent genomic landscapes with clinical implications in biliary tract cancers.

600 Background: Biliary tract cancers (BTC), including cholangiocarcinomas and gallbladder adenocarcinomas, remain poorly understood. Stratifying this heterogeneous group of diseases based on molecular features may lead to novel precision treatments and yield urgently needed improvements in outcomes. Methods: We performed whole-genome and transcriptome sequencing of 165 laser-capture microdissected BTC tumors with paired normal tissues, representing 165 patients. First, we assigned all samples to 28 gene expression-based BTC signatures from nine previously published classifiers to create a signature similarity network. Using Markov clustering on the optimized network, we identified broad consensus molecular subtypes. Next, we used non-negative matrix factorization to decompose the consensus molecular subtype signatures into underlying components to identify associated cell types and processes. Finally, we integrated subtypes with de novo derived mutational signatures and drivers as well as electronic health records to search for prognostic and outcome implications. Results: Published subtyping classifications reflected only two underlying consensus molecular subtypes, which we named BTC-CMS-A and BTC-CMS-B. BTC-CMS-A tumors represented two thirds of intrahepatic cholangiocarcinomas and all mixed hepatocellular carcinomas, but no extrahepatic BTCs or gallbladder cancers, while BTC-CMS-B tumors were found along the whole biliary tract. BTC-CMS-B components exhibited markers of common bile duct epithelium but also shared expression profiles with intestinal Goblet cells, suggestive of an intestinal metaplasia histopathology. In contrast, BTC-CMS-A expressed hepatoblast markers while also exhibiting upregulation of pathways associated with kidney cell lineage. These results were validated by projecting all CMS-associated expression components onto two public single-cell RNAseq datasets. Of 16 drivers identified de novo from WGS, BAP1 and TP53 mutations were most frequent, mutually exclusive, and each was restricted to BTC-CMS-A and BTC-CMS-B, respectively. Mutational signature extraction revealed BTC-CMS-B tumors had increased tumor mutational burden and APOBEC-associated mutational signatures, whereas BTC-CMS-A was associated with increased non-homologous end-joining. Clinically, hepatitis B was more frequent in BTC-CMS-A cases, while patients with primary sclerosing cholangitis exclusively had BTC-CMS-B tumors. BTC-CMS-A was associated with significantly longer overall survival compared to CMS-B, after adjusting for clinically relevant covariates. This finding was validated in three external cohorts. Conclusions: BTC-CMS unify previous BTC classifications and display distinct clinical and genomic profiles and therefore should guide the development of targeted therapies and biomarker-driven trials.