28Biliary tracts cancer consensus molecular subtypes improve outcome stratification over anatomy

Abstract Background & Objectives Biliary tract cancer (BTC) groups a family of rare cancers with high recurrence rates after surgery and poor survival, especially in advanced disease. Several molecular classification systems based on transcriptomics demonstrate prognostic relevance, however clinical implementation has been limited by a lack of consensus or elucidation of their biological underpinnings. Methods We performed whole genome and transcriptome sequencing of 169 tumors enriched with laser capture microdissection and annotated with clinical records. Results Network integration and clustering on existing classifiers revealed that BTC transcriptomes can be summarized into two consensus molecular subtypes (BTC-CMS). BTC-CMS-A tumors almost exclusively carry the clinically actionable alterations characteristic of BTC (IDH1 mutations and FGFR2 fusion). These tumors are restricted to the liver and confer superior survival after surgery (P = 9.5e-05) and in advanced disease (P = 0.02) in cohorts without precision therapy treatments. BTC-CMS-B tumors span the biliary tree and are associated with chronic inflammation risk factors including primary sclerosing cholangitis (P = 0.016), fluke infection (P = 1.8e-10) and cholecystitis (P = 0.0029). These tumors exhibit mutations characteristic of pancreatic cancer (TP53, SMAD4, CDKN2A and KRAS), a connection further supported by similarities in their transcriptomic profiles. BTC-CMS can also be reliably differentiated by tumour proteomes and methylomes, offering multiple modalities for tumor classification. de novo expression signature extraction by non-negative matrix factorization and subsequent projection of these signatures onto single cell RNAseq data supports that individual cells from a given tumour are exclusively one subtype, with divergent copy number and expression profiles. Furthermore, we show patient tumors are restricted to one subtype across metastases and through progression or recurrence, suggesting these are immutable programmes. Conclusion The BTC-CMS robustly defines two distinct molecular lineages of BTC that improve clinical and biological stratification over anatomy atone.