Shared evolutionary trajectories underlying carcinogenesis and metastasis in biliary tract cancer

Abstract The evolutionary dynamics of biliary tract cancer (BTC)—from precancerous transformation through metastatic dissemination and recurrence—remain poorly understood. We performed multi-regional and longitudinal whole-exome sequencing of 55 tumor specimens and 17 matched normals from 17 patients to reconstruct patient-specific phylogenetic trees and mutational signatures. TP53 (41.2%), KRAS (29.4%), and SMAD4 (23.5%) were the most frequently altered genes in primary tumors. Precancerous biliary intraepithelial neoplasia already harbored canonical driver mutations such as TP53 and KRAS , showing early branching and selective sweeps during malignant transition. During metastasis, distinct subclones expanded more than fivefold in 15 of 17 patients, frequently harboring driver-like alterations (e.g., NFKB1 , MAPK7 , PIK3C2A ). Postoperative recurrences retained the phylogenetic structure of the primary tumor, supporting polyclonal rather than single-cell seeding. Mutational signatures associated with 5-fluorouracil (SBS17b) and platinum (SBS35) were enriched in recurrent/metastatic lesions, suggesting that chemotherapy may shape the tumor mutational landscape. Druggable targets—including ERBB2 amplification and KRAS mutations—were maintained through recurrence and metastasis when present at diagnosis. These findings reveal a consistent evolutionary program in BTC, encompassing early clonal diversification, subclonal expansion during metastasis, and polyclonal recurrence, underscoring the need for evolution-informed therapeutic strategies that address both clonal and subclonal alterations.