Abstract
Cholangiocarcinoma (CCA) is a lethal cancer of the bile duct and is a major health concern in several parts of the world, including northeastern Thailand, where CCA incidence is the highest due to the endemic liver fluke Opisthorchis viverrini. Multiple studies have characterised genomic alterations in CCA tumours, and specific chromosomal alterations can predict prognosis. However, it is not known whether chromosomal instability (CIN), ongoing genomic alteration characteristic of most cancer types, is present in CCA tumours. In this study we leveraged a panel of cancer cell lines derived from fluke-positive CCA patients, as well as a matched normal cholangiocyte line as a control, to characterise CIN in CCA. We observed elevated rates of chromosome segregation errors compared to normal cells, although overall CIN rates were lower than those for highly genomically unstable cancers, such as colorectal or ovarian cancer. Chromosome segregation errors in CCA cell lines were potentially driven by elevated DNA replication stress and centrosome duplication. Single-cell genome sequencing and karyotyping of the cell lines showed extensive structural and numerical chromosomal aberrations, as well as copy number alterations that were heterogeneous between individual cells, supporting the presence of ongoing CIN in these cell line models. Low-pass whole-genome sequencing of 33 CCA tumour samples with matched normal tissue from northeastern Thailand, a liver fluke-endemic region showed increased whole and subchromosomal level alterations, with a higher extent of genomic alterations in intrahepatic tumours compared to extrahepatic. Eight tumours carried focal amplifications and/or deletions involving known cancer genes, as well as potential chromosomal instability-associated genes, including CCNE1 amplifications and a rare amplification of BRCA1. This study provides increased understanding of the rate and potential mechanisms of CIN in CCA that may inform new therapeutic strategies that synergise with specific ongoing CIN mechanisms. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.