Next generation sequencing mutation analysis on biliary brush cytology for differentiation of benign and malignant strictures in primary sclerosing cholangitis

Open AccessPublished:October 14, 2022DOI:
      This paper is only available as a PDF. To read, Please Download here.


      Background and aims

      Differentiation of benign and malignant biliary tract strictures on brush material remains highly challenging but is essential for adequate clinical management of patients with primary sclerosing cholangitis (PSC). In this case-control study, biliary brush cytology samples from PSC patients with cholangiocarcinoma (PSC-CCA) were compared to samples from PSC patients without CCA (PSC-controls) using next generation sequencing (NGS).


      Cells on archived slides were dissected for DNA extraction. NGS was performed using a gene panel containing 242 hotspots in 14 genes. Repeated brushes from the same patient were analyzed to study the consistency of NGS results. In PSC-CCA cases that underwent surgical resection, molecular aberrations in brushes were compared to NGS data from subsequent resection specimens.


      A total of 40 patients (20 PSC-CCA/20 PSC-controls) were included. The gene panel detected 22 mutations in 15/20 PSC-CCA brushes, including mutations in TP53 (8 brushes), KRAS (5), GNAS (3), ERBB2 (1), APC (1), PIK3CA (1) and SMAD4 (1). One GNAS and three KRAS mutations were found in 3/20 PSC-control brushes. The sensitivity of the NGS panel was 75% (95% CI=62%-80%), and specificity 85% (95% CI=64%-95%). Repeated brushes showed identical mutations in 6/9 cases. Three repeated brushes demonstrated additional mutations as compared to the first brush. In 6/7 patients, mutations in brush samples were identical to mutations in subsequent resection specimens.


      NGS mutation analysis of PSC brush cytology detects oncogenic mutations with high sensitivity and specificity and seems to constitute a valuable adjunct to cytological assessment of brush samples.

      Graphical abstract



      CCA (Cholangiocarcinoma), cfDNA (Cell-free DNA), ctDNA (Circulating tumor DNA), ERCP (Endoscopic retrograde cholangiopancreatography), FISH (Fluorescence in situ hybridization), H&E (Hematoxylin and eosin), IPMN (Intraductal papillary mucinous neoplasm), IPNB (Intraductal papillary neoplasm of the bile duct), LOD (Limit of detection), MGG (May Grunwald-Giemsa), NGS (Next generation sequencing), PSC (Primary sclerosing cholangitis), PSC-CCA (Primary sclerosing cholangitis associated cholangiocarcinoma), VAF (Variant allele frequency)