Detection of human aspartyl (asparaginyl) beta-hydroxylase and homeobox B7 mRNA in brush cytology specimens from patients with bile duct cancer

Background and Study Aims: The diagnosis of bile duct cancer is hampered by the low sensitivity of intraductal brush cytology and forceps biopsy. In the present study real-time reverse transcription polymerase chain reaction (RT-PCR) assays for the detection of human aspartyl (asparaginyl) beta-hydroxylase (HAAH) and homeobox B7 (HoxB7) mRNA from intraductal brush cytology specimens were established. Both markers are overexpressed in biliary cancer cell lines and possibly involved in the pathogenesis of bile duct cancer. Patients and Methods: RT-PCR assays were validated for detection limit, in-assay variability, and inter-assay variability. Target gene expression was determined in brush cytology specimens from 16 patients with biliary strictures (11 with histologically proven cholangiocarcinomas and five with benign biliary strictures). Results: The assay was quick (about 3 h), highly sensitive (with detection limits between 3 and 106 molecules), and reproducible (maximum in-assay variability 10.3%, maximum inter-assay variability 11.8 %). The sensitivity of routine brush cytology alone was 36% (four of 11 cases), with 100% specificity. A combination with detection of HoxB7 and HAAH mRNA increased the overall diagnostic sensitivity to 82%. Conclusions: Detection of these markers using the RT-PCR assays from brush cytology specimens described here may prove to be a useful additional tool for the diagnosis of bile duct carcinoma.