Verification of Wisteria floribunda agglutinin-positive glycoproteins as a cholangiocarcinoma marker

Cholangiocarcinoma (CC) is a lethal malignancy which exhibits asymptomatic growth infiltrating the surrounding structures, and thus,CC is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing the surgical resection. Since the definitive CC diagnosis relies on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. In the previous meeting, we reported that Wisteria floribunda agglutinin (WFA) is the best probe lectin which reliably distinguishes between CC and normal bile duct epithelia in tissue sections. Moreover, L1 cell adhesion molecule (L1CAM), CA125, and maspin were assigned as the reliable CC marker candidates by WFA-assisted glycoproteomics and immunohistochemistry. In this meeting, we will introducethe verification and validation process in one of the above candidates, L1CAM. Since the serum concentration of L1CAM was low as described in other reports (< 5 ng/mL), we firstly constructed a highly-sensitive detection system to confirm the existence of L1CAM in both bile and serum of CC patients with immunoprecipitation and western blotting. We then performed highly-sensitive glycan profiling with antibody-assisted lectin microarray (limit of detection: 25 pg) and confirmed WFA-positivity of biliary L1CAM from the CC patients. The subsequent validation study using bile samples from CC patients (n = 29) and patients with benign bile duct diseases (n = 29) showed that WFA-positive L1CAM distinguished CC from the benigndiseases with good specificity (sensitivity = 0.66, specificity = 0.93, overall accuracy = 0.79, area under the receiver operating curve [AUC] = 0.82). The combined use of the L1CAM assay with the highly-sensitive assay detecting WFA-positive sialylated mucin 1 (WFA-sialyl MUC1), a reliable CC marker (Matsuda A., et al., Hepatology, 2010), sufficiently improved the diagnostic accuracy of CC (overall accuracy = 0.84, AUC = 0.93). This combination assay using WFA–L1CAM and WFA–sialyl MUC1 will possibly be a useful serological test with enhanced reliability.