Glycoproteomics-based cancer marker discovery adopting dual enrichment with Wisteria floribunda agglutinin for high specific glyco-diagnosis of cholangiocarcinoma

Cholangiocarcinoma (CC) is a lethal malignancy because it exhibits asymptomatic growth infiltrating the surrounding structures and therefore 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 relevant surgical resection. Since the definitive CC diagnosis depends on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. We therefore developed a CC marker with high specificity by the Wisteria floribunda agglutinin (WFA)-assisted glycoproteomics approach. WFA-positive glycoproteins were enriched by the direct dissection of the WFA-stained CC tissue region and following WFA-agarose column chromatography. Subsequent analysis by mass spectrometry identified 71 proteins as candidate markers. Screening of these candidates by gene expression profiling and immunohistochemistry resulted in the selection of L1 cell adhesion molecule (L1CAM) as the most specific CC marker. We confirmed the importance of WFA-positivity for L1CAM using both bile and serum of CC and benign bile duct disease patients. Specifically, WFA-positive L1CAM was enriched from serum by the WFA-assisted affinity capturing, with which CC was efficiently distinguished from benign. In the primary verification study using bile from CC patients (n = 29) and that of benign bile duct disease (n = 29), WFA-positive L1CAM distinguished CC with high 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 WFA-positive L1CAM assay with the high sensitive assay detecting WFA-positive sialylated mucin 1 sufficiently improved the diagnostic accuracy of CC (overall accuracy = 0.84, AUC = 0.93). This combination will possibly be a precise procedure for CC diagnosis compared with conventional diagnostic techniques. Biological significance In this study, we constructed the system for verification of the candidate molecules that exhibit disease specific glyco-alterations and discovered a useful CC marker by the glycoproteomics-assisted strategy for biomarker discovery. Based on the strategy, we previously found that WFA is the best probe to detect CC-specific glycosylation and WFA-positive sialyl MUC1 as a possible biomarker candidate. While the diagnostic specificity of WFA-positive sialyl MUC1 was not superb, we proposed a new biomarker candidate WFA-positive L1CAM with high specificity in bile and serum to complement the previous one. We proved that the novel combination assay of WFA-L1CAM and WFA-sialyl MUC1 selected based on our strategy has the possibility to become a reliable serological test. This study represents application of our strategy, which can be extrapolated to discovery of marker candidates for other diseases. (C) 2013 Elsevier B.V. All rights reserved.