A proteomics-based approach for the identification of biliary markers of cholangiocarcinoma

Prognosis for patients with cholangiocarcinoma (CCA) continues to be poor as a result of the difficulty in distinguishing malignant from benign bile duct disease, late stage diagnosis and a lack of sufficiently sensitive and specific diagnostic markers. These factors underlie the pressing clinical need for novel disease biomarkers. The utility of bile as a proximal fluid for biomarker discovery (compared to serum) was investigated using two dimensional difference gel electrophoresis (2D DIGE). Significant differences between the proteomes of bile and serum were identified, supporting the hypothesis that bile offers a potentially enriched microenvironment of proteins shed/secreted by tumour. However, as with serum, a few major abundant proteins dominate the bile proteome, therefore an albumin/IgG depletion technique was optimised to improve biomarker identification. The bile proteome was initially characterised in samples from patients with hilar CCA. A protein mastermap was generated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) and a catalogue of proteins by liquid chromatography – tandem mass spectrometry (LC-MS/MS), which identified 80 and 813 unique proteins respectively. This represents one of the largest compendiums to date and forms a basis for future proteomic-based biomarker studies. A comparative analysis of biliary proteins in CCA and benign biliary disease was performed using a label-free proteomic approach to identify potential diagnostic biomarker(s). Comparative analysis of bile protein profile in 5 patients with CCA versus benign biliary disease identified 13 proteins which were at higher levels in malignant disease of which metalloproteinase-9 (MMP-9), Rho GDP-dissociation inhibitor 2 (also known as Ly-GDI), Annexin A3 and pre-B-cell colony-enhancing factor (PBEF) were taken forward in immunoblotting-based validation. MMP-9 was shown to be overexpressed in bile of CCA and represents a potential diagnostic marker. In addition analysis of bile samples showed lipocalin-2 and its complex with MMP-9 were present in greater amounts in CCA compared to benign biliary disease.