Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation

Background Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. Methods Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. Results Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. Conclusions EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.