Bile extracellular vesicles for early cholangiocarcinoma detection in primary sclerosing cholangitis.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a major complication of primary sclerosing cholangitis (PSC), with a 20-year incidence of ∼15%. Early detection is challenging, as current surveillance rarely identifies CCA at a resectable stage. This study investigated bile-derived extracellular vesicles (EVs) as a liquid biopsy source for protein biomarkers enabling early diagnosis and prediction of CCA in PSC. METHODS: Bile samples were collected from PSC (n=52), PSC with concomitant CCA (PSC-CCA; n=14), and PSC patients who later developed CCA (PSC to CCA; n=8). EV proteomes were analyzed by mass spectrometry. Multivariable logistic regression models were used to evaluate diagnostic performance. Biomarker expression was assessed in CCA single-cell RNA sequencing (scRNA-seq) datasets, and selected proteins were validated in total serum by ELISA. RESULTS: Proteomic analysis identified 15 EV-proteins that distinguished PSC-CCA from PSC independently of demographic and clinical factors. A four-protein machine learning model (COPA, ATP5H, VTNC, PRDX2) combined with serum CA19-9 achieved high diagnostic performance for PSC-CCA in our cohort (AUC=0.993), outperforming CA19-9 alone (AUC=0.801). A separate three-protein panel (TM9S4, ATPO, KINH) showed potential for early identification of PSC patients who later developed CCA, prior to clinical or radiological detection, with an internally estimated sensitivity of 100% and specificity of 97%, whereas CA19-9 showed no predictive value. COPA, VTNC, and PRDX2 were validated in total serum by ELISA, and scRNA-seq supported the expression of these markers in malignant cholangiocytes. CONCLUSIONS: Bile EVs contain protein biomarkers with promising diagnostic value and potential utility for risk stratification of future CCA development in individuals with PSC. Pending further external validation, as bile collection may be performed during routine ERCP procedures, this liquid biopsy approach could facilitate earlier detection, timely interventions, and improved outcomes. IMPACT AND IMPLICATIONS: Primary sclerosing cholangitis (PSC)-associated cholangiocarcinoma (CCA) remains a major clinical challenge, burdened by ineffective surveillance strategies and poorly sensitive diagnostic tools. This exploratory study identifies bile extracellular vesicle (EV) protein signatures with promising diagnostic performance and potential value for short-term risk stratification or early detection, including biomarker panels that enhance standard CA19-9 performance and detect malignancy prior to clinical or radiological diagnosis. The detectability of some of these biomarkers by ELISA supports their technical feasibility and potential clinical applicability. These findings require validation in larger, independent cohorts before clinical implementation can be considered; if externally validated, bile EV-based biomarkers may represent a complementary tool during routine ERCP procedures, potentially supporting earlier identification of curative treatment candidates and strengthening the rationale for bile-based liquid biopsy approaches in PSC-associated CCA surveillance.