Serum proteomic profiling identifies pathways and protein signatures associated with early versus late recurrence in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by frequent postoperative recurrence, yet reliable biomarkers for distinguishing early from late recurrence remain limited. This study aimed to identify serum protein biomarkers associated with early versus late recurrence and develop a proteomic recurrence score (PRS) to distinguish these recurrence groups after curative resection. Serum proteomic profiling was performed using LC-MS/MS in patients with resected CCA. Candidate proteins were identified through integrated univariate and multivariate analyses, followed by pathway enrichment and protein-protein interaction analyses. Proteins with ≥50% detection frequency were refined for model development, and elastic net - based feature selection within a repeated nested cross-validation framework was used to derive the proteins for construction of the PRS. A total of 232 candidate proteins associated with recurrence were identified, highlighting enrichment of immune/inflammatory signaling, metabolic reprogramming, and tumor progression pathways. Refinement yielded 33 consistently detectable proteins. During nested model-size optimization, the 20-protein candidate model achieved a patient-level OOF ROC-AUC of 0.937 (95% CI, 0.881-0.979). Based on feature-selection stability, the 20 most stable proteins were subsequently prioritized as a fixed exploratory signature for PRS construction, achieving a patient-level OOF ROC-AUC of 0.948 (95% CI, 0.895-0.988). A PRS derived from an exploratory 20-protein signature was significantly associated with clinicopathological variables and independently associated overall survival in multivariable Cox regression. Comprehensive serum proteomic profiling identified biological pathways associated with recurrence timing and enabled the prioritization of an exploratory 20-protein PRS for distinguishing early from late recurrence among patients with CCA. The PRS demonstrated promising internal discrimination and was independently associated with overall survival. External validation in independent cohorts is required before clinical application.