Targeted isolation of tumor-derived extracellular vesicles using a cocktail of magnetic nanospheres for minimally invasive pancreatic cancer diagnosis

Pancreatic ductal adenocarcinoma (PDAC) has a poor survival rate, largely due to late detection and a lack of effective blood-based diagnostics. Circulating extracellular vesicles (EVs) carry tumor-derived DNA, but efficient and specific isolation of cancer-relevant EVs from plasma is difficult. We developed a minimally invasive workflow to enrich PDAC-associated EVs from plasma. This method employes a three-antibody magnetic nanosphere cocktail (CA19-9, EpCAM, mesothelin) to capture PDAC-associated EVs, and interrogates EV-DNA for KRAS mutations by droplet digital PCR (ddPCR). We evaluated the diagnostic performance of this method for PDAC in two independent cohorts. In a training cohort of 20 PDAC patients (predominantly stage IV) and 10 healthy donors (HD), KRAS mutations were detected with 100% sensitivity, 90% specificity, and an area under the ROC curve (AUC) of 0.91. In an independent validation cohort of 26 PDAC patients spanning stages I–IV and 8 HD, KRAS mutations were detected with 76.9% sensitivity, 87.5% specificity, and AUC 0.84. Stage-stratified pooled analysis showed early-stage performance of KRAS mutations with 78.9% sensitivity, 88.9% specificity, and AUC 0.795, and advanced-stage performance of KRAS mutations with 92.6% sensitivity, 88.9% specificity, and AUC 0.93. The higher EV-DNA yield and KRAS mutant allele fraction in advanced-stage disease support the biological plausibility of our findings. This antibody-cocktail nanosphere approach enables sensitive and minimally invasive detection of tumor-derived EV-DNA from plasma, with promising potential for PDAC diagnosis and monitoring. Larger prospective studies are warranted to validate early-stage detection performance and assess integration with imaging and CA19-9 for PDAC detection and longitudinal monitoring.