Cancer-associated venous thrombosis (VTE) increases mortality and morbidity. However, limited tools are available to identify high risk patients. Upon activation, neutrophils release their content through different mechanisms, thereby prompting thrombosis. We explored plasma microRNAs (miRNAs) and neutrophil activation markers to predict VTE in pancreatic ductal adenocarcinoma (PDAC) and distal extrahepatic cholangiocarcinoma (DECC). Twenty-six PDAC and 6 DECC patients recruited at cancer diagnosis, were examined for deep vein thrombosis and pulmonary embolisms, and were then followed-up with clinical examinations, blood collections, and biCUS. Ten patients developed VTE and were compared with 22 age- and sex-matched controls. miRNA expression levels were measured at diagnosis and right before VTE, and neutrophil activation markers (cell-free DNA, nucleosomes, calprotectin, and myeloperoxidase) were measured in every sample obtained during follow-up. We obtained a profile of 7 miRNAs able to estimate the risk of future VTE at diagnosis (AUC = 0.95; 95% Confidence Interval (CI) (0.987, 1)) with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. Seven miRNAs were up- or down-regulated before VTE compared with diagnosis. We obtained a predictive model of VTE with calprotectin as predictor (AUC = 0.77; 95% CI (0.57, 0.95)). This is the first study that addresses the ability of plasma miRNAs and neutrophil activation markers to predict VTE in PDAC and DECC.

Upon activation, neutrophils release their content through different mechanisms like degranulation and NETosis, thus prompting thrombosis. The natural anticoagulant activated protein C (APC) inhibits neutrophil NETosis and, consequently, this may lower the levels of neutrophil activation markers in plasma, further diminishing the thrombotic risk exerted by this anticoagulant. We aimed to describe the status of markers of neutrophil activation in plasma of patients with venous thrombosis, their association with the thrombotic risk and the potential contribution of APC. We quantified three markers of neutrophil activation (cell-free DNA, calprotectin, and myeloperoxidase) in 253 patients with venous thromboembolism (VTE) in a stable phase (192 lower extremity VTE and 61 splanchnic vein thrombosis) and in 249 healthy controls. In them, we also quantified plasma APC, soluble endothelial protein C receptor (EPCR), and soluble thrombomodulin (TM), and we genotyped two genetic regulators of APC: the EPCR gene (PROCR) haplotypes (H) and the TM gene (THBD) c.1418C>T polymorphism. We found a significant increase in plasma cell-free DNA (p < 0.0001), calprotectin (p = 0.0001) and myeloperoxidase (p = 0.005) in VTE patients compared to controls. Furthermore, all three neutrophil activation markers were associated with an increase in the thrombotic risk. Cell-free DNA and calprotectin plasma levels were significantly correlated (Spearman r = 0.28; p < 0.0001). As expected, the natural anticoagulant APC was significantly decreased in VTE patients (p < 0.0001) compared to controls, what was mediated by its genetic regulators PROCR-H1, PROCR-H3, and THBD-c.1418T, and inversely correlated with cell-free DNA levels. This is the largest case-control study that demonstrates the increase in markers of neutrophil activation in vivo in VTE patients and their association with an increased thrombotic risk. This increase could be mediated by low APC levels and its genetic regulators, which could also increase NETosis, further enhancing thrombosis and inflammation.

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