Tissue Factor Positive Extracellular Vesicles and Risk of Thromboembolism in Diffuse Large B-Cell Lymphoma: A Prospective Exploratory Study

Abstract

Cancer is associated with an increased risk of venous thromboembolism (VTE). Cancer patients experience hypercoagulability, which increases the risk of VTE. Thrombus formation in VTE is initiated by tissue factor (TF), located in the blood vessels, and exposed in vascular injury. TF is also found on the surface of tumor cells. Extracellular vesicles (EVs) are small membrane vesicles formed from activated or apoptotic cells. In cancer, TF+ and TF- EVs can be observed in the blood, where the TF+ EV number is higher in cancer patients than in healthy controls. The association of EVs with VTE in patients with lymphoma has not been established. The present study was designed to explore the role of TF+ EVs in the development and prediction of VTE in a homogenous lymphoma patient population, i.e., diffuse large B-cell lymphoma (DLBCL). The concentration of total EVs and EVs-expressing tissue factor (CD142) was determined with a protocol developed by Karolinska Institute, Sweden. In brief, samples were stained with Annexin V-FITC and CD142-APC (Thermo Fisher Scientific, Waltham, MA, USA). Megamix-Plus SSC (BioCytex, Marseille, France) was used to gate the EVs' size; samples were analyzed on the BD FACSCanto™. EVs were defined as vesicles ≤1.0 µm in size and positive for Annexin V. Flow cytometry data were evaluated with FlowJo software (version 10.8.1, BD, Ashland, OR, USA). Peripheral blood samples obtained pre-treatment from 62 newly diagnosed or relapsed patients with DLBCL have been collected at Clinic for Hematology at the University Clinical Center of Serbia. The clinical and routine laboratory data have been collected from hospital records. Patients who had completed a minimum of one chemotherapy cycle were followed for all VTE events from the time of diagnosis to 3 months after the last cycle of therapy. VTE was diagnosed objectively based on radiographic studies (duplex venous ultrasound, contrast-enhanced thoracic computed tomography scan, magnetic resonance imaging - for central nervous system thrombosis), clinical examination, and laboratory evaluation. All probable cases of VTE were reviewed by a final diagnosis committee composed of two specialists (internist and radiologist). The median patients' age was 59 years (range, 20-87 years); 51.6% were males. Most patients were newly diagnosed, 38.7% had advanced stage (III and IV) disease, and 47.8% had ECOG performance status >1. Total concentration of EVs and TF+ EVs were higher in DLBCL patients compared to healthy volunteers (448 [167-868] x109 EVs/L (median [IQR]) vs 159 [151-159], p=0.009 and 140 [50-272] vs 13 [12-23], p<0.001). The concentration of EVs and TF+ EVs was not different between patients who developed VTE during follow-up (n=11) and those who did not (n=51) (530 [167-1318] (median [IQR]) vs (447 [163-822], p>0.05 and (214 [33-385]) vs 140 [50-255], p>0.05). After a median follow-up of 32 months, neither EVs nor TF+ EVs were associated with overall survival in DLBCL patients in Cox regression analysis. In conclusion, while the concentration of TF+ EVs is increased in patients with DLBCL, it is not significantly associated with VTE incidence. The role of TF+ EVs activity in VTE in DLBCL patients remains to be established in future studies. Vladimir Otasevic and Charlotte Gran equally contributed as first authors Darko Antic and Jovan Antovic equally contributed as senior authors