PB0777 - The Risk of Thrombosis in Patients with Acute Lymphoblastic Leukemia may be Explained by the Impact of Peripheral Blasts on Endothelial Cells: An In-vitro study

Background: Thrombosis is a well-known complication in children with acute lymphoblastic leukemia (ALL) with a reported frequency of up to 36.7% in the PARKAA trial. Children with ALL were found to have persistent increased thrombin generation following diagnosis, yet, the pathogenesis and impact of therapy on thrombin activation are still unknown. We previously demonstrated an association of peripheral lymphoblasts with increased levels of von Willebrand Factor (VWF) and parameters of thrombin generation. Further studies were required to confirm the hypothesis that peripheral lymphoblasts activate the endothelium, leading to the elevation of VWF; and causing the prothrombotic state seen in children with ALL.

Aims: The primary aim of the study is to explore the in vitro effects of coculturing peripheral lymphoblasts with endothelium by evaluating markers of endothelial activation. The secondary aim is to investigate the effects of methyl-prednisolone therapy on this coculture system.

Methods: Human umbilical vein endothelial cells and peripheral blood-derived lymphoblasts were obtained from Lonza and ATCC, respectively. In our coculture system, confluent endothelium was exposed to lymphoblasts in serum-free media during the experimental period. Biomarkers of endothelial activation were evaluated by antigenic and cell-based ELISA. Thrombin generation on experimental endothelium was evaluated using a plasma recalcification assay.

Results: The presence of leukemic lymphoblasts significantly increased endothelial VWF, plasminogen activator inhibitor-1 (PAI-1) secretion, and stimulated vascular adhesion molecule-1 (VCAM-1) production (Fig.1). Leukemic blasts also elevate endothelial tissue factor level in cell lysates and E-selectin cell surface expression. Additionally, plasma recalcification time was decreased in the coculture system. Methyl-prednisolone treatment resulted in a modest reduction in endothelial VWF and PAI-1 secretion (Fig.2).

Conclusion(s): The clinical observation was confirmed in vitro. The presence of leukemic blasts induces endothelial activation, as demonstrated by increased release of endothelial acute phase reactants and prothrombotic phenotype of endothelium. Treatment with methyl-prednisolone alleviates the effect of lymphoblasts on the endothelium.