Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus

Background: Thrombosis is a common complication for breast cancer patients receiving chemotherapy. However, the mechanisms by which breast cancer chemotherapeutic agents increase this risk are largely uncharacterized. Nucleic acids released by injured cells may enhance coagulation via the activation of the contact pathway. Objectives: In this study, we examined the effects of breast cancer chemotherapy agents on the release of cell-free DNA (CFDNA) and its relationship to thrombin generation using in vitro and in vivo methods. Methods: CFDNA release and thrombin-antithrombin (TAT) levels were measured in plasma of breast cancer patients and healthy mice receiving chemotherapy. Venous whole blood and cultured cells were exposed to chemotherapy and CFDNA release and levels of DNA-histone complexes were measured. The procoagulant activity of isolated CFDNA was measured with calibrated, automated thrombin generation. Results: Breast cancer patients receiving chemotherapy had elevated levels of CFDNA 24 h post-chemotherapy, a time-point at which elevated thrombin-antithrombin levels have been previously reported. Treatment of healthy mice with doxorubicin, epirubicin and 5-fluorouracil increased CFDNA release, with a corresponding elevation in TAT complex formation. Venous whole blood and neutrophils incubated with chemotherapeutic agents had elevated CFDNA in plasma or cell supernatants. In addition, incubation of venous whole blood with chemotherapy decreased histone-DNA complex levels. CFDNA released from epirubicin-treated whole blood significantly elevated thrombin generation in a dose-dependent manner, and involved activation of the contact pathway. Conclusions: Release of CFDNA from chemotherapy-injured cells may represent a novel mechanism by which thrombosis is triggered in cancer patients.