Neutrophil Extracellular Traps Enhance Procoagulant Activity and Predict Poor Prognosis in Patients With Metastatic Breast Cancer

Abstract

Objective: Neutrophil extracellular traps (NETs) are associated with poor prognosis and an increased risk of venous thromboembolism (VTE) in metastatic breast cancer (MBC). This study aims to determine whether NETs promote hypercoagulability and if NETs and plasma hypercoagulability markers are biomarkers of survival in MBC.

Methods: Circulating levels of neutrophil extracellular trap (NET) markers and hypercoagulability markers (TAT, fibrinogen, and D-dimer) were assessed in 112 MBC patients before treatment, compared to 55 healthy controls. Stratified by NET levels and plasma TAT, fibrinogen, and D-dimer, the correlation with overall survival was analyzed. The NET procoagulant activity was evaluated using fibrin and purified coagulation complex production assays, and by measuring coagulation time (CT).

Results: MBC patients exhibited significantly elevated plasma NET levels compared to healthy controls (all P<0.05), circulating MPO-DNA and NE-DNA levels were positively correlated with plasma TAT, fibrinogen, D-dimer, CT, FVIIIa, and platelet (PLT) counts. Additionally, we observed a significant increase in NETs formation in control neutrophils exposed to MBC plasma compared to those exposed to control plasma. NETs from MBC neutrophils significantly increased the potency of control plasma to generate thrombin and fibrin, effects that were notably attenuated by DNase I. Plasma TAT and D-dimer levels were significantly higher in MBC patients who died within three years post-recruitment compared to those who survived beyond three year. Plasma TAT and D-dimer were inversely correlated with survival. High plasma levels of MPO-DNA were associated with significantly worse overall survival (HR: 2.445, 95% CI: 1.255-4.762, P=0.007). MBC patients with both high D-dimer and high MPO-DNA had significantly reduced survival (HR: 2.450, 95% CI: 1.332-4.488, P=0.002).

Conclusion: Our results highlight the increased release of NETs in MBC patients and reveal that NET formation enhances hypercoagulability and cancer progression. Targeting NETs may be a potential therapeutic strategy to inhibit MBC progression and mitigate thrombotic complications in MBC.

Keywords: hypercoagulability; metastatic breast cancer; neutrophil extracellular traps; procoagulant activity; prognosis.

Figure 1

MBC patients exhibit increased circulating NETs. The expression of NETs markers MPO-DNA (A), NE-DNA (B), and CitH3 (C) in plasma of MBC patients (n=112) and healthy control patients (n=55) were examined by flow cytometry and these NETs markers were significantly elevated in MBC patients. **** P<0.0001.

Figure 2

MBC plasma primes neutrophils to form NETs. (A) Confocal results showed a significant increase in NETs formation in control neutrophils exposed to MBC plasma compared to those exposed to control plasma. (B) Flow cytometry results showed that control neutrophils exhibited a significant increase in MPO-DNA quantification following stimulation with MBC plasma. (C) The frequency of NET-releasing neutrophils was also higher when control neutrophils were treated with MBC patient plasma than when treated with control plasma. ** P<0.01, *** P<0.001.

Figure 3

Procoagulant activity of NETs released by neutrophils derived from patients with MBC. (A) TAT levels increased significantly after the addition of NETs isolated from MBC patients to control plasma compared to baseline. (B) NETs from MBC patient neutrophils significantly increased fibrinogen production in control plasma. (C) coagulation time was markedly reduced for NETs isolated from MBC patients compared to controls, while there was no significant difference in coagulation time when comparing control NETs. (D–F) DNase I significantly reduced the effect of NETs released by neutrophils derived from patients with MBC on control plasma generation of thrombin and fibrin. ** P<0.01, *** P<0.001.

Figure 4

Coagulation plasma markers TAT and D-dimer, and NETs marker MPO-DNA are associated with poorer survival in metastatic breast cancer. Coagulation markers TAT and D-dimer were quantified by immunoassay in metastatic breast cancer (MBC) patient plasma samples. (A) TAT, (C) D-dimer, (E) MPO-DNA: concentrations compared to survival groups, ****p<0.0001. (B) TAT, (D) D-dimer, (F) MPO-DNA: log-rank (Mantel–Cox) tests comparing survival in months from study entry in these coagulation and NETs markers dichotomised around the median values (TAT 571.24pg/mL, D-dimer 381.945ng/mL, MPO-DNA 162.91 ng/mL) were carried out.

Figure 5

The combined presence of a high D-dimer and circulating MPO-DNA is associated with reduced survival in metastatic breast cancer. Survival in patients with high MPO-DNA (≥162.91 ng/mL) and high D-dimer (≥381.94ng/mL) (red line, n=43) was compared to all other patients (blue line, n=69). A log-rank (Mantel–Cox) test comparing survival in months from study entry in the shown NETs/coagulation marker groups was performed.