NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation

Abstract

Introduction: Large elevations of high sensitive Troponin T (hsTnT) in ischemic stroke patients is associated with a poor outcome. In a pilot study we found a high prevalence of malignancies among these patients. Since neutrophil extracellular traps (NETs) have been linked to cancer-associated thrombosis, we hypothesized that the concomitant cerebral and myocardial ischemia could be the result of a NET-induced hypercoagulable state.

Materials and methods: Clinical assessments, plasma analyses and autopsies with histopathology (in cases of in-hospital mortality) were performed on ischemic stroke patients with high elevations of hsTnT (N=12) and normal hsTnT (N=19).

Results: Patients with hsTnT elevation had an unexpectedly higher prevalence of cancer (p=0.002), half of which were diagnosed post-mortem. Autopsies of these patients revealed widespread myocardial, cerebral and pulmonary microthrombosis with H3Cit in thrombi. A pro-coagulant state and an increase of the NET specific marker citrullinated histone H3 (H3Cit) was found in plasma of patients with elevated hsTnT compared to patients with normal levels (p<0.001). Plasma analyses in cancer patients showed even higher H3Cit levels (p<0.001), and an increase in granulocyte colony-stimulating factor, known to prime neutrophils towards NETosis. H3Cit correlated positively with thrombin-antithrombin complex (p=0.004) and soluble P-selectin (p<0.001), further linking NETosis to the pro-thrombotic state.

Conclusions: The high prevalence of known or occult cancer in our study suggests that cancer-associated arterial microthrombosis may be underestimated. By linking the thrombosis to NETs, we suggest markers of NETosis that could aid in revealing cancer in arterial microthrombosis as well as arterial microthrombosis in cancer.

Keywords: Cancer-associated microvascular thrombosis; Ischemic stroke; Neutrophil extracellular traps; Troponin elevation.

Fig 1. Histopathological investigations of cerebral, myocardial and pulmonary arterial microvascular thrombosis

A. Hematoxylin and eosin staining revealed microthrombosis in a small cerebral artery, in a small intramuscular coronary artery as well as in a small pulmonary artery. Small focal infarction was observed around the coronary microthrombi. Metastatic spread of prostate adenocarcinoma (small arrows) surrounding the pulmonary artery (arrow) was also present. B. Immunohistochemistry of H3Cit showed the presence of NETs in microthrombi of the brain, heart and lung. H3Cit positive cells as well as extracellular H3Cit was observed in all microthrombi. Granulocytes (blue nuclei stain) were also observed inside and around the coronary thrombus. C, D. Confocal microscopy revealed extracellular H3Cit which colocalised with DNA and confirmed the presence of NETs (arrows) in the cerebral, coronary and pulmonary microthrombi. The microthrombi were rich in von Willebrand factor (VWF). Smooth muscle actin (SMA) staining delineates the vessel wall.

Fig 2. Plasma markers of coagulation, platelet activation and NETosis

A. Plasma markers of coagulation, platelet activation and NETosis were significantly higher in stroke patients with hsTnT elevation (n=12) compared to stroke patients without hsTnT elevation (n=20). B. Comparison of these plasma markers between ischemic stroke patients with and without cancer. Even higher levels were seen in patients with cancer (n=8). C. Exclusion of patients with cancer in the group of ischemic stroke patients with troponin elevation. When excluding patients with cancer, the differences between patients with hsTnT elevation (n=5) and normal levels of hsTnT (n=19) were diminished, suggesting a link between NETosis, coagulation and cancer. Mann-Whitney U test was used to determine p-values.

Fig 3. Markers of NETosis correlate with neutrophil activation and a pro-thrombotic state

A. A positive correlation between the NET biomarker H3Cit and cfDNA, MPO, TAT, and soluble P-selectin further suggested a NET-induced pro-coagulant state. B. G-CSF also revealed a positive correlation with H3Cit, MPO, TAT and soluble P-selectin, strengthening the conclusion that the priming toward NETosis may drive the pro-thrombotic state. Notably, these correlations all remained significant when excluding the patient with the highest level of H3Cit (A. r=0.74, p<0.001 for the correlation between H3Cit and cfDNA; r=0.64, p<0.001 for the correlation between H3Cit and MPO, r=0.45, p=0.013 for the correlation between H3Cit and TAT; r=0.57, p=0.001 for the correlation between H3Cit and sP-selectin. B. r=0.57, p=0.001 for the correlation between G-CSF and H3Cit; r=0.51, p=0.004 for the correlation between G-CSF and MPO, r=0.38, p=0.038 for the correlation between G-CSF and TAT; and r=0.39, p=0.033 for the correlation between G-CSF and sP-selectin. C. r=0.64, p<0.001 for the correlation between H3Cit and MPO). Spearman's rank correlation was used for significance of correlations.