Neutrophil extracellular traps in cancer and cancer-associated thrombosis

Cancer is associated with a hypercoagulable state, and venous thromboembolism (VTE) may be the first sign of occult cancer. Cancer screening of all patients presenting with VTE would, however, overload the healthcare system and burden patients with unnecessary investigations. Current data suggest that neutrophil extracellular traps (NETs), prothrombotic nuclear content released by neutrophils upon strong stimulation, are central in cancer biology. This thesis aimed at a clinical investigation of the role of coagulation in advanced cancer and the role of NETs in cancer-associated thrombosis.

In Study I, we evaluated the recently developed RIETE risk score to identify patients presenting with VTE and a simultaneous high risk of occult cancer. The risk score failed to identify VTE patients with a high risk of occult cancer, illustrating the need for the development of risk score models in this population.

In Study II, we developed an enzyme-linked immunosorbent assay for the quantification of nucleosomal citrullinated histone H3 (H3Cit-DNA), a protein-DNA complex generated during NET formation. The assay was rigorously validated revealing high accuracy. All assay components are furthermore commercially available, enabling rapid dissemination and implementation of the assay within the field of NETs research.

Study III was an exploratory study investigating several biomarkers reflecting neutrophil activation, NET formation, coagulation, and fibrinolysis and their association with mortality in 106 terminal cancer patients. Markers of neutrophil activation and NETs were associated with mortality in univariate and multivariate Cox regression. Several prior studies have revealed that markers of coagulation and fibrinolysis are associated with prognosis in cancer patients. However, no studies have investigated terminal cancer patients, and to our surprise, we did not find an association between poor prognosis and markers of coagulation and fibrinolysis.

Study IV was a prospective cohort study of 500 patients presenting with acute VTE. Venous blood was sampled at the time of VTE, and markers of NETs and neutrophil activation were analyzed. H3Cit-DNA and cell-free DNA were associated with cancer diagnosis during a one-year follow-up in univariate analyses, but only H3Cit-DNA remained significant after adjustments in multivariate analyses, which could indicate a role of NETs in the development of cancer-associated thrombosis.

In summary, there are as of date no accurate risk scores identifying VTE patients with underlying cancer. Through the development of an assay quantifying the NET marker H3Cit- DNA in human plasma, we found that H3Cit-DNA is elevated in advanced cancer and in patients presenting with VTE and an underlying cancer, contributing to the growing evidence of the role of NETs in cancer and cancer-associated thrombosis. Further research will determine the diagnostic potential of NETs.

List of scientific papers

I. Rosell A, Lundström S, Mackman N, Wallén H, Thålin C. A clinical practicebased evaluation of the RIETE score in predicting occult cancer in patients with venous thromboembolism. J Thromb Thrombolysis. 2019 Jul;48(1):111- 118.
https://doi.org/10.1007/s11239-019-01822-z

II. Thålin C, Aguilera K, Hall NW, Marunde MR, Burg JM, Rosell A, Daleskog M, Månsson M, Hisada Y, Meiners MJ, Sun ZW, Whelihan MF, Cheek MA, Howard SA, Saxena-Beem S, Noubouossie DF, Key NS, Sheikh SZ, Keogh MC, Cowles MW, Lundström S, Mackman N, Wallén H, Johnstone AL. Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma. J Thromb Haemost. 2020 Oct;18(10):2732-2743.
https://doi.org/10.1111/jth.15003

III. Rosell A, Aguilera K, Hisada Y, Schmedes C, Mackman N, Wallén H, Lundström S, Thålin C. Prognostic value of circulating markers of neutrophil activation, neutrophil extracellular traps, coagulation and fibrinolysis in patients with terminal cancer. Sci Rep. 2021 Mar 3;11(1):5074.
https://doi.org/10.1038/s41598-021-84476-3

IV. Rosell A, Gautam G, Ng H, Gry H, Nordström E, Månsson M, Gabrielsson L, Söderberg M, Lundström S, Mackman M, Westerlund E, Wallén H, Thålin C. Nucleosomal citrullinated Histone H3, a marker of neutrophil extracellular trap formation, is associated with occult cancer in patients presenting with venous thromboembolism. [Manuscript]