Neutrophils, the predominant white blood cells in humans, hold a vital function within the immune response by serving as the initial line of defense during inflammation and against pathogens. A deficiency in neutrophil count heightens the risk of infections. They utilize mechanisms such as phagocytosis, granule release, and the creation of neutrophil extracellular traps (NETs) to counteract invading agents. Although the processes of phagocytosis and granule release were previously recognized, the discovery of NETs, which involve the expulsion of chromatin and granules to ensnare and neutralize bacteria, came to light in 2004 .
NETs are extracellular structures released by neutrophils in response to excessive or large pathogens. NETs tangle pathogens using DNA, histones, proteases, and inflammatory compounds. They are formed through a “suicidal” process as a last-resort defense mechanism against pathogens and can harm both pathogens and host tissues upon release [1, 2].
Sterile injuries also trigger NETs, although their purpose in this context is less clear. Inflammation mediators shared between sterile and non-sterile injuries induce NETs, suggesting coincidental deployment under sterile conditions. NETs possess anti-inflammatory functions like acting as a cytokine or chemokine degrading platforms and promoting T cell exhaustion through immunosuppressive ligands .
Neutrophils, NETs, cancer and thrombosis
Beyond their defensive role, recent research has illuminated the intricate roles of neutrophils in chronic diseases, particularly in cancer. Neutrophils are now acknowledged as significant participants within the tumor microenvironment, influencing all cancer stages. They drive tumor initiation through inflammation-induced reactive oxygen species and protease release. Neutrophils also exert a dual impact on tumor progression, either inhibiting metastasis through direct cytotoxicity or facilitating it by fostering immunosuppression, angiogenesis, cancer cell mobility, and epithelial-mesenchymal transition. The pro-tumor aspect dominates much of the research, substantiated by evidence linking intratumoral neutrophils to unfavorable prognoses in various cancers [1, 2].
In 2012, it was revealed that one of the mechanisms tying neutrophils to thrombosis and cancer involves NETs formation . NETs exhibit thrombotic properties and participate in both venous and arterial thrombosis. The presence of cancer enhances NET formation, likely contributing to the heightened thrombosis risk observed in these individuals .
Biomarkers for NETs in patients with VTE and cancer
Blood biomarkers indicating neutrophil activation and the creation of NETs are linked to acute venous thromboembolism (VTE) in symptomatic patients [4, 5]. However, commonly used markers of NET formation include granular proteins like neutrophil elastase (NE), myeloperoxidase, and cell-free DNA (cfDNA), and these may come from various sources, including apoptotic or necrotic cells and neutrophil activation, which can occur without NET formation . Citrullination of histone H3 mediates chromatin decondensation, which is a pre-requisite for NET formation . Citrullinated histone H3 (H3Cit) is thereby considered a NET specific biomarker. High levels of H3Cit in plasma are connected to an increased VTE risk in cancer patients and has been linked to arterial thrombosis in cancer patients . Moreover, plasma H3Cit levels are higher in patients with cancer compared to severely ill hospitalized patients without cancer . Elevated plasma H3Cit and nucleosomal H3Cit (H3Cit-DNA) levels are also linked to unfavorable outcomes in patients with advanced cancer [9, 10].
The Biomarkers In Thrombosis Study: an interim analysis.
The Biomarkers In Thrombosis Study (NCT03781531) is an ongoing prospective cohort investigation that seeks to recruit a cohort of 1000 patients who are currently presenting with acute VTE. The primary focus of this study is to discern the significance of specific blood biomarkers linked to neutrophil activation and NETs, aiming to identify individuals within the VTE spectrum who possess an elevated vulnerability to occult cancer .
Occult cancers typically denote advanced or metastatic tumors with an undisclosed primary source. Occasionally, they arise from unexplained symptoms or abnormal laboratory results that cannot be attributed to other causes. Occult cancer and unprovoked VTE showed an association.
An interim analysis of the Biomarkers In Thrombosis Study has been undertaken, concentrating on the first 500 enrolled patients with VTE. This analysis delves into the intricate interplay of bloodborne biomarkers linked to neutrophil activation and NETs, aiming to predict new cancer diagnoses within the following year. The study’s one-year follow-up period was chosen based on observations that most cancer diagnoses occurred within this timeframe .
During the year following VTE diagnosis, 7% (29 out of 417) of VTE patients were subsequently diagnosed with cancer, with half of these cases (14 out of 29) emerging within the initial 10 days post-VTE diagnosis. For patients eligible for cancer screening, the rate of new cancer diagnoses stood at 3.8% (15 out of 400). Common cancer sites were colorectal, lung, pancreatic, breast, prostate, and upper gastrointestinal, with 69% of metastatic disease cases. Remarkably, patients under 50 years old did not receive any cancer diagnoses .
Patients with cancer-associated thrombosis (CAT, including active cancer or cancer diagnosed during follow-up) exhibited elevated H3Cit-DNA and cfDNA levels but not NE, compared to those with VTE and no cancer. Adjustments for various factors showed that only H3Cit-DNA levels remained significantly associated with CAT. In univariate Cox regression analyses, elevated levels of H3Cit-DNA and cfDNA, but not NE, were associated with a higher likelihood of a cancer diagnosis within a year of VTE diagnosis .
Most notably, high levels of H3Cit-DNA and cfDNA were observed in patients with cancer cases that emerged shortly after VTE. H3Cit-DNA’s association with cancer persisted even after adjusting for known clinical risk factors, emphasizing it as an independent risk marker for occult cancer in acute VTE patients .
These findings affirm the role of NET formation in CAT and highlight circulating H3Cit-DNA’s association with hidden cancer in patients with VTE. Incorporating H3Cit-DNA into multianalyte cancer screening tests could enhance the sensitivity of cancer detection .
- Adrover JM, McDowell SAC, He XY, Quail DF, Egeblad M. NETworking with cancer: The bidirectional interplay between cancer and neutrophil extracellular traps. Cancer Cell. 2023;41(3):505-526. doi:10.1016/j.ccell.2023.02.001
- Herre M, Cedervall J, Mackman N, Olsson AK. Neutrophil extracellular traps in the pathology of cancer and other inflammatory diseases. Physiol Rev. 2023;103(1):277-312. doi:10.1152/physrev.00062.2021
- Demers M, Krause DS, Schatzberg D, et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A. 2012;109(32):13076-13081. doi:10.1073/pnas.1200419109
- van Montfoort ML, Stephan F, Lauw MN, et al. Circulating nucleosomes and neutrophil activation as risk factors for deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2013;33(1):147-151. doi:10.1161/ATVBAHA.112.300498
- Smith P, Rosell A, Farm M, et al. Markers of neutrophil activation and neutrophil extracellular traps in diagnosing patients with acute venous thromboembolism: A feasibility study based on two VTE cohorts. PLoS One. 2022;17(7):e0270865. Published 2022 Jul 28. doi:10.1371/journal.pone.0270865
- Rosell A, Gautam G, Wannberg F, et al. Neutrophil extracellular trap formation is an independent risk factor for occult cancer in patients presenting with venous thromboembolism [published online ahead of print, 2023 Jul 19]. J Thromb Haemost. 2023;S1538-7836(23)00565-2. doi:10.1016/j.jtha.2023.07.007
- Wang Y, Li M, Stadler S, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009;184(2):205-213. doi:10.1083/jcb.200806072
- Thålin C, Demers M, Blomgren B, et al. NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation. Thromb Res. 2016;139:56-64. doi:10.1016/j.thromres.2016.01.009
- Thålin C, Lundström S, Seignez C, et al. Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer. PLoS One. 2018;13(1):e0191231. Published 2018 Jan 11. doi:10.1371/journal.pone.0191231
- Mauracher LM, Posch F, Martinod K, et al. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. J Thromb Haemost. 2018;16(3):508-518. doi:10.1111/jth.13951