An impact of neutrophil extracellular traps to the prothrombotic state and tumor progression in gynecological cancer patients

Introduction / Введение

Hypercoagulation accompanies carcinogenesis, tumor progression, and metastasis. It is also associated with higher risk of venous thromboembolism (VTE) as the second leading cause of death in cancer patients [1]. On the other hand, thromboprophylaxis and prophylactic anticoagulant therapy in cancer patients can reduce thrombosis risk improving survival [2]. However, anticoagulant therapy is associated with VTE occurrence and high risk of bleeding [3]. Thus, a deeper insight into understanding of hypercoagulation pathogenesis in cancer patients is necessary to develop new antithrombotic strategies.

Recent studies have shown that activated neutrophils produce neutrophil extracellular traps (NETs), consisted of extracellular DNA, histones, cytoplasmic proteins, and neutrophil granule proteins. NETs are considered to be a part of a new procoagulant mechanism and a link between inflammation and thrombosis [4]. NETs extracellular DNA leads to coagulation activation [5], whereas histones induce platelet and erythrocyte activation. Inflammation always accompanies carcinogenesis [6]. One of the studies showed that neutrophils increased NETs production in mice with cancer and were directly involved in thrombogenesis [7]. In addition, NETs were shown to accompany cancer-associated organ failure [8]. Thus, we hypothesized that tumor growth stimulates neutrophils to release NETs in gynecological cancer patients resulting in activated coagulation and may be one of metastasis factors underlying metastasis that confirmed NET-related procoagulant activity in gynecological cancer patients.

Aim: to evaluate the NETs procoagulant activity in gynecological cancer patients.

Materials and Мethods / Материалы и методы

Study design / Дизайн исследования

Between April 2020 and October 2022, 120 women including 87 patients aged 34 to 72 years hospitalized for elective surgical treatment or chemotherapy at the University Clinical Hospital No 4 of Sechenov University and Petrovsky National Research Centre of Surgery (main group), and 33 medical staff members aged 32 to 68 years (control group) were enrolled in a prospective controlled interventional non-randomized study.

Inclusion and exclusion criteria / Критерии включения и исключения

Inclusion criteria (main group): age over 18; diagnosis at admission – ovarian cancer, uterine cancer, cervical cancer and mammary cancer, confirmed by instrumental, laboratory and clinical examination; signed informed voluntary consent to participate in the study.

Inclusion criteria (control group): age over 18; no active cancer and oncological diseases, thrombosis and thromboembolism, chronic inflammatory diseases in history; signed informed consent to participate in the study.

Exclusion criteria (for both groups): age under 18; active infectious and/or inflammation; cardiovascular diseases, severe course; decompensated diabetes mellitus; chronic diseases of the liver and kidneys in the acute stage; other concomitant oncological diseases; thromboembolic complications associated with coagulopathy and thrombocytopathy; anticoagulants and antiplatelet agents use; thrombotic or hemorrhagic syndrome at the time of examination; refusal to participate in the study.

Patients groups / Группы обследованных

The main group included breast and gynecologic cancer patients, stage I–III: uterine cancer (subgroup 1; n = 18, including 7 patients at stage 1TNM, 5 patients at stage 2TNM, 6 patients at stage 3TNM), ovarian cancer (subgroup 2; n = 26, 11 patients at stage 1TNM, 5 patients at stage 2TNM, 10 patients at stage 3TNM), cervical adenocarcinoma (subgroup 3; n = 13; all at stage 1TNM), breast cancer (subgroup 4; n = 30, 9 patients at stage 1TNM, 6 patients at stage 2TNM, 7 patients at stage 3TNM, 8 patients at stage 4TNM). The control group included 33 healthy women.

Study methods / Методы исследования

Plasma samples were obtained by venipuncture from all patients once upon admission (before surgical treatment, anticoagulants therapy, chemotherapy), centrifuged and stored at –80° C. Fasting blood samples were collected from the cubital vein into a plastic tube added with anticoagulant (sodium citrate solution 3.8 %) in a ratio of 9:1 using a dry sterile needle.

Assessing NETosis markers / Определение маркеров нетоза

To determine the human myeloperoxidase antigen (MPO:Ag) concentration in the blood plasma, a Hycult Biotech ELISA kit (Netherlands) was used, normal MPO:Ag reference level was 2.56 ± 0.33 ng/ml.

The citrullinated histone H3 (citH3) in blood plasma was measured using the Citrullinated Histone H3 ELISA Kit (Cayman Chemical, Ann Arbor, USA).

Assessing markers of thrombinemia and fibrin formation / Определение маркеров тромбинемии и фибринообразования

Thrombinemia markers – thrombin-antithrombin complexes (TAT) were determined by enzyme-linked immunoassay, using the Siemens Healthineers Enzygnost^TM TAT MicroKit (Siemens Healthcare Diagnostics Products GmbH, Germany) on a Boehnringer ELISA-Photometer spectrophotometer (Boehnringer, Germany).

D-dimer was measured using a commercial immunoassay (TECHNOLEIA^®, Austria, Techoclone reagent). According to the manufacturer's data, D-dimer concentration > 250 ng/mL was considered pathological.

Ethical aspects / Этические аспекты

Informed consent was obtained from all patients to participate in the study and to process personal data in accordance with the World Medical Association's Declaration of Helsinki (WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects, 2013).

Statistical analysis / Статистический анализ

Data processing was performed using a specialized software Statistica 7.0 (StatSoft, Inc., USA).

Statistical analysis included descriptive statistics: arithmetic mean (M), standard deviation (SD), minimum and maximum values of laboratory parameters.

The normality test was performed using the Jarque-Bera test. The null hypothesis H₀ that residual values of participant groups parameters have a normal distribution was rejectedat the significance level p ≤ 0.05.

Non-parametric test and inter-group parameter magnitude comparison was performed using the Mann-Whitney test for unrelated samples (Mann–Whitney U-test). The null hypothesis H₀ was proposed as the absence of differences between patient groups. H₀ was deviated in all cases at significance level of p ≤ 0.05.

When studying the relationships between variables in terms of reflecting the corresponding causal relationships between the MPO:Ag and citH3 values, we calculated the ρ-coefficient of Spearman Rank Order Correlations with fixing the coefficient significance level in the correlation matrix at the significance level p ≤ 0.05.

Results / Результаты

Сlinical and anamnestic characteristic / Клинико-анамнестическая характеристика обследованных пациенток

The clinical and anamnestic characteristics of individuals examined are presented in Table 1.

NETosis intensity in oncogynecological patients / Интенсивность нетоза у онкогинекологических пациенток

The NETosis intensity in oncogynecological patients was assessed based on the following markers (MPO:Ag and citH3) level. It was shown that both markers were significantly elevated in cancer patients than in healthy individuals (Table 1, Fig. 1, 2).

Hemostasis activation in oncogynecological patients / Активация гемостаза у онкогинекологических пациенток

While analyzing the D-dimer and TAT levels in oncogynecological patients, they were found to be significantly increased compared with control group, which continued to rise along with aggravated TNM stage (Fig. 3, 4).

Subsequent correlation analysis revealed a significant correlation between the increased levels of TAT complexes as well as citH3 and MPO:Ag (Tables 2, 3).

Discussion / Обсуждение

Hypercoagulation profoundly elevates mortality rate in cancer patients [9]. Armand Trousseau firstly described the relationship between idiopathic VTE and latent tumors highlighting the correlation between cancer and thrombosis. Recent studies have shown that NETs are involved in activating coagulation, promoting initiation and thrombosis progression. In addition, NETs contribution to tumor progression and metastasis is being actively studied

NETs were first described in 2004 [10]. NETs are produced by activated neutrophils during NETosis originally recognized as a host defense mechanism. NETs are derivatives of activated neutrophils consisting of DNA strands and histones that grab and hold various pathogens until they are destroyed by NET-associated enzymes [11].

However, NETosis began to be determined in aseptic inflammation [4][12][13]. The role of NETs in autoimmune diseases such as psoriasis, systemic lupus erythematosus, and rheumatoid arthritis has been demonstrated [14–16]. NETs are also involved in the pathogenesis of thrombosis in diabetes mellitus, atherosclerosis, and vasculitis [17–19].

NETs affect the hemostasis system in various ways, contributing to the procoagulant state and disrupting the anticoagulants functioning and fibrinolysis [20]. Neutrophils in mouse breast cancer and chronic myeloma leukemia stimulate increased NETs production with subsequent hemostasis activation [21].

The numerous NETs-related effects are summarized in Table 4.

In the current study, NETs-dependent role was demonstrated in the gynecological patients with uterine, cervical, ovarian and breast cancer resulting in activated coagulation system. There were assessed the concentrations of NETosis markers, such as MPO:Ag and citH3 in gynecological cancer patients with different tumors at different stages. Simultaneously, there were investigated D-dimer and TAT complexes levels. In our study, an increased NETs production was noted as a significantly increased MPO:Ag and citH3 levels in cancer patients, mostly at late TNM stages. These data provide indirect evidence that rise in the tumor load causes increased NETs production.

In parallel with elevated NETosis markers, depending on the disease stage, there was also observed increased hemostasis activation markers (D-dimer and TAT complexes). A significant correlation was found between the TAT and thecit H3 as well as MPO:Ag levels.

In recent years, the NETs-related role in tumor growth has been actively studied. It has been proven that cells participating in the inflammatory response and inflammatory mediators contribute to cancer induction, tumor growth, and metastasis [36]. Some NETs components exert a cytotoxic effect, e.g., MPO damages melanoma cells. Patients with MPO deficiency have a higher risk of tumor progression and recurrence [37]. NETs histones corrode the tumor vasculature, destroy epithelial cells, and promote tumor cell lysis [7][38].

At the same time, NETs proteases can destroy the extracellular matrix, triggering metastasis. NETs-released matrix metalloproteinase 9 (MMP-9) blocks tumor cell apoptosis and ensures migration, invasion, and metastasis, e.g., in lung cancer [39–41]. Moreover, NETs in patients with Ewing's sarcoma were shown to bear neutrophils actively releasing NETs in the tumor tissue. However, neutrophils and NETs were determined mainly in patients at late stages with metastases. The disease relapsed rapidly in patients with complete remission after chemotherapy [42].

Attaching to tumor cells, NETs facilitate the metastasis by binding the tumor cell and endothelium in the target organ of metastasis. In animal models it has been shown that after NETs fixation to the vascular endothelium, they begin to grab tumor cells via DNA strands from the bloodstream. Thus, the NETs production, tumor progression, and metastasis can create a vicious circle in cancer patients. The aforementioned mechanism allows to consider NETs as one of the players in the metastasis, and hence as a potential therapeutic target. It is potentially plausible to prevent the NETs-related effect on tumor progression by administering elastase or DNase inhibitors [43].

In our study along with assessing NETosis marker levels, there were analyzed hemostasis activation markers (TAT and D-dimer) so that all patients were found to have significantly activated hemostasis. However, while conducting a correlation analysis, a significant correlation was observed only between TAT complex and NETosis marker levels.

Animal cancer models were featured with increased tissue neutrophil infiltration in various organs paralleled with vascular damage and platelet-neutrophil complexes due to massive necrosis. Administering DNase had a positive effect in regression of such conditions again confirming the NETosis-related contribution to developing multiple organ failure in cancer patients [8]. Recent studies on animals have shown that the NETosis suppression lowers a rise in the prothrombotic state and metastasis [7][8][43].

Thus, NETs play one of the major roles in triggering blood clotting in cancer patients. Accordingly, NETs can act as a target for novel approaches to develop thromboprophylaxis [44]. Assessing NETosis markers represents a potential screening approach for initial hemostasis di- sorders when the first-line laboratory tests have not been changed yet.

Conclusion / Заключение

The study results indicate that the tumor tissue creates conditions stimulating neutrophils to release procoagulant extracellular traps, which, in turn, not only contribute to creating procoagulant state, but also ensure metastasis. Developing therapies targeting NETs may potentially affect hemostasis system in cancer patients and reduce magnitude of metastasis spread.