High Levels of Soluble P-selectin, Neutrophil Extracellular Traps, and Myeloperoxidase as Risk Factor of Deep Vein Thrombosis in Malignancy Patients Receiving Platinum-Based Chemotherapy

Characteristics of research subjects by case and control group

This study included 62 participants. Both the case and control groups included patients with malignancy who received platinum-based chemotherapy. The case group consisted of 31 patients with malignancy who received platinum-based chemotherapy and had deep vein thrombosis (DVT), while the control group consisted of 31 study subjects with malignancy who received platinum-based chemotherapy without deep vein thrombosis (DVT). The characteristics of the study participants are presented in Table 1.

Regarding the type of malignancy recorded in this study, the majority of cases were cervical, colorectal, or ovarian malignancies. The control group had nasopharyngeal and cervical malignancies ( Table 2). Most of the samples during the study had non-metastatic clinical stages, both in the case and control groups. This is because patients with metastatic stage were mostly not administered chemotherapy due to their performance status; therefore, patients with metastatic stage were not recruited in this study.

Results of soluble P-Selectin, Neutrophil Extracellular Traps (NET), Myeloperoxidase (MPO) levels

After examining the levels of Soluble P-Selectin, Neutrophil Extracellular Traps (NET) and Myeloperoxidase (MPO) in this study, then based on the Area Under Curve ROC (Receiver Operating Characteristic) as shown in Figure 3, the cut-off point of the three variables was obtained and grouped the data into two categories, high and low. Based on the ROC curve, the cut-off value used for Soluble P-Selectin was 82.84 ng/mL. The sensitivity and specificity of Soluble P-Selectin were 0.57 (95% CI = 0.30– 0.82, p < 0.001) and 0.73 (95% CI = 0.51–0.90, p < 0.001) and the Diagnostic Odds Ratio (DOR) was 4.31 (95% CI = 2.22–8.37, p < 0.01). The ROC curve showed significant accuracy of Soluble P-Selectin (AUC = 0.74, p = 0.05). For NET, a cut-off value of 5.89 was obtained with an AUC of 0.66, sensitivity of 0.61, and specificity of 0.64. Meanwhile, the cut-off value for MPO was 644.14 mg/mL, with an AUC of 0.54, sensitivity of 0.42, and specificity of 0.74.

Figure 3. Area under curve ROC (Receiver Operating Characteristic) levels of Soluble P-Selectin, Neutrophil Extracellular Traps (NET) and Myeloperoxidase (MPO).

The results of the examination of the levels of Soluble P Selectin, neutrophil extracellular traps (NET), and myeloperoxidase in this study were grouped based on the cut-off points obtained as shown in Table 3.

Based on Table 3, it was found that MPO did not have a significant difference between the case and control groups. This study also explored MPO as a continuous variable into its relationship with DVT that potentially reveals associations missed by categorizing it as high/low. The results of the MPO analysis as continuous data can be seen in Table 4. The data show that MPO has no significant impact on DVT even as numeric data (p = 0.588).

This study was then continued by conducting a multivariate analysis to assess variables including confounding variables to DVT. This study found that both Soluble P Selectin, Neutrophil Extracellular Traps, NET, MPO, and confounding variables such as gender, type of chemotherapy, and type of cancer did not have a significant impact on the incidence of DVT (p > 0.05). The results of multivariate analysis can be seen in Table 5.

Soluble P-Selectin as a risk factor for deep vein thrombosis

The results of this study support several previous studies regarding the role of Soluble P-Selectin in cancer patients as a risk predictor and as a diagnostic tool to detect the presence of VTE. Previous study showed 167 (93.82%) patients were suspected of having DVT and after a diagnostic examination, it was confirmed that 59 (35.33%) patients had DVT and 11 (6.18%) had pulmonary embolism. There was a significant difference in the mean levels of Soluble P-Selectin in this study (p < 0.001).⁵

A study by Setiawan et al. found that 12.5% ​​of patients experienced DVT. His study found that patients who experienced DVT had significant soluble P-selectin and von Willebrand factor. Soluble P-selectin concentrations were also significantly increased after chemotherapy (81.95 ng/mL vs 92.5 ng/mL, p < 0.001). The study found that both platelets and endothelial cells appear to be activated by chemotherapy.²⁰ The study by Vandy et al. reported that in a study conducted on all inpatients and outpatients for 3 years, 159 patients with confirmed DVT had statistically significant levels of Soluble P Selectin, D-dimer, CRP, and von Willebrand factor. In this study, the Soluble P-Selectin cut-off point used was > 90 ng/mL combined with Wells Score 2 as a DVT diagnostic tool with a specificity of 96%, positive predictive value of 89%, and negative predictive value of 77%.²¹

A study conducted by Antonopaulus et al. found that Soluble P-Selectin levels increased after the occurrence of VTE (OR = 2.89, 95% CI = 2.31–3.61, p < 0.001) or DVT only (OR = 2.64, 95% CI = 1.95-3.56, P < 0.001). This study excluded patients with solid tumors and HIV-positive patients with lupus anticoagulants. The aggregated sensitivity and specificity of Soluble P-Selectin were 0.57 (95% CI = 0.30–082, p < 0.001) and 0.73 (95% CI = 0.51-0.90, p < 0.001) and the Diagnostic Odds Ratio (DOR) 4.31 (95% CI = 2.22–8.37, p < 0.01). The ROC curve showed significant Soluble P-Selectin accuracy (AUC = 0.74, p = 0.05). This indicates that Soluble P-Selectin is significantly increased in patients with DVT, with or without PE complications, with high diagnostic performance.²²

Another study showed that there was a significant difference in Soluble P-Selectin levels between patients in the case and control groups, with a cut-off point of 70.5 ng/ml with a sensitivity of 98%, specificity of 100%, NPV of 96.8%, and PPV of 100%.²² Platinum-based chemotherapy induces thrombosis through several mechanisms including direct vascular endothelial damage, induction of cell apoptosis, and subsequent production of microparticles. In addition, platinum-based chemotherapy is also suspected to directly induce the activation of monocytes and macrophages that produce Tissue Factor (TF), and is able to induce an increase in von Willebrand factor.⁸ P-Selectin is one of a cell adhesion molecule (CAM) found on the surface of activated endothelial cells, which coat the inner surface of blood vessels and activated platelets. Soluble P-selectin is the soluble form of P-selectin in the circulation and is considered to be a key molecule in haemostasis and mediating the inflammatory process in thrombosis. Soluble P-selectin induces the production of procoagulant microparticles and increases fibrin deposition.²³ Soluble P-selectin and P Selectin Glycoprotein Ligan-1 (PSGL-1) play a role in the interaction of platelets with neutrophils in the innate immune system and in the formation of Neutrophil Extracellular Traps (NET), known as NETosis, an important process in the immuno-thrombosis system.

Theoretically, when viewed from the perspective of the Virchow Triad (stasis, endothelial injury, and hypercoagulability), in this study, an increase in Soluble P-Selectin levels in malignancy patients reflects a hypercoagulable (prothrombotic) condition, and the use of chemotherapy results in endothelial injury, thereby increasing the risk of thrombosis in cancer patients.⁵ The results of this study prove the hypothesis that high Soluble P-Selectin levels in malignancy patients using platinum-based chemotherapy can be used as a risk factor for DVT.

Neutrophil Extracellular Traps (NET) as a risk factor for deep vein thrombosis

The administration of platinum-based chemotherapy to cancer patients increases the risk of developing DVT. Through several mechanisms that have been described, platinum-based chemotherapy mostly induces the process of thrombosis formation through endothelial injury and increased expression of tissue factors, thereby increasing activated monocytes and neutrophils as well as through platelet activation mechanism, thereby inducing the emergence of a NETosis process so that NET is formed.

Neutrophil extracellular traps (NETs) are composed mostly of chromatin and neutrophil granular proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G. The results of several studies have indicated that neutrophils are a major component of the intravascular immune response to circulating blood pathogens. As host protection against pathogens, neutrophils secrete neutrophil extracellular traps (NETs). In addition to the capacity of intracellular mechanisms to fight infection, neutrophils also utilize extracellular devices to protect the host from cellular damage caused by infection. Several studies have suggested a role for NETs in tumor progression, metastasis, and cancer-associated thrombosis.^24,25

The role of NET in the mechanism of immune-thrombosis can occur through several pathways, such as direct activation of factor XII and binding to Von Willebrand factor in recruiting and activating more platelets. In addition, the enzymes contained in NET, such as neutrophil elastase (NE) and myeloperoxidase (MPO), are able to cause inactivation of endogenous anticoagulants, such as Tissue Factor Pathway Inhibitor (TFPI), and NET is able to bind directly to tissue factors and activate the extrinsic haemostasis system. The activation of intravascular blood coagulation and thrombus formation in microvessels (microvascular thrombosis) under certain conditions that support this intravascular immune mechanism is called immunothrombosis.²⁴

The main player in the release of neutrophils from the chromatin nucleus, known as neutrophil extracellular traps (NET), is citrullinated histone H3 (H3Cit). The increase in plasma H3Cit reported in several studies was found to be significant in thrombotic patients compared to that in healthy individuals. Neutrophil analysis in thrombotic patients showed a greater proportion of H3Cit intracellular neutrophils than in healthy individuals. The presence of plasma H3Cit in thrombotic patients correlates strongly with the activation of the markers of neutrophil elastase (NE), myeloperoxidase (MPO), and the inflammatory cytokines IL-6 and IL-8, known as NETosis. H3Cit can be detected in the nuclei of neutrophils at the time of stimulation and released into the blood circulation at the time of NETosis.²⁶

Thalin et al. suggested that H3Cit is a potential blood plasma marker for the diagnosis and prognosis of thrombosis and cancer. Examination of H3Cit in the circulating blood is crucial for diagnosis and prognosis.²⁶ In this study, the authors used the H3Cit marker to represent NET produced in patients with malignancy undergoing platinum-based chemotherapy. The results of this study are similar to those of other studies that have been carried out, such as the cohort study by Mauracher et al., who found that high plasma NET levels, identified by the H3Cit biomarker, are a fairly specific prognostic indicator of an increased risk of VTE 2 years after diagnosis or recurrence in cancer patients. An increase in H3Cit to 100 ng/mL is known to increase the risk of VTE by 13%.¹²

In a cohort study followed for 2 years, it was found that cancer patients had elevated NET biomarkers, and 2.3% of them had arterial thromboembolism (ATE). An increase in H3Cit levels was also associated with an increased risk of developing ATE. This study also found that increasing H3Cit up to 100 ng/mL is known to increase the hazard ratio by 1.1 higher for mortality in patients with cancer.²⁷

Myeloperoxidase (MPO) as a risk factor for deep vein thrombosis

The studies were grouped into high and low, with a cut-off point of 644.14 ng/mL. High MPO levels were observed between the case and control groups with an Odds Ratio of 2.08 with a 95% Confidence Interval of 0.709 – 6.085. Myeloperoxidase (MPO) is a peroxidase enzyme that is synthesized and stored in the cytoplasm of neutrophils. MPO is mostly expressed in immune cells, such as leukocytes, polymorphonuclear neutrophils, lymphocytes, monocytes, and macrophages. MPO is secreted under certain conditions such as chronic inflammation, infection, and other conditions that involve the immune system. Activated neutrophils, monocytes, and macrophages release MPO at sites of inflammation.²³ Previous study examining the relationship between plasma DNA (including MPO) and DVT biomarkers (Von Willebrand Factor (VWF)), D-dimer, and P-selectin found a unidirectional relationship between plasma DNA levels compared to other DVT biomarkers.²⁸ In the pathogenesis of VTE, it is known that one is caused by damage to the vascular endothelium, causing the induction of tissue factor and F.VII; thus, venous thrombosis is associated with the inflammatory process. Research using a murine model supports this by demonstrating the contribution of platelets, monocytes, and neutrophils to the process of thrombus initiation and propagation. Several recent studies have also shown a contribution to the formation of Neutrophil Extracellular Traps (NET) in venous thrombosis, and MPO is suspected that MPO is a component of NET.²⁶ After inflammation due to vascular endothelial damage, endothelial cells secrete massive amounts of Von Willebrand Factor (VWF) and P-selectin. Secretion of these two components enhances platelet adhesion and leukocyte withdrawal and induces NET production. In addition, the production of cytokines by endothelial cells (IL-1β, IL-8, and ROS) accelerates NET formation.²⁹

Another mechanism underlying the relationship between MPO and VTE is the increased consumption of Nitric Oxide (NO) that occurs as a result of the inflammatory process. MPO itself binds to NO in the process, resulting in an increase in NO consumption both through the inflammatory process and by MPO itself. This condition is thought to cause inflammation of the blood vessel endothelial cells, which worsens.²⁹

MPO is a biomarker of neutrophil activation and is involved in the pathogenesis of immune-thrombosis. Neutrophil activation, degranulation, and markers of MPO release are now widely used as biomarkers for unstable angina and acute myocardial infarction, which are thought to be proatherogenic and play a role in destabilizing atherosclerotic plaques. MPO is widely recommended as an early biomarker for patients with acute coronary syndrome. Venous thromboembolism (VTE) is a condition associated with excessive free radical production in a homeostatic system that has not shown much efficiency.²⁹

This study showed that high MPO levels are not proven to be a risk factor for DVT in malignancy patients receiving platinum-based chemotherapy. One factor that can affect the results of the examination of MPO levels is that the MPO substrate is the same as the general peroxidase substrate. Hemoglobin is known to exhibits pseudoperoxidase activity, which can interfere with assays measuring true peroxidase enzymes such as myeloperoxidase (MPO). Hemoglobin’s intrinsic peroxidase-like activity may contribute to background signal or assay interference, potentially leading to underestimation or overestimation of MPO levels.^30,31 Based on the ELISA kit, there are no further cross-reactivity data in human samples, but the kit states that recombinant mouse MPO cross-reacts at approximately 0.4% while neutrophil elastase cross-reacts at approximately 0.25%. However, in this study, we did not assess and analyze hemoglobin levels in both case and control groups. We acknowledge this as a limitation and will consider reviewing and adjusting for hemoglobin levels in future studies to determine whether MPO retains prognostic relevance when hemoglobin-related interference is accounted for. Additionally, myeloperoxidase can be detected by cytometry, immunohistochemistry, or cytochemical staining. Some literature states that from existing studies comparing different levels of myeloperoxidase tests have not obtained satisfactory results, so further standardization and validation are needed.^29,31

The relationship between various types of cancer and Soluble P Selectin, NET, and MPO levels is currently still being developed. P-selectin interacts with various cancer types through its ligands on tumor cells. Previous studies document the binding of P-selectin to various cancer-derived cell lines, which shows its role in facilitating metastatic behavior and cancer cell adhesion. However, specific statements regarding the effect of cancer types on soluble P-selectin have not been further studied.^32,33 Meanwhile, NET generated from activated neutrophils have been implicated in many types of cancer progression both solid tumor and hematological malignancy. The specific studies on the direct correlation between NETs and cancer type are also limited. On the other hand, the presence of MPO as inflammatory mediators and markers is often increased in cancerous conditions. Increased MPO levels in cancer patients are thought to correlate with disease activity and poor prognosis, especially among myeloid malignancies and solid tumors. This study focuses on how Soluble P Selectin, NET, and MPO affect DVT in platinum-based chemotherapy with various types of cancer. There is not much information about the type of cancer that can affect these levels.³⁴ Therefore, further research is expected to assess the effect of cancer type on Soluble P Selectin, NET, and MPO levels.

One factor that can affect the results of the examination of MPO levels is that the MPO substrate is the same as the general peroxidase substrate. Additionally, myoglobin and haemoglobin are known to exhibit peroxidase activity, which may impair the results. Myeloperoxidase can be detected by cytometry, immunohistochemistry, or cytochemical staining. Some literature states that from existing studies comparing different levels of myeloperoxidase tests have not obtained satisfactory results, so further standardization and validation are needed.²⁹ The results of myeloperoxidase (MPO) examination in this study were grouped into high and low, with a cut-off point of 644.14 ng/mL. High MPO levels between the case and control groups were not proven to be a risk factor for DVT in malignancy patients receiving platinum-based chemotherapy.¹³

Therefore, Soluble P-Selectin and Neutrophil Extracellular Traps (NET) play a role in the pathophysiology of Deep Vein Thrombosis (DVT) in malignant patients who receive platinum-based chemotherapy. The administration of anticoagulants as VTE thromboprophylaxis can be considered according to risk stratification based on existing recommendations. The pathomechanism is thought to be an immune thrombosis process, which is a new concept that is being developed. The presence of endothelial injury and activation of monocytes and neutrophils due to platinum-based chemotherapy induces the formation of NETosis, which is the initial initiation of thrombosis.