Patients with ovarian cancer have a high rate of venous thromboembolism (VTE). Ovarian clear cell carcinoma (OCCC) and high-grade serous ovarian carcinoma (HGSOC) are the 2 subtypes associated with the highest risk of VTE. For instance, the prevalence of VTE in OCCC is much higher than the rate in other subtypes of epithelial ovarian cancers (27.3% vs 6.8% in 1 study) [1].
To date, the pathological mechanisms underlying VTE formation in cancer patients have not been elucidated. One protein that has attracted much attention is tissue factor (TF). Binding of factor (F)VII/VIIa to TF on cell surfaces triggers the activation of coagulation. Many cancer types express high levels of TF. In addition, cancer cells release TF on extracellular vesicles (EVs), which are small membrane vesicles. We found that human pancreatic cancer cell lines expressed high levels of TF and released TF-positive EVs [2]. These TF-positive EVs activate coagulation and platelets and enhance venous thrombosis in mice [2]. Similarly, Sasano et al. [3] showed that human ovarian cancer cell lines expressed TF and released TF-positive EVs that activated platelets and enhanced thrombosis in mice.
Several studies have investigated the relationship between TF expression in biopsy samples from tumors and VTE in patients with ovarian cancer [[4], [5], [6]]. Uno et al. [4] collected tumor biopsy samples from 32 patients with ovarian cancer, 10 of which had VTE. Five of the 11 patients with OCCC had VTE. A high number of tumors expressed TF (24 of 32) and levels of TF correlated with VTE, D-dimer, and OCCC. Another study by the same group collected tumor biopsies from 128 patients with ovarian cancer [5]. In this study, tumor TF expression was an independent risk factor for VTE. Abu Saadeh et al. [6] collected tumor biopsies from 99 patients with ovarian cancer. Nineteen (19%) of the patients had VTE. Tumor TF protein expression was measured by immunohistochemical analysis and enzyme-linked immunosorbent assay, and TF mRNA was measured by real-time PCR. OCCC expressed the highest level of TF mRNA and protein among the different types of ovarian cancer. The study also found that tumor TF expression was significantly higher in patients with ovarian cancer with VTE than in patients without VTE. A high level of TF mRNA expression was also associated with lower patient survival.
In a previous study, we determined if levels of circulating EV-TF activity were associated with the occurrence of future and previous VTE within 9 months of cancer surgery in patients with ovarian cancer [7]. We retrospectively collected tumor and plasma samples from 105 patients with OCCC or HGSOC. Thirty-five (33%) of the patients developed VTE within 9 months of surgery. Patients with VTE had a worse overall survival compared with patients without VTE. Tumor TF expression was significantly higher in patients with OCCC than in patients with HGSOC. However, there was no difference in the level of EV-TF activity in patients with VTE compared with patients without VTE. In another study, we determined if levels of EV-TF activity in plasma were associated with VTE in a 6-week period before and after surgery [8]. EV-TF activity was measured using a fibrin generation assay with or without an anti-TF antibody. Thirty-eight patients with ovarian cancer were studied and 6 patients developed VTE, 2 preoperatively and 4 postoperatively. There was no difference in the levels of EV-TF activity in patients with or without VTE.
In this study, we prospectively recruited 66 patients with epithelial ovarian cancer between May 2017 and June 2019 at the University of North Carolina. Four of these patients had OCCC. Six (9%) of the patients developed VTE during the 2-year follow-up. VTEs occurred 5, 50, 113, 254, 479, and 640 days after sample collection. The Table shows the characteristics of patients with or without VTE. The median age of patients with VTE was higher than patients without VTE, but this difference was not statistically significant (P = .0959, Mann–Whitney test). Similarly, there was not statistical significance in the stage of cancer in patients with VTE compared with patients without VTE using Chi-square test for trend (Cochran–Armitage test for trend) (P = .8961). Consistent with previous studies, we observed a higher rate of VTE in patients with OCCC than in patients with other types of ovarian cancer (25% vs. 8%). Venous blood samples were collected in sodium citrate before surgery from the patients. All patients provided written consent for the study using a protocol approved by the University of North Carolina at Chapel Hill Institutional Review Board (11-1201). Platelet-poor plasma was prepared by centrifuging blood at 1500 x g for 15 minutes at room temperature. Levels of TF activity in EVs isolated from plasma were measured using an in-house assay. There was no difference in the levels of EV-TF activity in patients with VTE compared with patients without VTE (Figure A). The level of EV-TF activity in patients with ovarian cancer was very low (0.24 ± 0.34, mean ± SD, n = 66). This is similar to the level of EV-TF activity in a previous study with healthy controls (0.21 ± 0.11, mean ± SD). Surprisingly, patients with OCCC had low levels of EV-TF activity (0.24 ± 0.18 pg/mL, n = 4). A subset of the samples was used in a previous study to evaluate the ability of a new assay to measure levels of TF-positive EVs in plasma [9].
Table.
Patient characteristics.
| Variable | Non VTE (n = 60) | VTE (n = 6) |
|---|---|---|
| Age, y | 61 (54-68) | 71 (58-75) |
| BMI, kg/m2 | 26 (24-49) | 28 (25-55) |
| Stage | ||
| I | 9 (15%) | 1 (17%) |
| II | 6 (10%) | 1 (17%) |
| III | 25 (42%) | 1 (17%) |
| IV | 12 (20%) | 3 (50%) |
| Recurrent | 8 (13%) | 0 (0%) |
| Grade | ||
| 1 | 2 (3%) | 0 (0%) |
| 2 | 3 (5%) | 0 (0%) |
| 3 | 44 (73%) | 6 (100%) |
| 4 | 1 (2%) | 0 (0%) |
| N/A | 10 (17%) | 0 (0%) |
| Histology | ||
| Serous | 41 (68%) | 5 (83%) |
| Serous LMP | 4 (7%) | 0 (0%) |
| Clear cell | 3 (5%) | 1 (17%) |
| Other | 12 (20%) | 0 (0%) |
| Death | 23 (38%) | 4 (67%) |
Figure.
Measurement of different biomarkers in patients with ovarian cancer with or without venous thromboembolism. Patients with divided into 2 groups: no venous thromboembolism (VTE) (n = 60) and with VTE (n = 6). We measured various biomarker in the plasma from the patients (A) extracellular vesicle tissue factor activity (EV-TF activity), (B) citrullinated histone H3 (H3Cit)-DNA complexes, (C) white blood cell counts (WBC), (D) IL-6, (E) thrombopoietin (TPO), and (F) platelets. The median of each group is shown with a blue line.
Next, we measured additional biomarkers. Increased plasma levels of the neutrophil extracellular trap marker citrullinated histone H3 (H3Cit) have been shown to be associated with venous thrombosis in patients with cancer. We measured levels of H3Cit-DNA complexes in the plasma using an in-house assay. There was no difference in levels of H3Cit-DNA complexes in patients with VTE compared with patients without VTE (Figure B). We also found no difference in white blood cell counts in patients with VTE compared with patients without VTE (Figure C). Paraneoplastic thrombocytosis occurs in patients with ovarian cancer via a mechanism that involves tumor expression of IL-6 that increases hepatic thrombopoietin expression and an increased platelet count [10]. Therefore, we measured plasma levels of IL-6 and thrombopoietin using commercial enzyme-linked immunosorbent assays (R&D Systems, DY206, and DTP00B). We did not observe any increase in either IL-6 or thrombopoietin in patients with VTE compared with patients without VTE (Figure D and E). Moreover, there was no difference in platelet counts between the 2 groups (Figure F).
Our data indicate that EV-TF activity is not associated with VTE in patients with ovarian cancer. This result is consistent with our earlier study with a different cohort of patients with ovarian cancer [7]. In contrast, 3 studies have concluded that tumor TF expression was associated with VTE in patients with ovarian cancer [[4], [5], [6]]. We believe that the measurement of circulating levels of EV-TF activity is a more accurate and practical method to measure thrombotic risk in patients with ovarian cancer compared with tumor TF expression. Because thrombi in the deep veins are at a distant site to the tumor itself, tumors must release procoagulant substances, such as TF-positive EVs, into the circulation to trigger thrombosis. Interestingly, we have found that EV-TF activity is associated with VTE in patients with pancreatic cancer but not with other types of cancer, including glioblastoma. We could not identify other biomarkers that were increased in patients with ovarian cancer with VTE. The main limitation of this current study is the small number of patients with only 6 VTE events. We did not collect race/ethnicity of the patients used in the present study, but we do not think that this impacts the conclusions of the study.
Acknowledgments
Author contributions
N.M., V.B.-J, and Y.H. designed the experiments. Y.H. and N.M. wrote the manuscript and the coauthors provided helpful comments. M.N., S.P., and V.B.-J. collected the samples from the patients. Y.H., S.A., and R.C. measured the different biomarkers in the plasma samples. Y.H., S.A., V.B.-J., and N.M. analyzed the data.
Funding
This work was supported by the NIH NHLBI R35HL155657 (N.M.) and the John C. Parker professorship (N.M.). We would like to thank the patients that donated blood for this study.
Relationship disclosure
V.B.-J. receives grants from Merck, Genentech, Chimerix, and Epirium. V.B.-J. serves as a consultant for Eisai. The other authors have no relevant conflicts of interest to disclose.
Informed patient consent
Patients provided informed consent. The study’s protocols were approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol 11-1201).
Footnotes
Handling Editor: Cihan Ay
References
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