In this study, we have examined the hypothesis that the pathogenesis of thrombosis that complicates many forms of cancer is due to high levels of tissue factor associated with circulating tumor-derived microparticles. To test this hypothesis, we had four requirements: (1) to develop a method for reliably detecting, sizing and quantitating tissue factor antigen-bearing microparticles in platelet-poor plasma, without isolation and manipulation of microparticles; (2) to survey patients with various forms of cancer to approximate the prevalence of tissue factor-bearing microparticles in their plasma; (3) to design and execute a case-control study to determine whether the detection of tissue factor-bearing microparticles is more closely associated with cancer patients with acute thrombosis than with cancer patients without thrombosis; (4) to determine whether tissue factor-bearing microparticles associated with malignancy are tumor-derived.
In order to address the analytical issues of identifying and quantitating microparticles in platelet-poor plasma, we adapted a flow cytometric methodology capable of particle size measurements based upon impedance. Triggering on fluorescence, the diameter, in micrometers, of each fluorescent particle was determined. We demonstrated that this system was capable of reproducibly determining both the size and number of fluorescent microspheres to an accuracy of ± 5%. This method demonstrates marked improvement over light scattering-based flow cytometry, a non-optimal technique for estimating microparticle size and number.
In the current work, we have shown a significant association between the presence of tissue factor-bearing microparticles and acute cancer-associated thrombosis. In contrast, acute thromboembolic disease in the absence of malignant disease was not characterized by a similar increase in levels of tissue factor-bearing microparticles. The cancer patients who presented with an acute VTE were similar to a cancer-no VTE population for multiple factors including age, sex, cancer diagnosis, current cancer stage, diabetes, and smoking. However, the cancer patients with VTE tended to have lower platelet counts or hemoglobin and were less likely to be receiving chemotherapy. The basis for the differences in blood counts and active therapy between the cancer VTE and cancer no VTE groups is likely due to differences in timing of study enrollment. The acute VTE group was identified at any point during the treatment cycle whereas control patients were enrolled at outpatient visits and were more likely to have recovered from chemotherapy-associated cytopenias. As both higher platelet counts and chemotherapy are considered contributing risk factors for cancer-associated VTE (9
), the risk of thrombosis attributed to tissue factor-bearing microparticles may in fact be greater than observed. In this case-control study, we established that tissue factor-bearing microparticles are four-fold more likely to be found in cancer patients with an acute thrombotic event than in cancer patients without an acute thrombotic event. These results extend previous observations that tissue factor-bearing microparticles can be found in the plasma of some patients with cancer, and demonstrate that tissue factor-bearing microparticles are a risk factor for the development of thrombosis.
To further explore the association of circulating tissue factor-bearing microparticles and VTE, we performed a review of all radiographic studies obtained following enrollment to assess for the development of proximal deep vein thrombosis or pulmonary emboli in cancer patients who had not incurred VTE at time of enrollment. Although this retrospective review was performed in a blinded manner, the patients were not systematically assessed for the development of VTE. However, the potential for systematic bias is low as this was a non-intervention trial and enrollment did not impact the subsequent management of these patients. Only those cancer patients with detectable tissue factor-bearing microparticles subsequently developed a thrombotic event in the year following enrollment. This analysis corroborated the association between tissue factor-bearing microparticles and cancer-associated thrombosis identified in the case-control study and suggests that these microparticles may be central to the pathogenesis of thromboembolic disorders associated with malignant disease. Due to the inherent limitations of small case control studies and retrospective cohort analysis, larger prospective studies with systematic monitoring for the development of VTE is required to confirm these findings.
Other groups have also observed that tissue factor-bearing microparticles can be measured in patients with advanced cancer (29
). Tesselaar and colleagues demonstrated an increase in tissue factor activity in platelet-poor plasma from patients with cancer compared with platelet-poor plasma from healthy controls (29
). Tissue factor activity associated with microparticles was measured in all seven metastatic cancer patients with a recent history of venous thromboembolic disease. However, the absolute number of tissue factor-bearing microparticles measured by flow cytometry was not significantly different in cancer patients compared with healthy controls (29
). Tesselaar et al using light scattering-based flow cytometry reported a median of 460 tissue factor-bearing microparticles per μl of platelet-poor plasma in pancreatic cancer patients (range: 240-1550/μl)(29
), or less than one tissue factor-bearing microparticle per leukocyte, whereas the tissue factor-bearing microparticle concentrations in our cancer patients varied from 70,000 per μl to 3,200,000 per μl. This is a discrepancy of from three to four orders of magnitude, and raises issues of the sensitivity for the identification of tissue factor-bearing microparticles by light scattering. Hron et al reported that the median number of tissue factor-bearing microparticles was significantly greater in twenty cancer patients with colorectal cancer compared with controls while the individuals' history of VTE was not specified (30
). More recently, Khorana and colleagues observed that two patients with pancreatic cancer with increased levels of plasma tissue factor subsequently developed a venous thromboembolic event.(39
We present two independent arguments that the tissue factor-bearing microparticles are largely derived from the tumor itself rather than from inflammatory cells. First, in three patients with pancreatic carcinoma undergoing pancreatectomy with curative intent, the microparticle concentration before surgery was reduced to very low or unmeasurable levels about a month after surgery. Second, in three patients with pancreatic carcinoma, we have demonstrated that about 50% of the tissue factor-bearing microparticles co-express MUC-1, a tumor marker for pancreatic carcinoma. Tumor cells express tissue factor (12
), the expression of tissue factor activity on tumor cells correlates with thrombotic risk in pancreatic cancer patients (40
), cancer cells in culture shed procoagulant vesicular structures (34
), and circulating tumor-derived tissue factor-bearing microparticles can be detected in mice xenografted with human pancreatic tumors (41
). These results provide compelling evidence that tissue factor-bearing microparticles are derived from certain tumors. Indeed, we suspect that the variability of microparticle generation from different tumors, as well as the total tumor burden, is the basis for the variability of thrombotic events in cancer-associated thrombosis. However, the presence of circulating tissue factor bearing microparticles is not unique to cancer patients as high levels were identified in a subset of non-cancer controls as well as individuals with an idiopathic VTE. The clinical significance of these elevations in non-cancer populations is not known.
Venous thromboembolic disease is a leading cause of death in patients with malignant disease (42
) but recent randomized studies failed to demonstrate the benefit of prophylactic anticoagulation in cancer patients even in the presence of indwelling central catheters (43
). The methodologies and conclusions of this work provide a rationale basis to determine whether the detection of tissue factor-bearing microparticles in cancer patients and their use as a biomarker predict an increased risk of a thromboembolic event. If so, it is critical to determine whether patients identified with this biomarker will benefit from primary thromboprophylaxis.