VEGF , D-dimer and Coagulation Activation Markers in Indonesian Patients with Polycythemia Vera and Essential Thrombocythemia and Their Relation with Recurrence of Thrombosis

Results: The mean age for PV/ET was 51.7 ± 14.9 years. Thrombosis episode was recorded for 94.3% (33/35) patients. Twenty (57.1%) clinically stable and 15 (42.9%) patients had recurrence of thrombosis. D-dimer (P=<0.001), fibrinogen (P=0.005) were statistically significant and VEGF (P=0.06) were seen in recurrence of thrombosis compared to clinically stable patients who had normal D-dimer. Elevated D-dimer seen in recurrence thrombosis was significantly correlated with VEGF (P=0.002) levels. Elevated VEGF were seen in 45% of clinically stable patients and 73.3% in recurrence of thrombosis. VEGF, D-dimer and Coagulation Activation Markers in Indonesian Patients with Polycythemia Vera and Essential Thrombocythemia and Their Relation with Recurrence of Thrombosis ORIGINAL SrySuryani Widjaja1, Karmel L Tambunan2, Yahwardiah Siregar1, Rahajuningsih Dharma3, Stephen CL Koh4


Introduction
Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia negative myeloproliferative neoplasms (MPNs) with cardinal features of an increased red cell mass in PV and high platelet count in ET (1,2).They have high predisposition to thrombosis with a rate of major thrombosis as high as about 50% (3,4).It is associated with JAK2V617F somatic mutation which carries an enhanced risk of thrombosis (5) but their role in thrombotic complications is not entirely clear.Clinical factors (age, previous history of thrombotic events, JAK2 V617F somatic mutation (6) as well as leukocytosis, erythrocytosis and thrombocytosis is associated with enhanced risk of thrombosis (7).Blood hyperviscosity causes high shear stress of vessel wall and accounts for chronic endothelial dysfunction, platelet and leukocyte activation (8).Blood cell activation effects on the endothelium and plasma clotting factors are most likely the cause of thrombin formation in MPN have been reported (9).The mechanisms leading to thrombosis remain largely speculative, but are likely to be complex and multifactorial (10).Vascular endothelial growth factor (VEGF) a potent regulator of vascular permeability and angiogenesis is also an indirect procoagulant released by platelets (11,12).Increased angiogenic activity has been reported in PV and ET (13,14) and platelets are the major physiological transporter of VEGF in blood (15).VEGF induces platelet activation and elevated levels stimulate endothelial activation, enhance tissue factor synthesis and induce thrombin formation (16,17).Increased levels might be responsible for endothelium activation and for occurrence of a thrombotic event (18).The role of D-dimer assays in the diagnosis of patients with suspected deep vein thrombosis (DVT) has been extensively studied (19).It is a very useful diagnostic tool in the management of patients with suspected DVT and pulmonary embolism (PE) (20) and has been shown to have a high sensitivity and a high negative predictive value for DVT exclusion (21).It is a marker for hypercoagulability and links with venous as well as arterial thrombotic events and has been used to determine the hypercoagulable state leading to thrombosis in PV and ET (22,23).The main strategy of management in PV and ET is to prevent thrombosis besides treating the disease (24).
The aim of the study is to determine VEGF, D-dimer and coagulation activation markers in Indonesian patients with polycythemia vera and essential thrombocythemia and their relation to recurrence of thrombosis.

Subjects
The study received ethical approval from the Health Research Ethical Committee (197/KOMET/ FK/USU2010), Medical School, University of North Sumatra, Indonesia.Only patients diagnosed with PV and ET as defined by the WHO Protocol 2008 Conclusions: Elevated D-dimer and fibrinogen with higher mean VEGF levels were seen in recurrence of thrombosis.VEGF and D-dimer measurements have clinical use in determining the risk of patients with vascular complications.(25) and who gave signed informed consent were admitted to the study.Patients were recruited between August 2010 and January 2011 from the hospitals in Medan, Indonesia: Adam Malik Hospital, Dr Pirngadi Hospital, Herna Hospital, Imelda and Elizabeth Hospital.Recruitment of patients with either PV/ET on their visit to various hospitals and clinics was referred to the study.This include fourteen patients who were diagnosed with recurrence of thrombosis at admission to the study and one patient who developed recurrence of thrombosis three days later.
A total of 45 patients were recruited and only 35 patients (PV n=24 females n=3; ET n=11 females n=4) who fulfilled the inclusion criteria was admitted into the study.Their age ranged between 14 years and 79 years (mean 52.0 ± 15.1 years).Their characteristics are shown in Tables 1 and 2

Blood collection
Blood sampling was performed from a clean venepuncture using the Vacutainer system (Beckton Dickenson, New Jersey, USA).About 8 mL of blood was collected and 3 mL gently mixed into citrate tubes (sodium citrate 0.109M) and the remainder blood into plain tubes for serum collection.The citrated blood samples were double spun for 15 min each time at 2000g within an hour of blood collection.Serum and plasma samples were aliquoted and kept at -70° C until assayed.Blood sampling was performed only at admission to the study; in recurrence of thrombosis blood sampling was performed post-diagnosis at between 1 to 5 days (n=8), 10-20 days (n=5), and 30 days (n=1) except for one blood sampling was performed 3 days before diagnosis.Most of the patients were on aspirin treatment regime.

Laboratory assays
The following assays were performed: JAK2 V617F mutation (IPSOGEN JAK2 MutantQuant Kit) was performed at the Molecular Diagnostic Centre, National University Health System (NUHS), Yong Loo Lin School of Medicine, National University of Singapore.Serum VEGF Immunoassay (R&D systems, Minneapolis, USA) and beta Thromboglobulin (βTG) (Asserachrome Kit, Stago, France).Von Willebrand Factor (VWF), in-house modified Elisa method using International Standard from National Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, UK, Fibrinogen (Clauss method) using international standard from NIBSC, plasma D-dimer (Zymu D-dimer Kit, France).The above tests were performed at the Coagulation Laboratory, Department of Obstetrics & Gynaecology, NUHS.

Statistical Analysis
The Statistical Package for Social Sciences (SPSS17; SPSS Inc, Chicago, Il, USA) was used to perform the statistical analysis.Non-parametric Mann Whitney U test was performed together with independent t-test to evaluate patients under treatment who were clinically stable and those who developed clinical symptoms of recurrence of thrombosis at the time of admission to the study.Pearsons correlation analysis was used to determine for correlation between elevated D-dimer against VEGF seen in recurrence of thrombosis.A P value of less than 0.05 was considered statistically significant.

Results
In total, 35 patients who fulfilled the WHO defined criteria for PV/ET was studied.Their characteristics with history of thrombotic episodes are shown in Tables 1 and 2, VEGF and haemostatic parameters in Table 3.The statistics of evaluating PV/ET patients who were clinically stable and those who developed clinical symptoms of recurrence of thrombosis are shown in Table 4.The scatter plots of VEGF and D-dimer levels between clinically stable and recurrent thrombosis patients are shown in Figure 1 and the correlation between elevated D-dimer and VEGF levels in patients who developed recurrence of thrombosis in Figure 2.

Characteristics of patients studied
The mean age of the patients was 51.7 ± 14.9 years and ranged between 14 and 79 years.The ages between PV and ET with their history of thrombotic episodes are shown in Table 1.All patients were diagnosed with PV/ET between six months and ten years (n=29) and greater than ten years (n=6) (Table 2).Past history of thrombosis was recorded between one to seventeen years post diagnosis of PV/ ET, one to five years ago in PV (100%, 24/24) and ET (81.8%, 9/11), of patients except for 2 patients (ET) who were clinically stable and diagnosed two and six years ago.Twenty patients were considered clinically stable (PV n=12; ET n=8) and 15 developed clinical symptoms of recurrence of thrombosis (PV n=12, ET n=3) at the time of study: microvascular

VEGF and haemostatic parameters
No statistically significant differences in the parameters studied between PV and ET were seen and therefore their data were combined for anal-  ysis.Elevated levels of VEGF (mean 202.4±164.1 pg/mL), TAT-complex (mean 2.80±1.74ug/mL)and β-TG (mean 799.3± 883.8 IU/mL) were evident in PV/ET patients compared to normal reference.Although D-dimer, VWF and fibrinogen mean levels were within normal reference range their ranges were widely variable (  3).
Statistics of parameters studied in PV/ET patients who were clinically stable (n=20) were compared to those who had developed clinical symptoms of recurrence of thrombosis (n=15) at admission to the study (Table 4).Recurrence of thrombosis occurred in 13.3% (2/15) of patients at 65 years old and above.The mean age was higher for patients who experienced a recurrence of thrombosis (mean 57.1 ±7.5 years vs mean 47.7 ±17.7 years) relative to those that were clinically stable but this observation did not reach statistical significance (P=0.07).Statistically significant elevated D-dimer (P= <0.001) and fibrinogen (P=0.005) were seen in patients with recurrence of thrombosis compared to patients who were clinically stable.Higher mean trends were seen for TAT-complex (P=0.07) and VWF (P=0.05) in recurrence of thrombosis but they did not reach statistical significance.No significant differences were also seen for VEGF and Beta-TG levels even though the levels were elevated above the normal reference in both groups (Table 4).Elevated VEGF levels were seen in 45.0% (9/20) of clinically stable patients and 73.3% (11/15) in patients with recurrence of thrombosis.Clinically stable patients had no elevated D-dimer levels whilst in the recurrence of thrombosis group 66.7% (10/15) patients had elevated levels (>400ng/mL).The scatter plot for VEGF and D-dimer in both groups is shown in Fig. 1.

Discussion
The thrombotic risk in PV and ET is more pronounced in those over 60 years old and with a history of previous thrombosis as has been previously reported (3,4).PV and ET have high predisposition to thrombosis with a rate of major thrombosis as high as about 50% (3,4).Incidence of recurrent thrombosis was 5% in those below 65 years old and 10.9% in those above 65 years old (26) whilst another retrospective study the rate was 15% (27).Overall incidence of recurrence is 5.9% for those below 60 years old and risk factor of 1.7 fold increase in those greater than 60 years old (28).In our study recurrence occurred in 42.9% (15/35) of which 14.7% (2/15) were over 65 years of age.VEGF measurement have been suggested to assess high and low risk PV patients (18).In this retrospective study of 35 patients diagnosed with PV/ET, 100% patients with PV had history of thrombosis between year one to seventeen while in ET it was 81,8% between year one to twelve from diagnosis, the incidence for PV/ET was 94.3% which was much higher than reported (3,4).At admission to the study, 15 patients (42.9%),PV n=12, ET n=3 were reported to have developed recurrence of thrombosis between one and five years from last thrombotic episode whilst the remaining 20 patients were clinically stable including two (ET) patients with no history of thrombosis.This recurrence of thrombosis was higher than reported (26,8).A hypercoagulable state for this population was evident from elevated D dimer and fibrinogen levels seen in patients with recurrence of thrombosis, which is in agreement with the study of Wautier and co-workers (9).In support of this observation, D-dimer levels were not elevated in clinically stable patients.VEGF levels were elevated in 45.0% of patients who were clinically stable and 73.3% in patients who experienced recurrence of thrombosis.The elevated levels are of concern as they are known as a pro-coagulant, inducing endothelial activation and thrombin formation (16,17).Markedly increased D-dimer was found in PV/ET and no differences of coagulation activation markers was found in relation to JAK2-V617F mutational status (29).JAK2 V617F mutation was confirmed in 54.2% PV and 54.5% ET patients.We did not find any significant differences in the plasma levels of D-dimer and coagulation activation markers between JAK2 V617F positive and negative patients even though JAK2 V617F mutation associated with thrombosis (16,17).Considerable variation of JAK2 V617F mutation frequency have been reported with PV of between 65-97% and ET 23-57% (30) and PV 95%, ET 50-60% (31).The frequency of the mutation in PV patients (54.2%) in our study is rather low, whilst it was comparable to the published literature in ET patients (54.5%).The published reports for JAK2 V617F frequency are for Caucasian population and the frequency in Asians especially with PV need to be further investigated.Possible variations in mutation discrepancies observed in PV include assay sensitivity, diagnostic accuracy and treatment effect (32) have been suggested.

Conclusion
In conclusion, plasma VEGF levels were elevated (57.1%) in PV/ET patients relative to controls, although the difference was not significant.Similarly, plasma D-dimer and fibrinogen levels were significantly higher in patients with recurrence of thrombosis relative to clinically stable patients, whereas plasma VEGF levels were non-significantly elevated.Elevated plasma D-dimer seen in patients with recurrence of thrombosis was found to correlate significantly with elevated plasma VEGF levels.VEGF and D-dimer measurements have been used in clinical practice to determine the risk of PV/ET patients with vascular complications.
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Figure 1 :
Figure 1: D-dimer and VEGF levels in recurrence of thrombosis (n=15) and clinically stable states (n=20) in patients with PV/ET.

Figure 2 :
Figure 2: Correlation of elevated D-dimer against VEGF in recurrence of thrombosis.

Table 1 .
Characteristics of patients with PV and ET

Table 2 .
Duration of diagnosis, JAK2 V617F mutation and history of thrombosis in clinically stable patients (A) and those who developed recurrent thrombosis (B).

Table 4 .
Statistics of parameters studied in PV/ET patients who are clinically stable (A) compared to those who developed recurrent thrombosis (B).