Thrombosis Research 135 (2015) 588–593
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Regular Article
Thrombotic events in acute promyelocytic leukemia☆ Mirjana Mitrovic a,⁎, Nada Suvajdzic a,b, Ivo Elezovic a,b, Andrija Bogdanovic a,b, Valentina Djordjevic c, Predrag Miljic a,b, Irena Djunic a, Maja Gvozdenov c, Natasa Colovic a,b, Marijana Virijevic a, Danijela Lekovic a, Ana Vidovic a,b, Dragica Tomin a,b a b c
Clinic of Hematology CCS, Belgrade, Koste Todorovica 2 Serbia Faculty of Medicine, University of Belgrade, Dr Subotica 8 Belgrade, Serbia Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444aBelgrade, Serbia
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 24 October 2014 Accepted 30 November 2014 Available online 4 December 2014 Keywords: Acute promyelocytic leukemia Thrombotic events ATRA Risk factors PAI-1 4G/5G polymorphism
a b s t r a c t Introduction: Thrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to10-15%. Materials and Methods: We retrospectively analyzed the data on TE appearance in 63 APL patients. Results: TE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P = 0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P = 0.046), PT (P = 0.022), aPTT (P = 0.044), ISTH DIC score (P = 0.001), bcr3 (P = 0.02) and FLT3-ITD (P = 0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3ITD mutation (P = 0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P = 0.05). Regarding risk factors for arterial TE we failed to identify any. Conclusions: We have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score b 5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G. © 2014 Elsevier Ltd. All rights reserved.
Introduction Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a life-threatening coagulopathy leading to a high hemorrhagic early death (ED) rate of up to 29% [1–4]. In addition, thrombotic events (TE) appear to be more common in APL than in other acute leukemias, with reported prevalence of both arterial and vein thrombosis ranging from 2 to10-15% [1,5–11]. Deep vein thrombosis (DVT) is the most common form of APL-related vein thrombosis, but clots may occur in unusual sites such as cerebral venous sinuses, portal and hepatic veins [6,7]. Moreover, thrombosis in APL may present with peripheral artery occlusion, myocardial infarction or ischemic stroke [6,7]. The potential mechanisms involved in the pathogenesis of APLrelated TE are most likely associated with both the inherent characteristics and the treatment of the disease [1,7]. Previous studies indicated
☆ Manuscript was presented on XXIV Congress of the International Society of Thrombosis and Haemostasis, Amsterdam, 29.07.-04.07.2013. ⁎ Corresponding author at: Clinic of Hematology CCS, Koste Todorovica 2, 11000 Belgrade, Serbia. Tel.: +381 63314278; fax: +381 633065112. E-mail address: [email protected] (M. Mitrovic).
http://dx.doi.org/10.1016/j.thromres.2014.11.026 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
several risk factors for TE development such as: high WBC, CD2/CD15 positivity, FLT3-ITD positivity; low fibrinogen level, APL-variant type, differentiation syndrome (DS) and high platelet count, hypoalbuminemia, male sex, poor performance status [5,10,11]. However, data on the pathogenesis, epidemiology, risk factors and clinical outcome of APL-related TE are controversial [1,7]. Concerning this issue, the aim of our single-center retrospective study was to report on TE in an unselected group of 63 de novo APL patients and to evaluate the associated clinical and laboratory parameters. Patients and Methods Patients, Diagnostic Procedures and Treatment During the period 2004 - 2013, 63 consecutive de novo APL patients were diagnosed at the Clinic of Hematology. The diagnosis was confirmed in all patients by detection of t(15;17)(q22;q12) or PML-RARA rearrangements. The diagnostics comprised cytomorphology, cytogenetics, molecular genetics and immunophenotyping of bone marrow (BM) or peripheral blood. Morphologic diagnosis was made according to the French–American–British classification [12]. Conventional G-band karyotyping was employed for cytogenetic analysis [13]. The PML-RARA
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fusion transcript was detected using a reverse transcriptase polymerase chain reaction (RT-PCR) [14]. The presence of FLT3-internal tandem duplication (ITD) mutations was investigated in 48 cases using PCR and PCR-RFLP methods [15]. Immunophenotyping was performed by flow cytometry using a direct multicolor immunofluorescent technique applied to whole BM specimens with a wide panel of monoclonal antibodies (CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD22, CD24, CD33, CD34, CD36, CD41a, CD45, CD56, CD64, CD66b, CD117, CD235a, HLA-DR, CD3, MPO, CD79a, and TdT [16]. All patients were treated with ATRA + anthracyclines (PETHEMA APL 99 and PETHEMA APL 2005 protocols) [17,18]. Platelet counts were maintained N 30 × 109/L by platelet transfusions; fresh-frozen plasma was administered when the fibrinogen level was b 1.0 g/L until resolution of the coagulopathy according to the recommendations [18]. During the first 28 days of hospitalization we recorded the number of days spent with platelet counts and fibrinogen below recommended goal values, i.e. number of days in which platelet or fresh-frozen plasma transfusions were applied. Antifibrinolytics and anticoagulant therapy were not used. Central venous catheters (CVC) were not inserted before coagulopathy had ceased. Hemostasis Related Parameters Activated prothrombin time (aPTT), prothrombin time (PT) and fibrinogen were measured using an ACL-Automated Coagulation Laboratory (IL, Italy). D-dimer was assessed immunoturbidimetrically by the BCS XP System (Siemens, Germany). Normal ranges were as follows: for PT 75-120%, for aPTT 25-35 s, for fibrinogen 2-4 g/L and for D-dimer b 250 μg/L. A diagnosis of disseminated intravascular coagulation (DIC) was made according to the recommendations of the International Society on Thrombosis and Hemostasis (ISTH). ISTH DIC score was calculated before therapy initiation. Patients with ISTH DIC score ≥ 5 were considered to have overt DIC [19]. Antithrombin (AT), Protein C (PC) and Lupus anticoagulant (LA) were determined with reagents from Siemens (Antithrombin III, Protein C, LA1 and LA2 Reagents) on Sysmex Ca-1500 instrument, according to manufacturer's instructions. Protein S (PS) was determined by coagulation method, using "ProS reagent" on ACL-7000 instrument (IL, Italy). Genotyping of factor (F) V Leiden, prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 (PAI-1) 4G/5G gene variants was carried out in 12 patients with TE and 25 patients without TE using PCR [20]. For comparison purposes, blood samples from 126 healthy blood donors were analyzed for those mutations. The healthy individuals had no personal or family record of TE disease and were free of any systemic, cardiovascular, malignant or inflammatory illness. Thrombotic and Bleeding Events Data Patients were assessed for TE and bleeding from the date of admission until the last day of follow-up. Central nervous system (CNS), pulmonary or gastrointestinal hemorrhage were considered as severe bleeding [4]. In patients with signs and symptoms of venous or arterial thrombosis specific imaging techniques were applied. Namely, DVT was established by Color Doppler ultrasonography. Budd-Chiari syndrome (BCS) was diagnosed by computed tomography (CT) and central retinal vein occlusion (CRVO) by ophthalmoscopy and fundus angiography. The diagnosis of cardiac ischemia was based on electrocardiographic and cardiac enzyme modifications. The diagnosis of ischemic stroke was based on neurologic examination and CT or MRI findings. D Dimer Monitoring Since 2011 D-dimer was assessed weekly after coagulopathy cessation during whole induction. In patients with recurring elevation
589
of D-dimer deep vein and CVC arm vein Color Doppler ultrasonography were performed. In patient N011 with D dimer recurring elevation and recent abnormal liver functional tests, hepatic vein Color Doppler ultrasonography was performed. Thrombotic Events Therapy Patients with venous TE were treated with low molecular weight heparin (LMWH) for 3 to 6 months. Dose of LMWH was reduced by half if the patients′ platelet counts were between 30 and 50 x 109/L. LMWH was discontinued at platelet count b 30 x 109/L. Arterial TE were treated with antiplatelet drugs at platelet count N 100x109/L. Recanalization of the veins was assessed at 3 and 6 months after anticoagulant therapy initiation by Color Doppler ultrasonography (patients with DVT and BCS) and ophthalmoscopy (CRVO). Statistical Analysis Chi-square and Fisher exact tests were used to analyze the association between variables and the probability P ≤ 0.05 was considered statistically significant. Survival curves were calculated by the KaplanMeier method and differences between groups were assessed by the log-rank test. Univariate and multivariate COX proportional regression models were employed for identification of risk factors. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the Statistical Package for Social Science. Results The study population consisted of 63 patients, 30 (48%) males and 33 (52%) females, of median age 44 years (range 19–78). Between September 2004 and August 2010, 42 de novo APL patients were treated according to the PETHEMA APL 99 protocol, while 21 patients received PETHEMA APL 2005 from September 2010 to May 2013. Bleeding was registered in 56/63 (89%) cases, severe in 14/63 (22.2%) with hemorrhagic early death rate of 8/63 (12.7%). All hemorrhagic deaths occurred during the first week of hospitalization with the median time of two days after therapy initiation. Thirteen (20.6%) patients, without previous history of thrombosis, developed TE, four arterial (6.3%) and nine venous (14.3%). In only one patient (N01), severe bleeding and TE developed concomitantly. The main clinical and biological characteristics of these patients are shown in Table 1. Arterial Thrombotic Events in APL Patients Arterial TE developed in 4/63 (6.3%) patients, with a female/male ratio of 3/1 and a median age of 50 years (range 38-64). Arterial TE encompassed acute myocardial infarction (AMI) in two cases and cerebrovascular accident (CVA) in two cases. CVA in patient N01 was the initial APL symptom, while the remaining three patients developed TE during induction. The median time from therapy initiation to arterial TE occurrence was 23 days (range 22-26). In patient N0 3 with AIM occuring during induction, percutaneous coronary intervention with stent implantation was performed immediately after BM recovery, followed by dual antiplatelet therapy with aspirin and clopidrogel for 6 months. The remaining three patients with arterial TE were not treated with antiplatelet drugs due to thrombocytopenia. In patient N0 2, CVA became stable during induction with remaining discrete rightsided hemiparesis. Patients N0 1 and 4 died within 28 days after therapy initiation: patient N0 4 due to AIM and patient N0 1 due to intracranial hemorrhage (Table 1). The mortality rate of patients with arterial TE was 50%. However, arterial TE did not have a significant impact on ED rate or 4-year overall survival (OS; Table 2).
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Table 1 Demographic, clinical and biological characteristic of the APL patients with thrombosis. Pts No
Age years/ sex
WBC x10 9/L
Plt count x109/L
Sanz risk score
ISTH score
CD2/ CD15
PML-RARα
1
38/f
8.6
47
low
7
-/ND
2 3
54/f 46/m
1.1 21.6
25 39
inter high
5 6
4
64/f
0.9
39
inter
5 6
64/m 51/m
1.4 1.8
93 45
7
31/m
88
8
46/m
9
FLT3 -ITD
DS
ND
ND
−/+ −/−
bcr3 bcr3
6
−/−
low low
4 5
23
high
5.6
48
45/m
1.3
10
52/m
11 12 13
49/f 53/m 26/f
Thrombosis
Thrombophilia
Risk factors
Outcome
yes
ND
no
yes no
PAI-1 4G/5G MTHFR hetero PAI-1 4G/4G PAI-1 4G/5G
smoking DM hyperten hyperten
Early death CR CR
PAI-1 4G/5G lupus anticoagulant MTHFR hetero MTHFR hetero PAI-1 4G/4G MTHFR hetero FV Leiden hetero MTHFR hetero PAI-1 4G/4G PAI-1 4G/4G PAI-1 4G/5G Prothrombin 20210 hetero MTHFR hetero PAI-1 4G/4G
no no
Early death CR CR
smoking
CR
no
CR
no
CR
smoking
CR
smoking smoking no
CR CR CR
Site
Time
DIC
yes
CVA
-
no no
CVA AIM
pre therapy induction induction
bcr1
+
no
AIM
induction
no
−/− −/−
bcr3 bcr1
ND
yes no
DVT DVT
induction induction
no no
5
−/+
bcr3
-
no
induction
no
inter
4
+/−
ND
+
no
DVT CVC DVT
induction
no
135
low
2
+/−
bcr1
ND
no
CRVO
mainten
no
28.4
15
high
5
+/−
bcr3
+
no
induction
yes
2.9 1.3 1.3
95 51 92
Low low low
5 5 5
+/− +/− −/−
bcr3 bcr1 bcr3
+ + +
No no yes
DVT CVC BCS DVT DVT CVC
induction induction induction
no no yes
N- number, f - female, m - male, WBC – with blood cells, Plt – platelets, ISTH score - International Society on Thrombosis and Hemostasis score, DIC - Disseminated Intravascular Coagulation, ND - not done, CVA - cerebrovascular accident, DVT – deep vein thrombosis, CVC – central vein catheter, AIM - acute myocardial infarction, CRVO – central retinal vein occlusion, BCS - Budd-Chiari syndrome, mainten - maintenance therapy, hetero – heterozygous, DM – diabetes mellitus, hyperten – hypertension, CR – complete remission.
Neither thrombophilic mutations nor clinical and biological features of our APL patients apeared predictive for ATE development (Tables 2 and 3).
Table 2 Clinical characteristics of the APL patients with and without arterial thrombosis. Parameter
With arterial thrombosis (4 pts)
Without thrombosis (50 pts)
P
Age (years) Sex (male/female) Smokers Diabetes mellitus Hypertension Bleeding Sever bleeding WBC count (x109/L) Platelet count (x109/L) Sanz risk score low (N0) intermediate high Fibrinogen (g/L) PT (%) aPTT (s) D-dimer (μg/L) ISTH DIC score CD2 expression (N0) CD15 expression (N0) bcr1-2/bcr3 FLT3-ITD N0 of days with platelets b 30x109/L N0 of days spent with fibrinogen b 1 g/L Differentiation syndrome (N0) Early death 4-year overall survival
50 (38-64) 1/3 ¼ (25%) 1 (25%) 2/4 (50%) 4/4 (100%) ¼ (25%) 1 (0.9-21.6) 32 (1-47) 1/4 (25%) 2/4 (50%) 1/4 (25%) 2.425 (0.86-3.9) 35 (21-75) 28.75 (23.5-36) 3157 (3045-6000) 6 (5-7) ¼ (25%) 1/3 (33.3%) 1(50%)/6(50%) 0/3 (0%) 14 (1-18)
41 (19-78) 20/30 20/50 (40%) 4/50 (8%) 17/50 (34%) 45/50 (90%) 12/50 (24%) 3.6 (0.5-183.2) 28 (4-101) 10/50 (20%) 21/50 (42%) 19/50 (38%) 2.69 (0.57-6.23) 62 (21-124) 26.1 (21.2-35.5) 2332 (624-11340) 6 (3-7) 9/42 (21.4) 15/43 (34.9%) 11(61%)/7(39%) 8/36 (22.2%) 12 (3-22)
0.247 0.492 0.678 0.08 0.234 0.567 0.781 0.552 0.775 0.24
0.674 0.277 0.757 0.264 0.459 0.503 0.726 0.32 0.444 0.628
1(0-2)
2 (0-5)
0.671
1/4 (25%)
11/50 (22%)
0.63
2/4 (50%) 17.5 (0-63)
12/50 (24%) 25 (0-104)
0.274 0.342
Pts – patients, WBC – white blood cells, N0 – number, ISTH score - International Society on Thrombosis and Hemostasis score.
Venous Thrombotic Events in APL Patients Venous TE developed in 9/63 (14.29%) patients, with a female/male ratio of 2/7 and median age of 47.5 years (range 26-64). Venous TE encompassed DVT in 7 cases (CVC associated in 3 cases), CRVO and BCS in one case each. The majority of the venous TE occurred during induction (8/9 patients; Table 1). The median time from therapy initiation to TE occurrence was 14 days (range 9-26). Only 4/13 (31%) patients displayed DIC at the time of venous TE occurrence. All 9 patients with venous TE were treated with anticoagulants resulting in complete vein recanalization. It is of note that venous TE did not have an impact on either ED or 4-year OS. The clinical and biological features of our APL patients with and without venous TE are shown in Table 4. The two groups were significantly different regarding female/male ratio (P = 0.046), PT (P = 0.022), aPTT (P = 0.044), ISTH DIC score (P = 0.001), bcr3 (P = 0.02) and FLT3-ITD (P = 0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate Cox's proportional regression was FLT3-ITD mutation (P = 0.034, RR 10.036, 95% CI 1.197-84.123). (See Table 5.) Concerning thrombophilia testing AT, PC and PS deficiencies were not identified. Only one patient (N0 6) was LA positive. Genotype frequencies are presented in Table 3. Thrombophilic gene mutations were present in 8/9 (88.9%) patients: FV Leiden heterozygous mutation and FII G20210A mutation in one (8.3%) patient each, PAI-14G/4G in 5/9 (55.6%) patients and PAI-1 4G/5G in 1/9 (8.3%) patients. Combined thrombophilia was registered in 4/9 (44.4%) patients. FV Leiden heterozygous mutation, prothrombin 20120 and PAI-1 4G/4G were three, three and five times more frequent in APL patients with venous TE when compared to patients without TE, with significant differences for PAI 4G/4G (P = 0.05).
D Dimer Monitoring During the period 2011 - 2013, 18 consecutive de novo APL patients underwent regular weekly D-dimer monitoring. In the one patient D
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Table 3 Allele and genotype frequencies in APL patients with and without arterial thrombotic event and healthy controls. APL patients with ATE (3 pts)
APL patients without TE (25 pts)
Healthy controls
ORa 95%CIa
ORb 95%CIb
Pa
Pb
heterozygous GA homozygous AA heterozygous GA homozygous AA heterozygous 4G/5G
0% 0% 0% 0% 2/3 (66.7%)
1/25 (4.0%) 0% 1/25 (4.0%) 0% 14/25 (56.0%)
4.2% 0% 5.8% 0% 46.2%
0.49
1/3 (33.3%)
5/25 (20.0%)
34.8%
0.60
0.96
heterozygous CT
1/3 (33.3%)
8/25 (32.0%)
39.2%
0.96
0.84
homozygous TT
0%
6/25 (24.0%)
11.5%
2.33 0.21-26.09 0.94 0.08-10.52 0.78 0.07-8.81 -
0.73
homozygous 4G/4G
1.57 0.13-19.67 2.00 0.15-26.74 1.06 0.08-13.52 -
-
-
Gene variant F II G20210A F V Leiden PAI – 1 4G/5G
MTHFR C677T
APL- acute promyelocytic leukemia, ATE - arterial thrombotic event, TE thrombotic event, Pts – patients, F – factor. a – APL patients with ATE and without TE comparison. b – APL patients with ATE and healthy controls comparison.
dimer monitoring was not performed due to hemorrhagic ED, sixth day after therapy initiation. Four (21%) patients were diagnosed with symptomatic TE. In four of the remaining 14 patients (28.6%) D dimer reelevation was seen. In three of those four patients a diagnosis of TE was made (DVT in patients N0 6 and 11; partial BCS in patient N0 10). One patient had falls positive result probably due to infection. TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P = 0.032).
Discussion Previous studies reported a rising incidence of APL-related TE from 2% in the pre-ATRA era to 4.5-15% in patients treated with a combination of ATRA and anthracyclines [1,5–11]. This could be ascribed to the higher level of vigilance and availability of advanced diagnostic tests, or alternatively to the new therapeutic approach [1,7]. Table 4 Baseline characteristics of the APL patients with and without venous thrombosis. Parameter
With thrombosis (9 pts)
Without thrombosis (50 pts)
P
Age (years) Sex (male/female) Bleeding Sever bleeding WBC count (x109/L) Platelet count (x109/L) Sanz risk score low (N0) intermediate high Fibrinogen (g/L) PT (%) aPTT (s) D-dimer (μg/L) ISTH DIC score ISTH DIC score b5 CD2 expression (N0) CD15 expression (N0) bcr1-2/bcr3 FLT3-ITD N0 of days with platelets b 30x109/L N0 of days spent with fibrinogen b 1 g/L Differentiation syndrome (N0) Early death 4-year overall survival
47.5 (26-64) 7/2 7/9 (78%) 1/7 (14.3%) 1.6 (1.3-88) 44 (15-135) 5/9 (55.6%) 2/9 (22.2%) 2/9 (22.2%) 3.9 (2.22-4.79) 74 (62-89) 31.76 (23.1-34.0) 2743 (551-6000) 5 (2-5) 4/9(44.4%) 5/9 (55.6%) 1/9 (11%) 3(30%)/6(70%) 5/9 (55.6%) 13 (5-20)
41 (19-78) 20/30 45/50 (90%) 12/50 (24%) 3.6 (0.5-183.2) 28 (4-101) 10/50 (20%) 21/50 (42%) 19/50 (38%) 2.69 (0.57-6.23) 62 (21-124) 26.1 (21.2-35.5) 2332 (624-11340) 6 (3-7) 5/50 (10%) 9/42 (21.4) 15/43 (34.9%) 11(61%)/7(39%) 8/36 (22.2%) 12 (3-22)
0.359 0.046 0.367 0.234 0.460 0.071 0.34
0.105 0.022 0.044 0.879 0.001 0.031 0.093 0.245 0.032 0.028 0.145
1 (0-3)
2 (0-5)
0.567
2/9 (22%) 0/9 32 (5-93)
11/50 (22%) 12/50 (24%) 25 (0-104)
0.93 0.181 0.883
Pts – patients, WBC – white blood cells, N0 – number, ISTH score - International Society on Thrombosis and Hemostasis score.
The main finding of this study is a higher incidence of APL-related thrombosis (20.9%) in comparison with previously published data [1, 5–11]. In addition, this is the first study addressing the issue of asymptomatic DVT in APL patients. Since 2011, when regular weekly D-dimer monitoring was initiated, TE have been diagnosed in three asymptomatic (3/14, 21.4%) and four (4/19, 21%) symptomatic patients. These results suggest that the true prevalence of venous TE in APL patients is underestimated. Concerning this issue recent studies suggest that the presence of both asymptomatic and symptomatic TE have a similar clinical impact on the cancer patients’ prognosis [21]. These findings emphasize need for VTE screening in APL patients after DIC cessation. Although not specific, D dimer is highly sensitive marker for VTE, with levels corresponding to the extent of thrombosis. The pathogenesis of the increased thrombotic risk in APL patients is not fully understood, and comprises a combination of variables related to the disease itself, treatment and host characteristics [1,5,7,9–11,22]. Notably, leukemic promyelocytes directly contribute to the thrombohemorrhagic tendency in APL via expression of tissue factor (TF) and cancer procoagulants [23,24]. Thus, DIC was registered in 87% of patients at diagnosis and in 30.7% of patients at the time of the TE occurrence. Concerning this issue, TE in APL patients might be part of the DIC. However, our patients with APL-related venous TE had significantly lower median initial ISTH scores compared to APL patients without thrombosis. Moreover, an initial ISTH DIC score ≤ 5 was predictive for venous TE. In our previous study ISTH DIC score ≥ 5 was predictor for bleeding and ISTH DIC score ≥ 6 was the most significant predictor factor for hemorrhagic early death in APL patients [4]. Our results suggest that DIC appears the major cause of bleeding in APL patients [4]. Moreover, the degree of DIC may correlate with the risk of both bleeding and thrombosis [4]. In our study thrombosis typically developed after therapy initiation, which is consistent with other reports [5,6]. This finding suggests an association between TE and treatment [1]. Moreover, anthracyclinesinduced apoptosis might contribute to thrombogenesis in APL via increased cellular TF activity and the release of TF-bearing microparticles from the damaged leukemic cells [7]. Additionally, ATRA has a procoagulant effect due to enhanced expression of adhesive molecules on both promyelocytes and endothelium and increased production of cytokines [1,7,23]. Why some patients with APL develop thrombosis and some do not has not been clarified yet. Several host and disease related risk factors have been proposed, such as: male sex, worse PS, high WBC and platelet counts, low fibrinogen level, hypoalbuminemia, M3 variant type, CD2/ CD15 and FLT3-ITD positivity [1,5,6,9–11]. In our study arterial and venous TE were separated in the risk factor analysis. However, we failed to identify any risk factor for arterial TE development, probably due to the low number of patients. On the other
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Table 5 Allele and genotype frequencies in APL patients with and without venous thrombotic events and healthy controls. Gene variant
APL patients with VTE (9 pts)
APL patients without TE (25 pts)
Healthy controls
ORa 95%CIa
ORb 95%CIb
Pa
Pb
3.00 0.17-53.71 3.00 0.17-53.71 0.10 0.01-0.91 5.00 0.97-25.77 2.66 0.56-12.65 -
2.88 0.30-27.65 2.02 0.22-18.48 0.15 0.02-1.18 2.35 0.61-9.01 1.94 0.50-7.60 -
0.46
0.36
0.46
0.53
0.04
0.07
c
0.21
0.22
0.34
-
-
F II G20210A
heterozygous GA
1/9 (11.1%)
1/25 (4.0%)
4.2%
F V Leiden
homozygous AA heterozygous GA
0% 1/9 (11.1%)
0% 1/25 (4.0%)
0% 5.8%
PAI – 1 4G/5G
homozygous AA heterozygous 4G/5G
0% 1/9 (11.1%)
0% 14/25 (56.0%)
0% 46.2%
homozygous 4G/4G
5/9 (55.6%)
5/25 (20.0%)
34.8%
heterozygous CT
5/9 (55.6%)
8/25 (32.0%)
39.2%
homozygous TT
0%
6/25 (24.0%)
11.5%
MTHFR C677T
APL- acute promyelocytic leukemia, Pts - patients, VTE – venous thrombotic event, TE – thrombotic event, F – factor. a – APL patients with VTE and without TE comparison. b – APL patients with VTE and healthy controls comparison.
hand male sex, ISTH-DIC score b 5, bcr3 isoform and FLT3-ITD positivity were identified as predictive for venous TE occurrence in our APL patients. Furthermore, multivariate analysis indicated FLT3-ITD positivity as the most significant predictor for venous TE (P = 0.034). Regarding the FLT3-ITD mutation issue, a study on gene expression profile found that leukemic cells from FLT3-ITD + patients have an increased expression of genes regulating blood coagulation and cell adhesion [24]. Notably, underlying hereditary thrombophilia may contribute to venous TE development in cancer patients [25]. However, there are few studies on this topic in APL patients with controversial results. That is Dally et al. [9] reported that 50% of patients with APL- related TE had congenital thrombophilia, while Breccia et al. [5] and Rees et al. [26] failed to confirm this finding. In our study, 88.9% of the APL patients with associated TE had some thrombophilic mutation, while 44.4% of them had combined thrombophilia. Namely, the FV Leiden heterozygousity and FII 20210 mutations were three times more frequent in our APL patients with venous TE than in patients without it. This is the first study assessing the impact of PAI-1 mutational status on APL-related TE to the best of our knowledge. The PAI 4G/4G was five and two times more frequent in our patients with APL-related venous and arterial TE than in patient without TE. PAI-1 is a key regulating factor determining endogenous fibrinolytic activity [27]. A 4G/5G polymorphism of the PAI-1 gene has been reported to influence the levels of PAI-1. The 4G allele was found to be associated with higher plasma PAI-1 activity, which can lead in impaired fibrinolysis [26]. Moreover, it was shown that retinoids induce increased production of plasminogen activator inhibitors, including PAI-1 and PAI-2 [22]. Association of ATRA therapy and high PAI-1 levels due to PAI-1 gene 4G/4G polymorphism might lead to APL-related TE. Therapy of APL-related TE is controversial due to DIC, thrombocytopenia and high bleeding risk and should be patient-tailored [1,6,7]. Generally, antiplatelet drugs are strongly indicated to prevent progression of arterial thrombosis. However, use of antiplatelet drugs in APL patients may increase the risk of hemorrhage. Therefore, withholding antiplatelet drugs until improvement of thrombocytopenia and full recovery from laboratory coagulopathy is recommended [6]. The GIMEMA group demonstrated no benefit for the prevention of early hemorrhagic deaths after using heparin [28]. However, other groups reported benefits of LMWH therapy in decreasing the early hemorrhagic death rate [1,29]. Furthermore, heparin can reduce the adhesion of APL leukemic blasts during induction with ATRA with potential reduction of the TE rate [1]. Our study suggests a possible relationship between TE occurrence on the one hand and laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and thrombofilic mutation, especilally PAI 4G/ 4G polymorphism) on the other. However, there were several limitations, such as the small number of patients and the retrospective,
single-center design. Moreover, due to the retrospective character, testing for acquired and congenital thrombophilia was not performed in all APL patients without TE. Conclusions Our study demonstrates that TE rate in APL patients is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Treatment of APL-associated thrombosis remains an unsettled question due to concomitant DIC and low platelet count during induction treatment. Prophylactic use of heparin might be considered in patients with inital ISTH DIC score b 5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G polymorphism. Conflict of interest statement The authors declare no conflict of interests. Acknowledgments This study was supported by Grants from the Ministry of Education and Science, Republic of Serbia No 41004 and No 173008. We thank Ana Ivkovic, Senior Librarian, Institute for Oncology and Radiology of Serbia, who deserves special mention for arranging for the relevant articles. Natasa Tosic for her hepl with PCR samples. References [1] Breccia M, Lo Coco F. Thrombo-hemorrhagic deaths in acute promyelocytic leukemia. Thromb Res 2014;133(Suppl. 2):S112–6. [2] Lehmann S, Ravn A, Carlsson L, Antunovic P, Deneberg S, Möllgård L, et al. Continuing high early death rate in acute promyelocytic leukemia: a population-based report from the Swedish Adult Acute Leukemia Registry. Leukemia 2011;25:1128–34. [3] Park JH, Qiao B, Panageas KS, Schymura MJ, Jurcic JG, Rosenblat TL, et al. Early death rate in acute promyelocytic leukemia remains high despite all-trans retinoic acid. Blood 2011;118:1248–54. [4] Mitrovic M, Suvajdzic N, Bogdanovic A, Kurtovic NK, Sretenovic A, Elezovic I, et al. International Society of Thrombosis and Hemostasis Scoring System for disseminated intravascular coagulation ≥ 6: a new predictor of hemorrhagic early death in acute promyelocytic leukemia. Med Oncol 2013;30:478. [5] Breccia M, Avvisati G, Latagliata R, Carmosino I, Guarini A, De Propris MS, et al. Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features. Leukemia 2007;21:79–83. [6] Chang H, Kuo MC, Shih LY, Wu JH, Lin TL, Dunn P, et al. Acute promyelocytic leukemia-associated thrombosis. Acta Haematol 2013;130:1–6. [7] Rashidi A, Silverberg ML, Conkling PR, Fisher SI. Thrombosis in acute promyelocytic leukemia. Thromb Res 2013;131:281–9. [8] Kwaan HC. Double hazard of thrombophilia and bleeding in leukemia. Hematology Am Soc Hematol Educ Program 2007:151–7.
M. Mitrovic et al. / Thrombosis Research 135 (2015) 588–593 [9] Dally N, Hoffman R, Haddad N, Sarig G, Rowe JM, Brenner B. Predictive factors of bleeding and thrombosis during induction therapy in acute promyelocytic leukemia - a single center experience in 34 patients. Thromb Res 2005;116:109–14. [10] Montesinos P, de la Serna J, Vellenga E, Rayon C, Bergua J, Parody R, et al. Incidence and risk factors for thrombosis in patients with acute promyelocytic leukemia. Experience of the PETHEMA LPA96 and LPA99 protocols. Blood 2011;108(Suppl. 1):1503. [11] Rodriguez-Veiga R, Montesinos P, Holowiecka A, Vellenga E, Rayon C, Rivas C, et al. Incidence and risk factors for thrombosis in 921 patients with acute promyelocytic leukemia registered in the PETHEMA LPA2005 and LPA2012. 6th International Symposium on Acute Promyelocytic Leukemia; 2013. p. 28 abstract. [12] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985;103:620–5. [13] Shaffer LG, Slovak ML, Campbell LJ. An International System for Human Cytogenetic Nomenclature. Basel: Karger; 2009. [14] Van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease (Report of the BIOMED-1). Leukemia 1999;13:1901–28. [15] Kiyoi H, Naoe T, Yokota S. Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia 1997;11:1447–52. [16] Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA. ITS Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC Press; 2008. [17] Sanz MA, Martin G, Gonzalez M, Leon A, Rayon C, Rivas C, et al. Risk adapted treatment of acute promyelocytic leukemia with all trans retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA group. Blood 2004;103: 1237–43. [18] Sanz MA, Montesinos P, Rayon C, Holowiecka A, de la Serna J, Milone G, et al. Riskadapted treatment of acute promyelocytic leukemia based on all-trans retinoic acid and anthracycline with addition of cytarabine in consolidation therapy for high-risk patients: further improvements in treatment outcome. Blood 2010;115:5137–46. [19] Taylor FB, Toh CH, Hoots WK, Wada H, Levi M. Scientific Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis
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Regular Article
Thrombotic events in acute promyelocytic leukemia☆ Mirjana Mitrovic a,⁎, Nada Suvajdzic a,b, Ivo Elezovic a,b, Andrija Bogdanovic a,b, Valentina Djordjevic c, Predrag Miljic a,b, Irena Djunic a, Maja Gvozdenov c, Natasa Colovic a,b, Marijana Virijevic a, Danijela Lekovic a, Ana Vidovic a,b, Dragica Tomin a,b a b c
Clinic of Hematology CCS, Belgrade, Koste Todorovica 2 Serbia Faculty of Medicine, University of Belgrade, Dr Subotica 8 Belgrade, Serbia Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444aBelgrade, Serbia
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 24 October 2014 Accepted 30 November 2014 Available online 4 December 2014 Keywords: Acute promyelocytic leukemia Thrombotic events ATRA Risk factors PAI-1 4G/5G polymorphism
a b s t r a c t Introduction: Thrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to10-15%. Materials and Methods: We retrospectively analyzed the data on TE appearance in 63 APL patients. Results: TE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P = 0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P = 0.046), PT (P = 0.022), aPTT (P = 0.044), ISTH DIC score (P = 0.001), bcr3 (P = 0.02) and FLT3-ITD (P = 0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3ITD mutation (P = 0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P = 0.05). Regarding risk factors for arterial TE we failed to identify any. Conclusions: We have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score b 5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G. © 2014 Elsevier Ltd. All rights reserved.
Introduction Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a life-threatening coagulopathy leading to a high hemorrhagic early death (ED) rate of up to 29% [1–4]. In addition, thrombotic events (TE) appear to be more common in APL than in other acute leukemias, with reported prevalence of both arterial and vein thrombosis ranging from 2 to10-15% [1,5–11]. Deep vein thrombosis (DVT) is the most common form of APL-related vein thrombosis, but clots may occur in unusual sites such as cerebral venous sinuses, portal and hepatic veins [6,7]. Moreover, thrombosis in APL may present with peripheral artery occlusion, myocardial infarction or ischemic stroke [6,7]. The potential mechanisms involved in the pathogenesis of APLrelated TE are most likely associated with both the inherent characteristics and the treatment of the disease [1,7]. Previous studies indicated
☆ Manuscript was presented on XXIV Congress of the International Society of Thrombosis and Haemostasis, Amsterdam, 29.07.-04.07.2013. ⁎ Corresponding author at: Clinic of Hematology CCS, Koste Todorovica 2, 11000 Belgrade, Serbia. Tel.: +381 63314278; fax: +381 633065112. E-mail address: [email protected] (M. Mitrovic).
http://dx.doi.org/10.1016/j.thromres.2014.11.026 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
several risk factors for TE development such as: high WBC, CD2/CD15 positivity, FLT3-ITD positivity; low fibrinogen level, APL-variant type, differentiation syndrome (DS) and high platelet count, hypoalbuminemia, male sex, poor performance status [5,10,11]. However, data on the pathogenesis, epidemiology, risk factors and clinical outcome of APL-related TE are controversial [1,7]. Concerning this issue, the aim of our single-center retrospective study was to report on TE in an unselected group of 63 de novo APL patients and to evaluate the associated clinical and laboratory parameters. Patients and Methods Patients, Diagnostic Procedures and Treatment During the period 2004 - 2013, 63 consecutive de novo APL patients were diagnosed at the Clinic of Hematology. The diagnosis was confirmed in all patients by detection of t(15;17)(q22;q12) or PML-RARA rearrangements. The diagnostics comprised cytomorphology, cytogenetics, molecular genetics and immunophenotyping of bone marrow (BM) or peripheral blood. Morphologic diagnosis was made according to the French–American–British classification [12]. Conventional G-band karyotyping was employed for cytogenetic analysis [13]. The PML-RARA
M. Mitrovic et al. / Thrombosis Research 135 (2015) 588–593
fusion transcript was detected using a reverse transcriptase polymerase chain reaction (RT-PCR) [14]. The presence of FLT3-internal tandem duplication (ITD) mutations was investigated in 48 cases using PCR and PCR-RFLP methods [15]. Immunophenotyping was performed by flow cytometry using a direct multicolor immunofluorescent technique applied to whole BM specimens with a wide panel of monoclonal antibodies (CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD22, CD24, CD33, CD34, CD36, CD41a, CD45, CD56, CD64, CD66b, CD117, CD235a, HLA-DR, CD3, MPO, CD79a, and TdT [16]. All patients were treated with ATRA + anthracyclines (PETHEMA APL 99 and PETHEMA APL 2005 protocols) [17,18]. Platelet counts were maintained N 30 × 109/L by platelet transfusions; fresh-frozen plasma was administered when the fibrinogen level was b 1.0 g/L until resolution of the coagulopathy according to the recommendations [18]. During the first 28 days of hospitalization we recorded the number of days spent with platelet counts and fibrinogen below recommended goal values, i.e. number of days in which platelet or fresh-frozen plasma transfusions were applied. Antifibrinolytics and anticoagulant therapy were not used. Central venous catheters (CVC) were not inserted before coagulopathy had ceased. Hemostasis Related Parameters Activated prothrombin time (aPTT), prothrombin time (PT) and fibrinogen were measured using an ACL-Automated Coagulation Laboratory (IL, Italy). D-dimer was assessed immunoturbidimetrically by the BCS XP System (Siemens, Germany). Normal ranges were as follows: for PT 75-120%, for aPTT 25-35 s, for fibrinogen 2-4 g/L and for D-dimer b 250 μg/L. A diagnosis of disseminated intravascular coagulation (DIC) was made according to the recommendations of the International Society on Thrombosis and Hemostasis (ISTH). ISTH DIC score was calculated before therapy initiation. Patients with ISTH DIC score ≥ 5 were considered to have overt DIC [19]. Antithrombin (AT), Protein C (PC) and Lupus anticoagulant (LA) were determined with reagents from Siemens (Antithrombin III, Protein C, LA1 and LA2 Reagents) on Sysmex Ca-1500 instrument, according to manufacturer's instructions. Protein S (PS) was determined by coagulation method, using "ProS reagent" on ACL-7000 instrument (IL, Italy). Genotyping of factor (F) V Leiden, prothrombin (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 (PAI-1) 4G/5G gene variants was carried out in 12 patients with TE and 25 patients without TE using PCR [20]. For comparison purposes, blood samples from 126 healthy blood donors were analyzed for those mutations. The healthy individuals had no personal or family record of TE disease and were free of any systemic, cardiovascular, malignant or inflammatory illness. Thrombotic and Bleeding Events Data Patients were assessed for TE and bleeding from the date of admission until the last day of follow-up. Central nervous system (CNS), pulmonary or gastrointestinal hemorrhage were considered as severe bleeding [4]. In patients with signs and symptoms of venous or arterial thrombosis specific imaging techniques were applied. Namely, DVT was established by Color Doppler ultrasonography. Budd-Chiari syndrome (BCS) was diagnosed by computed tomography (CT) and central retinal vein occlusion (CRVO) by ophthalmoscopy and fundus angiography. The diagnosis of cardiac ischemia was based on electrocardiographic and cardiac enzyme modifications. The diagnosis of ischemic stroke was based on neurologic examination and CT or MRI findings. D Dimer Monitoring Since 2011 D-dimer was assessed weekly after coagulopathy cessation during whole induction. In patients with recurring elevation
589
of D-dimer deep vein and CVC arm vein Color Doppler ultrasonography were performed. In patient N011 with D dimer recurring elevation and recent abnormal liver functional tests, hepatic vein Color Doppler ultrasonography was performed. Thrombotic Events Therapy Patients with venous TE were treated with low molecular weight heparin (LMWH) for 3 to 6 months. Dose of LMWH was reduced by half if the patients′ platelet counts were between 30 and 50 x 109/L. LMWH was discontinued at platelet count b 30 x 109/L. Arterial TE were treated with antiplatelet drugs at platelet count N 100x109/L. Recanalization of the veins was assessed at 3 and 6 months after anticoagulant therapy initiation by Color Doppler ultrasonography (patients with DVT and BCS) and ophthalmoscopy (CRVO). Statistical Analysis Chi-square and Fisher exact tests were used to analyze the association between variables and the probability P ≤ 0.05 was considered statistically significant. Survival curves were calculated by the KaplanMeier method and differences between groups were assessed by the log-rank test. Univariate and multivariate COX proportional regression models were employed for identification of risk factors. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the Statistical Package for Social Science. Results The study population consisted of 63 patients, 30 (48%) males and 33 (52%) females, of median age 44 years (range 19–78). Between September 2004 and August 2010, 42 de novo APL patients were treated according to the PETHEMA APL 99 protocol, while 21 patients received PETHEMA APL 2005 from September 2010 to May 2013. Bleeding was registered in 56/63 (89%) cases, severe in 14/63 (22.2%) with hemorrhagic early death rate of 8/63 (12.7%). All hemorrhagic deaths occurred during the first week of hospitalization with the median time of two days after therapy initiation. Thirteen (20.6%) patients, without previous history of thrombosis, developed TE, four arterial (6.3%) and nine venous (14.3%). In only one patient (N01), severe bleeding and TE developed concomitantly. The main clinical and biological characteristics of these patients are shown in Table 1. Arterial Thrombotic Events in APL Patients Arterial TE developed in 4/63 (6.3%) patients, with a female/male ratio of 3/1 and a median age of 50 years (range 38-64). Arterial TE encompassed acute myocardial infarction (AMI) in two cases and cerebrovascular accident (CVA) in two cases. CVA in patient N01 was the initial APL symptom, while the remaining three patients developed TE during induction. The median time from therapy initiation to arterial TE occurrence was 23 days (range 22-26). In patient N0 3 with AIM occuring during induction, percutaneous coronary intervention with stent implantation was performed immediately after BM recovery, followed by dual antiplatelet therapy with aspirin and clopidrogel for 6 months. The remaining three patients with arterial TE were not treated with antiplatelet drugs due to thrombocytopenia. In patient N0 2, CVA became stable during induction with remaining discrete rightsided hemiparesis. Patients N0 1 and 4 died within 28 days after therapy initiation: patient N0 4 due to AIM and patient N0 1 due to intracranial hemorrhage (Table 1). The mortality rate of patients with arterial TE was 50%. However, arterial TE did not have a significant impact on ED rate or 4-year overall survival (OS; Table 2).
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Table 1 Demographic, clinical and biological characteristic of the APL patients with thrombosis. Pts No
Age years/ sex
WBC x10 9/L
Plt count x109/L
Sanz risk score
ISTH score
CD2/ CD15
PML-RARα
1
38/f
8.6
47
low
7
-/ND
2 3
54/f 46/m
1.1 21.6
25 39
inter high
5 6
4
64/f
0.9
39
inter
5 6
64/m 51/m
1.4 1.8
93 45
7
31/m
88
8
46/m
9
FLT3 -ITD
DS
ND
ND
−/+ −/−
bcr3 bcr3
6
−/−
low low
4 5
23
high
5.6
48
45/m
1.3
10
52/m
11 12 13
49/f 53/m 26/f
Thrombosis
Thrombophilia
Risk factors
Outcome
yes
ND
no
yes no
PAI-1 4G/5G MTHFR hetero PAI-1 4G/4G PAI-1 4G/5G
smoking DM hyperten hyperten
Early death CR CR
PAI-1 4G/5G lupus anticoagulant MTHFR hetero MTHFR hetero PAI-1 4G/4G MTHFR hetero FV Leiden hetero MTHFR hetero PAI-1 4G/4G PAI-1 4G/4G PAI-1 4G/5G Prothrombin 20210 hetero MTHFR hetero PAI-1 4G/4G
no no
Early death CR CR
smoking
CR
no
CR
no
CR
smoking
CR
smoking smoking no
CR CR CR
Site
Time
DIC
yes
CVA
-
no no
CVA AIM
pre therapy induction induction
bcr1
+
no
AIM
induction
no
−/− −/−
bcr3 bcr1
ND
yes no
DVT DVT
induction induction
no no
5
−/+
bcr3
-
no
induction
no
inter
4
+/−
ND
+
no
DVT CVC DVT
induction
no
135
low
2
+/−
bcr1
ND
no
CRVO
mainten
no
28.4
15
high
5
+/−
bcr3
+
no
induction
yes
2.9 1.3 1.3
95 51 92
Low low low
5 5 5
+/− +/− −/−
bcr3 bcr1 bcr3
+ + +
No no yes
DVT CVC BCS DVT DVT CVC
induction induction induction
no no yes
N- number, f - female, m - male, WBC – with blood cells, Plt – platelets, ISTH score - International Society on Thrombosis and Hemostasis score, DIC - Disseminated Intravascular Coagulation, ND - not done, CVA - cerebrovascular accident, DVT – deep vein thrombosis, CVC – central vein catheter, AIM - acute myocardial infarction, CRVO – central retinal vein occlusion, BCS - Budd-Chiari syndrome, mainten - maintenance therapy, hetero – heterozygous, DM – diabetes mellitus, hyperten – hypertension, CR – complete remission.
Neither thrombophilic mutations nor clinical and biological features of our APL patients apeared predictive for ATE development (Tables 2 and 3).
Table 2 Clinical characteristics of the APL patients with and without arterial thrombosis. Parameter
With arterial thrombosis (4 pts)
Without thrombosis (50 pts)
P
Age (years) Sex (male/female) Smokers Diabetes mellitus Hypertension Bleeding Sever bleeding WBC count (x109/L) Platelet count (x109/L) Sanz risk score low (N0) intermediate high Fibrinogen (g/L) PT (%) aPTT (s) D-dimer (μg/L) ISTH DIC score CD2 expression (N0) CD15 expression (N0) bcr1-2/bcr3 FLT3-ITD N0 of days with platelets b 30x109/L N0 of days spent with fibrinogen b 1 g/L Differentiation syndrome (N0) Early death 4-year overall survival
50 (38-64) 1/3 ¼ (25%) 1 (25%) 2/4 (50%) 4/4 (100%) ¼ (25%) 1 (0.9-21.6) 32 (1-47) 1/4 (25%) 2/4 (50%) 1/4 (25%) 2.425 (0.86-3.9) 35 (21-75) 28.75 (23.5-36) 3157 (3045-6000) 6 (5-7) ¼ (25%) 1/3 (33.3%) 1(50%)/6(50%) 0/3 (0%) 14 (1-18)
41 (19-78) 20/30 20/50 (40%) 4/50 (8%) 17/50 (34%) 45/50 (90%) 12/50 (24%) 3.6 (0.5-183.2) 28 (4-101) 10/50 (20%) 21/50 (42%) 19/50 (38%) 2.69 (0.57-6.23) 62 (21-124) 26.1 (21.2-35.5) 2332 (624-11340) 6 (3-7) 9/42 (21.4) 15/43 (34.9%) 11(61%)/7(39%) 8/36 (22.2%) 12 (3-22)
0.247 0.492 0.678 0.08 0.234 0.567 0.781 0.552 0.775 0.24
0.674 0.277 0.757 0.264 0.459 0.503 0.726 0.32 0.444 0.628
1(0-2)
2 (0-5)
0.671
1/4 (25%)
11/50 (22%)
0.63
2/4 (50%) 17.5 (0-63)
12/50 (24%) 25 (0-104)
0.274 0.342
Pts – patients, WBC – white blood cells, N0 – number, ISTH score - International Society on Thrombosis and Hemostasis score.
Venous Thrombotic Events in APL Patients Venous TE developed in 9/63 (14.29%) patients, with a female/male ratio of 2/7 and median age of 47.5 years (range 26-64). Venous TE encompassed DVT in 7 cases (CVC associated in 3 cases), CRVO and BCS in one case each. The majority of the venous TE occurred during induction (8/9 patients; Table 1). The median time from therapy initiation to TE occurrence was 14 days (range 9-26). Only 4/13 (31%) patients displayed DIC at the time of venous TE occurrence. All 9 patients with venous TE were treated with anticoagulants resulting in complete vein recanalization. It is of note that venous TE did not have an impact on either ED or 4-year OS. The clinical and biological features of our APL patients with and without venous TE are shown in Table 4. The two groups were significantly different regarding female/male ratio (P = 0.046), PT (P = 0.022), aPTT (P = 0.044), ISTH DIC score (P = 0.001), bcr3 (P = 0.02) and FLT3-ITD (P = 0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate Cox's proportional regression was FLT3-ITD mutation (P = 0.034, RR 10.036, 95% CI 1.197-84.123). (See Table 5.) Concerning thrombophilia testing AT, PC and PS deficiencies were not identified. Only one patient (N0 6) was LA positive. Genotype frequencies are presented in Table 3. Thrombophilic gene mutations were present in 8/9 (88.9%) patients: FV Leiden heterozygous mutation and FII G20210A mutation in one (8.3%) patient each, PAI-14G/4G in 5/9 (55.6%) patients and PAI-1 4G/5G in 1/9 (8.3%) patients. Combined thrombophilia was registered in 4/9 (44.4%) patients. FV Leiden heterozygous mutation, prothrombin 20120 and PAI-1 4G/4G were three, three and five times more frequent in APL patients with venous TE when compared to patients without TE, with significant differences for PAI 4G/4G (P = 0.05).
D Dimer Monitoring During the period 2011 - 2013, 18 consecutive de novo APL patients underwent regular weekly D-dimer monitoring. In the one patient D
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Table 3 Allele and genotype frequencies in APL patients with and without arterial thrombotic event and healthy controls. APL patients with ATE (3 pts)
APL patients without TE (25 pts)
Healthy controls
ORa 95%CIa
ORb 95%CIb
Pa
Pb
heterozygous GA homozygous AA heterozygous GA homozygous AA heterozygous 4G/5G
0% 0% 0% 0% 2/3 (66.7%)
1/25 (4.0%) 0% 1/25 (4.0%) 0% 14/25 (56.0%)
4.2% 0% 5.8% 0% 46.2%
0.49
1/3 (33.3%)
5/25 (20.0%)
34.8%
0.60
0.96
heterozygous CT
1/3 (33.3%)
8/25 (32.0%)
39.2%
0.96
0.84
homozygous TT
0%
6/25 (24.0%)
11.5%
2.33 0.21-26.09 0.94 0.08-10.52 0.78 0.07-8.81 -
0.73
homozygous 4G/4G
1.57 0.13-19.67 2.00 0.15-26.74 1.06 0.08-13.52 -
-
-
Gene variant F II G20210A F V Leiden PAI – 1 4G/5G
MTHFR C677T
APL- acute promyelocytic leukemia, ATE - arterial thrombotic event, TE thrombotic event, Pts – patients, F – factor. a – APL patients with ATE and without TE comparison. b – APL patients with ATE and healthy controls comparison.
dimer monitoring was not performed due to hemorrhagic ED, sixth day after therapy initiation. Four (21%) patients were diagnosed with symptomatic TE. In four of the remaining 14 patients (28.6%) D dimer reelevation was seen. In three of those four patients a diagnosis of TE was made (DVT in patients N0 6 and 11; partial BCS in patient N0 10). One patient had falls positive result probably due to infection. TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P = 0.032).
Discussion Previous studies reported a rising incidence of APL-related TE from 2% in the pre-ATRA era to 4.5-15% in patients treated with a combination of ATRA and anthracyclines [1,5–11]. This could be ascribed to the higher level of vigilance and availability of advanced diagnostic tests, or alternatively to the new therapeutic approach [1,7]. Table 4 Baseline characteristics of the APL patients with and without venous thrombosis. Parameter
With thrombosis (9 pts)
Without thrombosis (50 pts)
P
Age (years) Sex (male/female) Bleeding Sever bleeding WBC count (x109/L) Platelet count (x109/L) Sanz risk score low (N0) intermediate high Fibrinogen (g/L) PT (%) aPTT (s) D-dimer (μg/L) ISTH DIC score ISTH DIC score b5 CD2 expression (N0) CD15 expression (N0) bcr1-2/bcr3 FLT3-ITD N0 of days with platelets b 30x109/L N0 of days spent with fibrinogen b 1 g/L Differentiation syndrome (N0) Early death 4-year overall survival
47.5 (26-64) 7/2 7/9 (78%) 1/7 (14.3%) 1.6 (1.3-88) 44 (15-135) 5/9 (55.6%) 2/9 (22.2%) 2/9 (22.2%) 3.9 (2.22-4.79) 74 (62-89) 31.76 (23.1-34.0) 2743 (551-6000) 5 (2-5) 4/9(44.4%) 5/9 (55.6%) 1/9 (11%) 3(30%)/6(70%) 5/9 (55.6%) 13 (5-20)
41 (19-78) 20/30 45/50 (90%) 12/50 (24%) 3.6 (0.5-183.2) 28 (4-101) 10/50 (20%) 21/50 (42%) 19/50 (38%) 2.69 (0.57-6.23) 62 (21-124) 26.1 (21.2-35.5) 2332 (624-11340) 6 (3-7) 5/50 (10%) 9/42 (21.4) 15/43 (34.9%) 11(61%)/7(39%) 8/36 (22.2%) 12 (3-22)
0.359 0.046 0.367 0.234 0.460 0.071 0.34
0.105 0.022 0.044 0.879 0.001 0.031 0.093 0.245 0.032 0.028 0.145
1 (0-3)
2 (0-5)
0.567
2/9 (22%) 0/9 32 (5-93)
11/50 (22%) 12/50 (24%) 25 (0-104)
0.93 0.181 0.883
Pts – patients, WBC – white blood cells, N0 – number, ISTH score - International Society on Thrombosis and Hemostasis score.
The main finding of this study is a higher incidence of APL-related thrombosis (20.9%) in comparison with previously published data [1, 5–11]. In addition, this is the first study addressing the issue of asymptomatic DVT in APL patients. Since 2011, when regular weekly D-dimer monitoring was initiated, TE have been diagnosed in three asymptomatic (3/14, 21.4%) and four (4/19, 21%) symptomatic patients. These results suggest that the true prevalence of venous TE in APL patients is underestimated. Concerning this issue recent studies suggest that the presence of both asymptomatic and symptomatic TE have a similar clinical impact on the cancer patients’ prognosis [21]. These findings emphasize need for VTE screening in APL patients after DIC cessation. Although not specific, D dimer is highly sensitive marker for VTE, with levels corresponding to the extent of thrombosis. The pathogenesis of the increased thrombotic risk in APL patients is not fully understood, and comprises a combination of variables related to the disease itself, treatment and host characteristics [1,5,7,9–11,22]. Notably, leukemic promyelocytes directly contribute to the thrombohemorrhagic tendency in APL via expression of tissue factor (TF) and cancer procoagulants [23,24]. Thus, DIC was registered in 87% of patients at diagnosis and in 30.7% of patients at the time of the TE occurrence. Concerning this issue, TE in APL patients might be part of the DIC. However, our patients with APL-related venous TE had significantly lower median initial ISTH scores compared to APL patients without thrombosis. Moreover, an initial ISTH DIC score ≤ 5 was predictive for venous TE. In our previous study ISTH DIC score ≥ 5 was predictor for bleeding and ISTH DIC score ≥ 6 was the most significant predictor factor for hemorrhagic early death in APL patients [4]. Our results suggest that DIC appears the major cause of bleeding in APL patients [4]. Moreover, the degree of DIC may correlate with the risk of both bleeding and thrombosis [4]. In our study thrombosis typically developed after therapy initiation, which is consistent with other reports [5,6]. This finding suggests an association between TE and treatment [1]. Moreover, anthracyclinesinduced apoptosis might contribute to thrombogenesis in APL via increased cellular TF activity and the release of TF-bearing microparticles from the damaged leukemic cells [7]. Additionally, ATRA has a procoagulant effect due to enhanced expression of adhesive molecules on both promyelocytes and endothelium and increased production of cytokines [1,7,23]. Why some patients with APL develop thrombosis and some do not has not been clarified yet. Several host and disease related risk factors have been proposed, such as: male sex, worse PS, high WBC and platelet counts, low fibrinogen level, hypoalbuminemia, M3 variant type, CD2/ CD15 and FLT3-ITD positivity [1,5,6,9–11]. In our study arterial and venous TE were separated in the risk factor analysis. However, we failed to identify any risk factor for arterial TE development, probably due to the low number of patients. On the other
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M. Mitrovic et al. / Thrombosis Research 135 (2015) 588–593
Table 5 Allele and genotype frequencies in APL patients with and without venous thrombotic events and healthy controls. Gene variant
APL patients with VTE (9 pts)
APL patients without TE (25 pts)
Healthy controls
ORa 95%CIa
ORb 95%CIb
Pa
Pb
3.00 0.17-53.71 3.00 0.17-53.71 0.10 0.01-0.91 5.00 0.97-25.77 2.66 0.56-12.65 -
2.88 0.30-27.65 2.02 0.22-18.48 0.15 0.02-1.18 2.35 0.61-9.01 1.94 0.50-7.60 -
0.46
0.36
0.46
0.53
0.04
0.07
c
0.21
0.22
0.34
-
-
F II G20210A
heterozygous GA
1/9 (11.1%)
1/25 (4.0%)
4.2%
F V Leiden
homozygous AA heterozygous GA
0% 1/9 (11.1%)
0% 1/25 (4.0%)
0% 5.8%
PAI – 1 4G/5G
homozygous AA heterozygous 4G/5G
0% 1/9 (11.1%)
0% 14/25 (56.0%)
0% 46.2%
homozygous 4G/4G
5/9 (55.6%)
5/25 (20.0%)
34.8%
heterozygous CT
5/9 (55.6%)
8/25 (32.0%)
39.2%
homozygous TT
0%
6/25 (24.0%)
11.5%
MTHFR C677T
APL- acute promyelocytic leukemia, Pts - patients, VTE – venous thrombotic event, TE – thrombotic event, F – factor. a – APL patients with VTE and without TE comparison. b – APL patients with VTE and healthy controls comparison.
hand male sex, ISTH-DIC score b 5, bcr3 isoform and FLT3-ITD positivity were identified as predictive for venous TE occurrence in our APL patients. Furthermore, multivariate analysis indicated FLT3-ITD positivity as the most significant predictor for venous TE (P = 0.034). Regarding the FLT3-ITD mutation issue, a study on gene expression profile found that leukemic cells from FLT3-ITD + patients have an increased expression of genes regulating blood coagulation and cell adhesion [24]. Notably, underlying hereditary thrombophilia may contribute to venous TE development in cancer patients [25]. However, there are few studies on this topic in APL patients with controversial results. That is Dally et al. [9] reported that 50% of patients with APL- related TE had congenital thrombophilia, while Breccia et al. [5] and Rees et al. [26] failed to confirm this finding. In our study, 88.9% of the APL patients with associated TE had some thrombophilic mutation, while 44.4% of them had combined thrombophilia. Namely, the FV Leiden heterozygousity and FII 20210 mutations were three times more frequent in our APL patients with venous TE than in patients without it. This is the first study assessing the impact of PAI-1 mutational status on APL-related TE to the best of our knowledge. The PAI 4G/4G was five and two times more frequent in our patients with APL-related venous and arterial TE than in patient without TE. PAI-1 is a key regulating factor determining endogenous fibrinolytic activity [27]. A 4G/5G polymorphism of the PAI-1 gene has been reported to influence the levels of PAI-1. The 4G allele was found to be associated with higher plasma PAI-1 activity, which can lead in impaired fibrinolysis [26]. Moreover, it was shown that retinoids induce increased production of plasminogen activator inhibitors, including PAI-1 and PAI-2 [22]. Association of ATRA therapy and high PAI-1 levels due to PAI-1 gene 4G/4G polymorphism might lead to APL-related TE. Therapy of APL-related TE is controversial due to DIC, thrombocytopenia and high bleeding risk and should be patient-tailored [1,6,7]. Generally, antiplatelet drugs are strongly indicated to prevent progression of arterial thrombosis. However, use of antiplatelet drugs in APL patients may increase the risk of hemorrhage. Therefore, withholding antiplatelet drugs until improvement of thrombocytopenia and full recovery from laboratory coagulopathy is recommended [6]. The GIMEMA group demonstrated no benefit for the prevention of early hemorrhagic deaths after using heparin [28]. However, other groups reported benefits of LMWH therapy in decreasing the early hemorrhagic death rate [1,29]. Furthermore, heparin can reduce the adhesion of APL leukemic blasts during induction with ATRA with potential reduction of the TE rate [1]. Our study suggests a possible relationship between TE occurrence on the one hand and laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and thrombofilic mutation, especilally PAI 4G/ 4G polymorphism) on the other. However, there were several limitations, such as the small number of patients and the retrospective,
single-center design. Moreover, due to the retrospective character, testing for acquired and congenital thrombophilia was not performed in all APL patients without TE. Conclusions Our study demonstrates that TE rate in APL patients is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Treatment of APL-associated thrombosis remains an unsettled question due to concomitant DIC and low platelet count during induction treatment. Prophylactic use of heparin might be considered in patients with inital ISTH DIC score b 5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G polymorphism. Conflict of interest statement The authors declare no conflict of interests. Acknowledgments This study was supported by Grants from the Ministry of Education and Science, Republic of Serbia No 41004 and No 173008. We thank Ana Ivkovic, Senior Librarian, Institute for Oncology and Radiology of Serbia, who deserves special mention for arranging for the relevant articles. Natasa Tosic for her hepl with PCR samples. References [1] Breccia M, Lo Coco F. Thrombo-hemorrhagic deaths in acute promyelocytic leukemia. Thromb Res 2014;133(Suppl. 2):S112–6. [2] Lehmann S, Ravn A, Carlsson L, Antunovic P, Deneberg S, Möllgård L, et al. Continuing high early death rate in acute promyelocytic leukemia: a population-based report from the Swedish Adult Acute Leukemia Registry. Leukemia 2011;25:1128–34. [3] Park JH, Qiao B, Panageas KS, Schymura MJ, Jurcic JG, Rosenblat TL, et al. Early death rate in acute promyelocytic leukemia remains high despite all-trans retinoic acid. Blood 2011;118:1248–54. [4] Mitrovic M, Suvajdzic N, Bogdanovic A, Kurtovic NK, Sretenovic A, Elezovic I, et al. International Society of Thrombosis and Hemostasis Scoring System for disseminated intravascular coagulation ≥ 6: a new predictor of hemorrhagic early death in acute promyelocytic leukemia. Med Oncol 2013;30:478. [5] Breccia M, Avvisati G, Latagliata R, Carmosino I, Guarini A, De Propris MS, et al. Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features. Leukemia 2007;21:79–83. [6] Chang H, Kuo MC, Shih LY, Wu JH, Lin TL, Dunn P, et al. Acute promyelocytic leukemia-associated thrombosis. Acta Haematol 2013;130:1–6. [7] Rashidi A, Silverberg ML, Conkling PR, Fisher SI. Thrombosis in acute promyelocytic leukemia. Thromb Res 2013;131:281–9. [8] Kwaan HC. Double hazard of thrombophilia and bleeding in leukemia. Hematology Am Soc Hematol Educ Program 2007:151–7.
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