Thrombosis in children with acute lymphoblastic leukemia

Thrombosis Research 111 (2003) 199 – 212

Review Article

Thrombosis in children with acute lymphoblastic leukemia Part II. Pathogenesis of thrombosis in children with acute lymphoblastic leukemia: effects of the disease and therapy Uma H. Athale a,*, Anthony K.C. Chan a,b a

Division of Pediatric Hematology/Oncology, Department of Pediatrics, 3N27D, HSC, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5 b Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada Received 3 October 2003; accepted 14 October 2003

Abstract At diagnosis, there is evidence of increased thrombin generation in children with acute lymphoblastic leukemia (ALL), the etiology of which is unclear. However, thromboembolism (TE) in children with ALL is most commonly reported after the initiation of antileukemic therapy indicating a possible interaction of the disease and therapy. Antileukemic therapy influences the haemostatic system either by direct effect of the chemotherapeutic agents or indirectly through the effect of supportive care, e.g. central venous line (CVL) or infectious complications secondary to immunosuppression. Asparaginase and steroids are shown to induce hypercoagulable state by suppression of natural anticoagulants, especially AT and plasminogen, and by elevations in F VIII/vWF complex, respectively. In addition, steroid therapy causes hypofibrinolytic state by dose-dependent increase in plasminogen activator inhibitor 1 (PAI-1) levels. Combination of these effects coupled with increased thrombin generation may be responsible for the increased incidence of TE observed with concomitant administration of asparaginase and steroids. Further studies to delineate the mechanism of increased thrombin generation in children with ALL and effects of various chemotherapeutic agents, in isolation and in combination, on haemostatic system are needed. D 2003 Elsevier Ltd. All rights reserved. Keywords: Acute lymphoblastic leukemia; Thromboembolism; Asparaginase; Steroids; Chemotherapy

1. Introduction Abbreviations: ALL, acute lymphoblastic leukemia; APTT, activated partial thromboplastin time; AT, antithrombin; APC-PCI, activated protein C-protein C inhibitor; APC-AT, activated protein C-a1 antitrypsin; a2-AP, a2-antiplasmin; a2-M, a2-macroglobulin; ARA-C, cytosine arabinoside; BFM, Berlin – Frankfurt – Munster; CVL, central venous line; CTX, cyclophosphamide; COALL, German Co-Operative Study Group for Childhood Acute Lymphoblastic Leukemia; CCSG, Children’s Cancer Study Group; CNS TE, central nervous system thromboembolism; DFCI, Dana-Farber Cancer Institute; DVT, deep venous thrombosis; FIB, fibrinogen; FDP, fibrinogen degradation product; FPA, fibrinopeptide A; HC II, heparin cofactor II; HMWK, high molecular weight kininogen; HDMP, high-dose methylprednisone; GM-CSF, granulocyte macrophage colony-stimulating factor; MTX, methotrexate; MTHFR, methylenetetrahydrofolate reductase; PT, prothrombin time; PK, prekallikrein; PF1.2, prothrombin fragment 1+2; PC, protein C; PS, protein S; TE, thromboembolism; TT, thrombin time; TAT, thrombin – antithrombin complex; TM, thrombomodulin; vWF, von Willebrand factor; VCR, vincristine. * Corresponding author. Tel.: +1-905-521-2100x73428; fax: +1-905521-1703. E-mail address: [email protected] (U.H. Athale). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.10.007

The review of epidemiological data highlights the importance of understanding the pathogenesis of thromboembolism (TE) in children with acute lymphoblastic leukemia (ALL). This knowledge will be important in developing strategies to prevent or effectively treat TE without compromising the therapy for ALL. In the following sections, we will review the available information regarding the effects of ALL, ALL therapy and supportive care on the haemostatic system and their likely contribution to the development of TE.

2. Effects of the disease Table 1 summarizes the studies evaluating coagulation and fibrinolytic parameters at the time of diagnosis of ALL in children [1 – 8]. Plasma concentrations of most coagula-

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Table 1 Evaluation of haemostatic system at the time of diagnosis in children with acute leukemia Authors (reference number)

Number of subjects

Tests or factors studied

Results

Episodes of TE/ phase of therapy

(1)

Priest et al. [1]

12 patients with ALL

Rodeghiero et al. [2] Abshire et al. [3]

14 patients with ALL 52 patients: 44 ALL, 8 ANLL

(4)

Mitchell et al. [4]

26 patients with ALL; 14 healthy controls

In vivo markers for generation of thrombin AT-IIa, HCII-IIa, a2-M-IIa, TAT, APC-AT, F1.2, APC-PCI, Factor II, anticoagulants: AT, a2-M, HCII, PC, total and free PS

(5)

Mitchell et al. [5]

30 patients with ALL; 163 healthy control

(6)

Oner et al. [6]

(7)

Giordano et al. [7]

(8)

Uszynski et al. [8]

19 patients with ALL; 12 healthy controls 32 patients with ALL; 80 healthy controls 23 patients with ALL; 11 healthy controls

Procoagulants: PK, HMWK, Factors XIIIa, XIIIs, XII, XI, X, IX, VIIIc, vWF, VII, V, II, FIB; anticoagulants: a2-M, HC II, a2AP, PC, PS; plasminogen, AT IIa generation PC, PS, HC II, PF1.2, TAT, vWF and TM

Increase in FIB levels and FDP in almost all patients. Levels of F V, AT, plasminogen and PT, PTT, TT within normal limits. 64% of patients had elevated FPA at diagnosis. f 50% with increased FDP and D-dimer; 27% with increased PT; 10% with prolonged PTT; 4% with increased Bh 1 – 42; 40% patients had normal coagulation studies. All markers for thrombin generation increased at diagnosis. Levels of F II, AT, a2-M, PC, and total PS were comparable to normal controls. HCII was significantly increased and free PS was decreased. In vitro capacity to inhibit thrombin was normal. Significantly increased levels of Factors VIII, vWF, IX, a2-M and PS. Significantly reduced levels of PC, PK, F XIIIA and S. Rest of the factors within normal limits for age. Levels TAT, PF1.2, TM were significantly elevated. PS levels were decreased. PC, HCII, vWF levels were unchanged. Patients had significantly higher TAT levels at diagnosis. Patients with T-ALL had significantly higher TAT levels. 16 (70%) patients had significantly elevated levels of TAT. 7 (30%) patients had values comparable to the control group

None

(2)

PT, PTT, TT, FDP, F V, FIB, plasminogen (functional, immunologic), AT (functional, immunologic), platelet counts PT, PTT, FIB, FDP, AT, PC, FPA

(3)

PT, PTT, FIB, D-dimers, TT, FDP, Factors II, VII, IX, X, Bh 1 – 42

TAT complex

TAT complex

2 of 14 patients (14.3%)/ remission-induction None

3 of 26 patients (11.5%) during consolidation therapy

3 of 30 (10.0%), phase of therapy unavailable

None

1 of 31 (3.1%)/ remission-induction None

Summary of studies evaluating coagulation and fibrinolytic parameters at the time of diagnosis of acute leukemia in children. Significant increase in factor VIII, von Willebrand Factor (vWF) and fibrinogen has been noted. Almost all studies evaluating parameters for thrombin generation have documented increased thrombin activation at diagnosis. PT = prothrombin time, PTT = partial thromboplastin time, TT = thrombin time, FIB = fibrinogen, FDP = fibrinogen degradation product, AT = antithrombin, HC II = heparin cofactor II, TAT = thrombin – antithrombin complex, a2-M = a2-macroglobulin, APC-AT = activated protein C-a1 antitrypsin, PK = prekallikrein, HMWK = high molecular weight kininogen, PF1.2 = prothrombin fragment 1 + 2, a2-AP = a2-antiplasmin, PC = protein C, PS = protein S, vWF = von Willebrand factor, TM = thrombomodulin, APC-PCI = activated protein C-protein C inhibitor, FPA = fibrinopeptide A.

tion factors and inhibitors are reported to be in the normal range at diagnosis except for significant increase in factor VIII, von Willebrand factor (vWF) and fibrinogen [1– 10]. This could be explained on the basis of inflammatory response. Mitchell et al. [5] reported significant increase in protein S (PS), a2-macroglobulin (a2-M) and marked reduction in protein C (PC), pre-kallikrein (PK) and F XIII. Recently, Oner et al. [6] documented that, at diagnosis, children with ALL had elevated levels of thrombomodulin and these levels increased significantly after high-dose methylprednisone. Almost all studies evaluating parameters for thrombin generation have documented increased thrombin generation at diagnosis (Table 1), indicating that increased thrombin generation may be an important factor in the pathogenesis of TE in association with ALL [1,2,4 –8]. However, none of the studies report TE at the time of diagnosis; three studies

report six events of TE during therapy [2,4,7]. Furthermore, the excess endogenous thrombin generation observed at diagnosis is shown to be present during first several months of therapy [2,4,11]. Interestingly, Giordano et al. [7] showed significantly higher levels of thrombin –antithrombin (TAT) complexes at diagnosis in patients with T-ALL (n = 7) compared to those with pre-B (n = 9) or common-ALL (n = 16). One of the thirty-two patients, a 9-year-old boy with T-ALL, developed saggital sinus thrombosis during remission induction therapy. He also was homozygous for methylenetetrahydrofolate reductase (MTHFR) mutation. The preliminary results of their multi-center retrospective analysis indicate that children with T-ALL have higher incidence of TE. Although several studies have reported elevated thrombin generation at diagnosis of ALL, there is little in vitro or in vivo evaluation for possible etiologic phenomenon

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for thrombin activation in children with ALL and its correlation with development of TE. Mitchell et al. [4,5] documented normal in vitro capacity of plasma to generate thrombin at diagnosis, indicating that the altered plasma concentration of some coagulation factors noted was probably not responsible for thrombin generation. Alessio et al. [12] reported elevated levels of procoagulant activity (PCA) in 64% of 25 ALL patients (age 3– 57 years); interestingly, two patients [producing very high levels of cancer procoagulant (CP)] developed TE during induction therapy with asparaginase. Recent studies, mainly in different types of cancers commonly seen in adults, have shown that malignant cells can directly interact with haemostatic system in various ways. Tumor cells can synthesize procoagulant molecules [CP, tissue factor (TF), F XIII like activity], fibrinolytic molecules [tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), urokinase plasminogen activator receptor (u-PAR), plasminogen activator inhibitor (PAI)] and inflammatory cytokines [interleukin-1h (IL1h), tumor necrosis factor-a (TNF-a), vascular endothelial growth factor (VEGF)]. In addition, tumor cells, through direct or indirect interaction, can activate endothelial cells, platelets and monocytes/macrophages resulting in downregulation of anticoagulant properties and upregulation of procoagulant properties [13,14]. In summary, at diagnosis and during first several months of therapy, there is evidence of increased thrombin generation in children with ALL. Although the exact etiology of thrombin activation is not known, it is likely to be secondary to a very complex interaction of malignant cells with other cellular elements leading to activation of coagulation. More studies are needed to delineate the mechanism of thrombin activation in association with childhood ALL.

3. Effects of therapy Despite the evidence of increased thrombin generation at diagnosis, TE in children with ALL is most commonly reported only after the initiation of antileukemic therapy indicating a possible interaction of the disease and therapy. This is in contrast to adult patients with cancer where TE could be a manifestation of undiagnosed or ‘‘occult’’ malignancy [14]. Chemotherapy influences the haemostatic system either by direct effect of the chemotherapeutic agents or indirectly through the influence of supportive care, e.g. central venous line (CVL) or complications like infections secondary to immunosuppression [9,10,14]. 3.1. Effects of chemotherapeutic agents Antileukemic therapy usually consists of four basic stages: remission-induction, intensification (or consolidation), central nervous system (CNS) prophylaxis, and maintenance (or continuation) therapy. Combinations of steroids,

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vincristine (VCR), anthracyclines with or without asparaginase form the mainstay of remission-induction therapy aimed to induce rapid complete remission. Intensification (or consolidation) therapy usually includes combination of several antileukemic agents (VCR, steroids, anthracyclines, methotrexate (MTX) and/or asparaginase) to improve the duration of complete remission and overall outcome [15,16]. Most of the studies evaluating the effects of chemotherapy on the coagulation and fibrinolytic parameters have done so during remission induction or consolidation phase of ALL therapy in children. This is probably a reflection of the phase of therapy when most episodes of TE occur. Several reports have shown that the chemotherapeutic agents used in therapy of ALL affect haemostatic proteins and interfere with the function of cellular components in the blood (e.g. platelets). Since most of these drugs are given in the form of combination chemotherapy, the effects of an isolated agent on coagulation system is difficult to assess. Of the various antileukemic agents, effects of asparaginase and steroids on coagulation system have been studied most extensively. 3.1.1. Asparaginase Asparaginase, a potent lympholytic agent, is an essential component of ALL therapy. It is a bacterially derived enzyme, which converts the amino acid asparagine to aspartic acid and ammonia. This leads to rapid depletion of extracellular pools of asparagine in the body. Unlike the normal cells, the lymphoblasts lack the enzyme asparagine synthatase and are dependent on exogenous asparagine supplies making them very susceptible to the cytotoxic effects of asparaginase [17,18]. The resultant inhibition of protein synthesis following asparaginase therapy is responsible for major toxicities related to asparaginase therapy [17,18]. Coagulopathy, a well-known complication of asparaginase, is characterized by thrombosis or hemorrhage; thrombosis being more common. Although the exact factors that tilt the balance towards either hemorrhage or thrombosis is still unknown, it probably is related to the accompanied drugs and the drug delivery sequence. For this review, we will focus on the thrombotic potential of asparaginase therapy. 3.1.1.1. Effects on haemostatic proteins. Most of the reports study the effects of asparaginase in association with combination chemotherapy [1,2,4 –6,9 – 11,17 – 37]. There is variability in results of these investigators probably related to the type of asparaginase preparation and dosage used, timing of sampling after the asparaginase, disease status, phase of therapy and effects of the dose and schedule of other chemotherapy agents. The most consistent findings are reduction in plasminogen, fibrinogen and antithrombin (AT) [4,9,15,17 – 26]. Furthermore, Nowak-Gottl et al. [38 – 40] showed that the level of reduction in coagulation and fibrinolytic proteins is related to the asparaginase activity

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achieved and levels of asparagine depletion. The importance of influence of other agents used in combination with asparaginase on the coagulation system is documented by Kirschke et al. [41]. They investigated the effects of two combinations of chemotherapeutic agents on natural anticoaguants, fibrinolytic agents and indicators of thrombin generation. Asparaginase given in combination with methotrexate (MTX) and cyclophosphamide (CTX) resulted in much lower and longer lasting decrease in plasminogen compared to the levels of plasminogen when asparaginase was given in combination with high-dose cytosine arabinoside (ARA-C) [41]. Asparaginase therapy is also reported to be associated with suppression of PC, PS and other haemostatic proteins [4,5]. However, the importance of these changes in the development of thrombosis is unclear. A case comparison study conducted by Pui et al. [23] showed that there was no definitive causative evidence of low levels of PC and/or PS in development of TE in children with ALL receiving threedrug (VCR, asparaginase and prednisone) induction therapy. They also noted a consistent qualitative abnormality in vWF [23,24]. There are only three studies evaluating the effects of asparaginase alone: two during consolidation phase and one at the beginning of remission-induction [4,26,27]. Miniero et al. [26] documented that asparaginase, as a single agent during consolidation phase, reduces the plasma levels of fibrinogen, AT, and factors IX and X with prolongation of APTT. However, remission-induction therapy with combination of asparaginase, VCR and prednisone, resulted in prothrombotic state with abnormal thromboelastogram, increase in platelet counts and F VIII levels. Homans et al. [27] showed significant reduction in the levels of AT and PC during asparaginase alone phase of consolidation. Although the changes in other coagulation parameters were significant, they were still within normal limits. Taking advantage of the window therapy with asparaginase as a single agent on Dana-Farber Cancer Institute (DFCI) ALL consortium study protocol 87-001, Mitchell et al. [4] showed that a single dose of asparaginase significantly reduced the plasma concentration of almost all coagulation proteins, especially of AT. Etiology of asparaginase-induced changes in coagulation factors with special reference to AT. Various mechanisms have been postulated to explain the changes in coagulation factors seen with asparaginase therapy [1,4,9 – 11,27,34 –36]. Depletion of coagulation factors is thought to be resulting, at least in part, by consumptive coagulation initiated by asparaginase [9,11,34]. However, there is not enough evidence to suggest that asparaginase therapy results in significant intravascular coagulation [11,34,37]. Several investigators have provided direct or indirect evidence to rule out the probability of ex vivo direct enzymatic degradation of coagulation proteins, especially AT, by asparaginase as suggested by Nowak-Gottl et al. [1,4,28,34– 36]. Bushman et al. [34] recently provided laboratory evidence

to support the widely accepted theory of hepatic inhibition of protein synthesis as a cause of depletion of coagulation proteins, including AT, after asparaginase therapy. Using HepG2 cell lines, they showed that treatment with 2000 IU/ ml of asparaginase for 24 h led to significant decrease in the amount of AT and heparin cofactor II (HC II) secretion. There was no alteration in the transcription of AT gene, suggesting that asparaginase probably suppresses AT synthesis at the level of protein translation or secretion. However, these experiments were performed using much higher concentration of asparaginase than that achieved in the clinic. They also noticed less than 50% cell viability following 24 h treatment with 1000 IU/ml of asparaginase. Thus, the reduction in the secretion of AT and HCII observed after treatment with 2000 IU/ml asparaginase may be a reflection of poor cellular viability rather than interference with protein translation or secretion. Etiologic role of asparaginase-induced AT suppression in the development of TE. Although there is no direct evidence, suppression of natural anticoagulants, especially AT and plasminogen, secondary to asparaginase therapy coupled with excess thrombin generation is thought to be a major contributing factor to the development of TE [9,10]. This presumption forms the foundation for various studies evaluating the role of AT replacement therapy, either with fresh frozen plasma (FFP) or with AT concentrates, as a prophylaxis against TE in children treated with asparaginase [42 – 49]. Zaunschiram and Muntean [42] used FFP (three times daily; dose individualized to raise the fibrinogen level > 120 mg/dl) and AT (continuous infusion over 24 h daily; dose individualized to maintain the AT levels >100% of normal) supplements in 13 patients with ALL. All of their patients needed supplementation during induction therapy with asparaginase; none of them developed any significant bleeding or TE. All the patients supplemented with FFP and AT achieved an increase in fibrinogen and AT as anticipated. Halton et al. studied effects of FFP (20 ml/kg) on coagulation parameters in eight children with ALL receiving asparaginase. They observed no clinical or statistical difference in coagulation proteins (prothrombin, fibrinogen), natural anticoagulants (AT, PC, PS, a2-m, HC-II) or the markers of endogenous thrombin generation (TAT, Ddimer) with FFP supplements. Two of the eight patients developed side-effects in the form of allergic reactions; strongly questioning the utility FFP for prophylaxis of TE in patients with ALL, especially without any associated beneficial effect [43]. Nowak-Gottl et al. [44] similarly showed ineffectiveness of FFP supplements to correct asparaginase-induced coagulation factor deficiency in children receiving ALL-induction therapy. In a case control study of AT supplementation to adult patients with ALL receiving L-asparaginase (20,000 IU/m2 on alternate days IV, total 6 doses), Gugliotta et al. [45] showed that AT supplementation, given as a bolus injection of 2000 IU (27 –35 IU/kg body weight) every other day for

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a total of 6 doses, effectively normalized the AT, TAT complexes and D-dimer indicating that AT supplementation reduced the parameters of hypercoagulability. However, it had no effect on the levels of fibrinogen, factor IX, PC and PS. Also, there was no evidence of clinical benefits of AT supplementation. Nowak-Gottl et al. [46] studied the effects of prophylactic supplementation of AT (median 30 IU/kg; range 20 –60 IU/kg body weight for median of 2 times; range 1 –4 times), to maintain AT concentration >60% of normal, in children receiving chemotherapy for ALL as per ALL-BFM-90 protocol. Fifteen of twenty-seven (55%) patients required AT supplementation. This resulted in normalization of AT levels and reduction of PF1.2, D-dimer and PAI-1. However, the sample size was too small to assess any clinical significance of AT supplementation in prevention of TE. Although the study was underpowered to test the clinical efficacy of AT concentrate in prevention of TE, the results of recently published prospective, randomized, controlled, phase II trial of AT replacement therapy (PARKAA) showed a trend towards efficacy and safety of AT concentrate [47]. In contrast to previous investigators, PARKAA study observed no significant differences in plasma markers of thrombin generation (e.g. TAT, F1.2, APC-a1 AT) in AT supplemented and nonsupplemented children [45 –49]. Thus, the available evidence regarding clinical benefits of AT supplements to reduce the incidence of TE in patients receiving therapy for ALL is inconclusive. Further, the role of AT supplements in correction of laboratory abnormalities of haemostatic proteins associated with chemotherapy is controversial. These studies indicate that the suppression of AT may not be the central event in the development of TE. 3.1.1.2. Effects of different types of asparaginase preparations. Various asparaginase preparations, derived from different biological sources, are available with resultant variation in pharmacokinetic properties, antileukemic efficacy, and side-effects. All of the approved and commercially available asparaginase preparations are derived either from various Escherichia coli strains (Asparaginase medacn, Crasnitinn, Elasparn, Oncosparn) or Erwinia chrysanthemi (Erwinasen) [18]. In addition, PEG-conjugated E. coli asparaginase (PEG asparaginase) is used as an alternative to the native form due to the lower immunogenecity. Various reports evaluating the effects of different types and preparations of asparaginase are published. None of the studies, however, report the effects of different preparations or types of asparaginase on the same patients. Nowak-Gottl et al. [38] compared two preparations of E. coli asparaginase: Crasnitin BayerR and Asparaginase medacR. Changes in coagulation parameters were more severe with Asparaginase medacR at a dose of 5000 IU/M2 compared to Crasnitin BayerR asparaginase with significant reduction in fibrinogen, plasminogen and a2-antiplasmin (a2-AP) and enhanced thrombin generation (increase

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in PF1.2). However, there was no difference between the levels of AT and D-dimer formation. Two out of twenty-five children receiving Asparaginase medacR developed CNS TE. These children also had F V Leiden mutation. Interestingly, asparaginase activity was significantly higher in Asparaginase medacR group compared to Crasnitin BayerR [38]. Data regarding the effects of Erwinia asparaginase on haemostatic proteins is controversial. Studies comparing the effects of E. coli and Erwinia asparaginase on NHL-ALLBFM-90 treatment protocol showed that compared to E. coli asparaginase, therapy with Erwinia asparaginase did not have any effect on plasma levels of fibrinogen, PC, AT and plasminogen [9]. O’Meara et al. [30] and Risseeuw-Appel et al. [31] showed no change in the AT level in patients receiving Erwinia asparaginase. In addition, RisseeuwAppel et al. showed that children receiving Erwinia asparaginase seemed to have lesser imbalance in coagulation system compared to those receiving E. coli asparaginase. Patients on Erwinia asparaginase had significant earlier rise in fibrinogen levels and lesser decline in PC concentration. However, in this study, the effects of E. coli asparaginase on coagulation proteins including AT and PC were much less severe compared to previous investigators. These studies raised the hopes that Erwinia asparaginase may be less allergic and hematological safer alternative to E. coli asparaginase prompting further evaluation of Erwinia asparaginase as an antileukemic agent. Carlson et al. [32] studied the effects of Erwinia asparaginase in 11 adults with ALL on multidrug induction therapy. Although they observed significant decrease in AT and fibrinogen, the extent of drop in AT was relatively much less compared to a historic series receiving E. coli asparaginase. Compared to patients receiving E. coli asparaginase, there was no change in the levels of vitamin Kdependent coagulation proteins, namely factors II, VII, IX and X. Despite this less severe effects on coagulation system, 1 of 11 patients in their series developed CNS TE during induction therapy with Erwinia asparaginase. Korte et al. [50], on the other hand, reported that Erwinia asparaginase induces hypercoagulable state, which resulted in TE in 3 of 21 patients. Nowak-Gottl et al. [39,40], comparing the effects of Asparaginase medacR, Crasnitin BayerR and ErwinaseR, showed significant reduction in fibrinogen, AT, plasminogen, a2-AP with Asparaginase medacR. However, patients treated with Crasnitin BayerR and ErwinaseR preparations showed significantly lower asparaginase activity and poorer asparagine depletion, indicating that suppression of haemostatic proteins is probably related to the level of asparaginase activity achieved. Recently, Albertson et al. [51] reported a more pronounced influence of Erwinia asparaginase on coagulation parameters compared to Asparaginase medacR. They also noticed high mean trough enzyme activity following Erwinia asparaginase compared to Asparaginase medacR during induction therapy.

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Recently, the European Organization for Research and Treatment of Cancer—Children’s Leukemia Group (EORTC-CLG) conducted a randomized trial using E. coli asparaginase and Erwinia asparaginase, at same dose and schedule, in 700 children with ALL [33]. The results of this trial showed that E. coli asparaginase was superior to Erwinia asparaginase for the treatment of childhood lymphoid malignancies, achieving higher complete remission and longer event free survival rates. The toxicity profile was similar except for coagulopathy. The coagulation abnormalities were much higher in patients treated with E. coli asparaginase compared to Erwinia asparaginase (30.2% vs. 11.9% p < 0.0001). However, the paper did not detail the coagulation abnormalities tested and its clinical correlation. Neither do we know if there was more bleeding tendency or more thrombotic tendency. In addition, the data regarding asparaginase activity and asparagine depletion, to correlate the efficacy or toxicity, in the two groups using E. coli asparaginase and Erwinia asparaginase in this study are not available [33]. In summary, the observed variability in the coagulation parameters following different asparaginase preparations may be a reflection of levels of asparaginase activity achieved rather than individual preparation. Further, at same dose and schedule, E. coli asparaginase is likely to be preferred over Erwinia asparaginase for ALL therapy because of its increased efficacy as an antileukemic agent. 3.1.1.3. Effects of asparaginase on cellular components. Semeraro et al. [25] documented that mononuclear cells obtained from children on day 14 after the start of Lasparaginase (6000 IU/m2/dose three times weekly) as a part of four-drug remission-induction therapy for ALL have an increased capacity to produce procoagulant activity (PCA) in response to endotoxin compared with cells before the start and 7 days after the completion of asparaginase therapy. However, in vitro treatment of cells with asparaginase failed to produce similar effect. Since theses patients were concomitantly receiving prednisone (40 mg/m2 daily, days 0 – 28), weekly VCR (2 mg/m2) and weekly intrathecal methotrexate, it is difficult to attribute the PCA response to asparaginase alone. Two studies report irreversible platelet aggregation in response to combination therapy [29,52]. Pui et al. [29] showed significant reduction in PTT and increase in coagulation factors V and VIII in association with irreversible platelet aggregation. Shapiro et al. [52] reported increased platelet aggregation to low molar ADP in three of four patients studied during multidrug induction therapy on Children’s Cancer Study Group (CCSG) protocol. One of these four patients developed CNS TE. In addition, they also noted that as a group all 15 patients studied had elevated levels of vWF; 58% of patients evaluated had abnormal collagen absorption, indicating a possible qualitative defect in vWF. In contrast, Homans et al. [27] failed to show significant changes in platelet counts or platelet functions

(as assessed by platelet adhesion, aggregation to ADP, collagen and epinephrine; and platelet factor 4) in patients with ALL receiving asparaginase alone during consolidation. This suggests that the platelet functional abnormalities noted by previous investigators may be related to the effects of other agents, especially steroids. 3.1.1.4. Is asparaginase the main culprit or a scapegoat? In summary, despite the various studies evaluating the effects of asparaginase on coagulation proteins, the clinical relevance of these coagulation abnormalities is unknown. So far, there is no conclusive evidence from human or animal studies to support the direct causative role played by asparaginase in the development of TE. The supportive evidence culminating asparaginase in the development of TE includes temporal relation of TEs to asparaginase therapy and coagulation abnormalities, especially acquired AT deficiency, during the therapy with asparaginase. Data from clinical trials aimed to correct AT deficiency have been inconclusive as regards the role of AT supplements in prevention of TE or even correction of surrogate markers of thrombosis. Besides, most of the studies reporting TE in association with asparaginase have used combination therapy with others agents being VCR, prednisone or dexamethasone with or without anthracycline, MTX or CTX. Very few studies use asparaginase as a single agent and when used as a single agent it is usually in the settings of complete remission. Studies using asparaginase either as a single agent or with MTX, CTX, etopocide, report very negligible incidence of TE. Furthermore, recent data indicates that TE is most likely to occur in the phase of therapy, which include both asparaginase and steroids [53 – 55]. Thus, asparaginase alone may not be a major determinant of TE and other agents like steroids are likely to modify the effects of asparaginase on haemostatic system to create a hypercoagulable state. 3.1.2. Corticosteroids Corticosteroids form the backbone of ALL therapy in children. Prednisone, prednisolone and dexamethasone are various corticosteroids commonly used in ALL therapy. Although the thrombogenic potentials of the steroids have been described in literature, the contribution of steroids in the pathogenesis of TE in association with ALL has not been adequately evaluated. 3.1.2.1. Effects of steroids on haemostatic proteins. The available information of effects of steroid therapy on haemostatic system comes from various studies conducted in different populations of mainly adult subjects. In addition to the wide range of diseases (which may primarily affect some of the haemostatic functions) studied, there is no consistency of the type of steroid preparation and the dose or duration of the steroids used [56 – 73] (Table 2). Very few studies report the effects of steroids on haemostatic system in children with ALL and like asparaginase these studies have

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mainly been conducted in the context of multi-agent chemotherapy [61,71,72]. Various investigators have consistently shown that prednisone therapy leads to elevation of factor VIII, vWF, prothrombin and AT, and decreases the levels of fibrinogen and plasminogen [56,58 –60,62,64]. The elevation of F VIII may explain the observation of decrease in clotting time, APTT and PT [57 – 60]. Isacson [56] treated 12 adult healthy volunteers with prednisone and noted that prednisone induces hypofibrinolytic state with reduction of a2-M and decrease in the content of venous wall plasminogen activator. Ueda et al. [62] studied a group of children with nephrotic syndrome receiving 60 mg/m2/day of prednisone and noted elevation of a2-M, along with increase in AT and PC. It is unclear if the changes in haemostatic proteins in this patient population represent control of primary disorder or direct effect of steroids. Recent studies indicate that elevations in F VIII/vWF complex and PAI1 levels are responsible for steroid induced hypercoagulable and hypofibrinolytic state described in patients with Cushing’s syndrome as well as in those on long-term steroid therapy after renal and heart transplantation [65 – 68]. Moreover, animal studies confirm that the effects of dexamethasone on fibrinolytic system are dosedependent. van Giezen et al. [69,70] observed inhibition of platelet aggregation and dose-dependent elevation in PAI-1 in rats, treated with once daily dose of dexamethasone for 5 days. Using an arterial thrombosis model they studied in vivo platelet aggregation. A dose of 0.1 mg/kg resulted in twofold decrease in arterial thrombosis. However, in vivo fibrinolytic activity was significantly decreased at higher dose of dexamethasone (3 mg/kg), but not at a dose of 1 mg/ kg. Thus, they conclude that decreased fibrinolytic activity related to higher dosage of dexamethasone neutralizes the inhibition of platelet aggregation observed at lower dosage of dexamethasone. In a study of 14 children with newly diagnosed ALL receiving VCR, doxorubicin and 60 mg/m2/day of prednisone, Suttor et al. [71] showed that the fibrinogen levels decreased whereas AT and PC increased. They attributed these changes to steroids since similar changes were observed in week 21 of ALL therapy with dexamethasone (10 mg/m2/day) during re-induction phase. Halton et al. [61] showed that the plasma concentration of coagulation proteins cycle in response to administration of prednisone every 3 weeks in children on maintenance therapy for ALL. Thomas et al. [72] showed that 12-day therapy with oral prednisone (60 mg/m2/day) in children with ALL resulted in the reduction of vWF antigen (Ag) and collagen-binding activity (CBA) compared to baseline, whereas combination of prednisone and asparaginase led to increase in vWF Ag and vWF CBA. In addition, the combination chemotherapy resulted in appearance of large multimers of vWF. 3.1.2.2. Effects of different types of steroids on haemostatic proteins. So far, there is no systematic study conducted to

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address the effects of different types of steroids on the haemostatic functions and the associated risk of TE. Recently, Nowak-Gottl et al. [74] reported that children receiving dexamethasone (10 mg/m2/day on days 8 –29 of induction; n = 56) in combination with E. coli asparaginase (5000 IU/m2 at 3-day interval starting day 12 through day 33, total 8 doses) on BFM 2000 protocol had significantly reduced risk of TE compared to children who received prednisone (60 mg/m2/day on days 1 – 36 of induction; n = 280) in combination with same dose and schedule of E. coli asparaginase on the earlier BFM 90/95 protocol (1.8% vs. 10.4% p = 0.028). This is the first report comparing the effects of dexamethasone and prednisone on the risk of TE in patients with ALL. However, these two studies were conducted in succession and it was not a randomized trial to compare the effects of two different types of steroids. Also, the number of patients on dexamethasone group was much smaller compared to that in prednisone group and patients received a total of 21 days of dexamethasone compared to 36 days of prednisone. Although the cumulative dose of these two steroid preparations is comparable, one wonders if the duration of steroid therapy is responsible for the observed difference in these two groups of patients since most of the episodes of TEs observed in prednisone group seemed to occur between days 25 and 35 of therapy. Of note these investigators previously reported 1.7% incidence of TE on re-induction therapy of BFM 90/95 protocol, which uses dexamethasone. Interestingly, this was following a 10% incidence of TE during induction therapy with prednisone [53]. Rodeghiero et al. [2] reported 14% incidence of symptomatic TE during remission induction therapy with dexamethasone 8 mg/m2 IV on days 1– 21 and Crasnitin BayerR asparaginase at a dose of 10,000 IU m2/ day IV on days 1 –14 along with VCR and doxorubicin. Thus, the current evidence regarding the effects of dexamethasone on the risk of TE in children with ALL is controversial. Certainly further studies will be required to define the role of dexamethasone in the pathogenesis of TE and the risk of TE in association with dexamethasone therapy. 3.1.2.3. Effects of corticosteroids on cellular components. Very few studies evaluate the effects of steroids on platelets. Most investigators showed increase the platelet counts following prednisone therapy [57,60,62,64]. The reported effects of steroids on platelet functions seem to be contradictory. Isacson failed to show any change in clotting time or platelet adhesiveness in healthy adults receiving oral prednisone. Of note, this study used much smaller dose of prednisone compared to ALL therapy [56]. Recently, van Giezen et al. [69,70], using an animal model, showed that oral dexamethasone therapy induces dosedependent suppression of platelet counts and platelet aggregation. Pui et al. [29], on the other hand, showed increased irreversible aggregation of platelets (secondary phase) in week 4 of induction therapy (three-drug induction with

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Table 2 Effects of steroids on haemostatic functions Steroid type, dose

Menczel, 1958 [57]

Patients with TE on Dicoumarol and prednisone (24) or Placebo (12)

Prednisone 10 mg PO
1 dose

Ozsoylu 1962 [58]

Cushing’s syndrome (1) Rheumatic fever (3) ITP (1) Classic hemophilia (3)

Isacson 1970 [56]

Effect on

Conclusion

Coagulation proteins

Anticoagulation Fibrinolytic system factors

Other

–

–

–

Significant # in whole blood clotting time at 2 and 4 h after prednisone therapy.

Corticosteroids produce a hypercoagulable state and can counteract the effects of oral anticoagulant therapy.

Pre-Sx Post-Sx Prednisone 60 mg/day
10 days Prednisone 60 mg/day
7 days

FVIII 420% – FVIII 110% z FVIII, FV, FVII, FX, _ z Fibrinogen z FVIII (mild>mod>severe)*, _ z FV, FVII, FX, and prothrombin

–

Quick time Pre-Sx# Post Sx N

High-dose corticosteroids increase the antihemophiliac factor levels considerably.

Healthy volunteers; age 18 – 36 years (12)

Prednisolone 10 mg PO BID
11 days

z FVIII, no change in FV or fibrinogen

# a2-M

Hypofibrinolytic stage. No Change in plasminogen and antiplasmin. # in the content of venous wall plasminogen activator.

No change in clotting time or platelet adhesiveness.

Interaction of steroids with plasminogen activator content in the vessel wall.

Zanon 1982 [59]

Cushing’s syndrome (15)

Pre and post therapy

z FVIII (VIII:C, VIII:Ag, VIII:vWF)

_

Low fibrinolytic activity # PTT and PT Normal ristocetin platelet aggregation.

High plasma cortisol levels lead to z FVIII production which in turn cause hypercoagulibility with z in the incidence of TE.

Jorgenson 1982 [60]

Patients with collagen (11) and hematological diseases (CLL 7, Lymphoma 2, Other 2)

Prednisone 30 – 40 mg/day
6 weeks

z FVII and II, z FVIII RA, # fibrinogen

z AT

# Plasminogen

# in fibrinogen probably secondary to control of primary pathology. Glucocorticoid therapy leads to increased activity of coagulation cascade which may be counterbalanced by z in AT

_ _

# APTT, no change in clot lysis time.

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Author, year Patient population (reference number) (number of patients)

z Platelet count, z Cholesterol

a2-antiplasmin level significantly and directly correlated with the dose of steroids. Steroids appear to be thrombogenic by inducing thrombocytosis, hyperlipidemia and # fibrinolysis.

–

z a2-antiplasmin, z Plasminogen

Significant correlation between mean arterial pressure and elevation in coagulation factors

Cushing’s syndrome induces hypercoagulable state.

_

_

z GM-CSF levels from 418 pg/ml prior to MP to 733 – 911 pg/ml 1 – 4 weeks after therapy

HDMP stimulates endogenous GM-CSF secretion

HDMP 20 – 30 mg/kg/day z FVIII:C and vWF

_

_

z platelet counts

z FVIII:C and vWF seem to be the effects of HMDMP and not related to underline pathology.

Prednisone 5 – 10 mg/day

–

Marked z in euglobin lysis time compared to controls; significant z in PAI-2 and tPA Ag

Increase in cardiac microthrombi formation in steroid treated patients.

Long-term steroid therapy induces prothrombotic state with elevation in F VIII, vWF and PAI-1.

MCNS (23) Healthy Controls (50)

Prednisone 60 mg/ m2/day
4 weeks

z Fibrinogen

Patrassi 1985 [67]

Cushing’s syndrome (9)

–

z F XII, XI, IX and VIII

Tuncer 1992 [63]

ALL (7) AML (3)

HDMP 20 – 30 mg/ kg/day
3 weeks

_

Ozturk 1994 [64]

Acute ITP (46); Chronic ITP (28); Non-ITP thrombocytopenia (10) Healthy controls (10)

Sartori 1999 [65]

Heart transplant patients (49): Receiving Cyclosporin and Azathioprine with (23 patients) or without (26) steroids

Significant z fibrinogen, vWF, and FVIII

z AT, z protein C, z a2-M

Summary of studies evaluating effects of steroids (endogenous or exogenous) on coagulation proteins, anticoagulation factors, fibrinolytic system and cellular components. Elevation of factor VIII, vWF, prothrombin, AT and platelet counts, and reduction in the levels of fibrinogen and plasminogen has been observed consistently. *= increase in F VIII in mild hemophilia>moderate hemophilia>severe hemophilia. Pre-Sx = pre-surgery, Post-Sx = post surgery, PO = per oral, BID = twice every day, a2-M = a2-macroglobulin, AT = antithrombin, APTT = activated partial thromboplastin time, PT = prothrombin time, MCNS = minimal change nephritic syndrome, HDMP = high-dose methylprednisone, GM-CSF = granulocyte macrophage colony-stimulating factor, Ag = antigen.

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z a2-antiplasmin, z plasminogen

Ueda 1990 [62]

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weekly VCR, L-asparaginase 10,000 IU/m2 twice weekly for first 2 weeks of induction; prednisone 40 mg/m2/day for 4 weeks) concomitant with decrease in PTT. Since the Lasparaginase effect disappears by 10 –12 days after the dose, the authors presumed that the platelet activation is probably secondary to steroid therapy. Korte et al. [50] suggested that recovery of platelet counts might play an important role in the development of TE. They documented elevated thrombin generation that coincided with fibrinogen recovery, cross-linked FDP and increase in platelet count following asparaginase therapy. Steroid therapy leads to an increase in absolute phagocytic count. Tuncer et al. [63] showed that high-dose methylprednisone therapy in patients with ALL stimulate endogenous granulocyte macrophage colony-stimulating factor (GM-CSF) secretion. GM-CSF leads to increase in numbers of neutrophils and monocyte/macrophage. In addition, it also enhances the phagocytic and tumoricidal activity of these cells [75]. Thus, it is likely that steroid therapy through endogenous GM-CSF secretion may cause enhancement of monocyte functions including activation of coagulation cascade. In conclusion, available evidence suggests that steroid therapy induces prothrombotic state mainly by elevations in F VIII/vWF complex and by dose-dependent increase in PAI1 levels. It is shown to affect platelet counts and functions. Despite this evidence, the effects of the dose or duration of steroid therapy on haemostatic system is unknown. Also, effects of different types of steroid preparations, especially dexamethasone, on the coagulation proteins and inhibitors in combination with other agents, namely asparaginase, are yet to be defined. This is particularly of interest to pediatric oncologist since dexamethasone is a more potent antileukemic agent, and recently, more frontline studies are using this agent for ALL therapy [73]. Further studies are needed to evaluate the effects of various types of glucocorticosteroids on haemostatic proteins and cellular components. 3.1.3. Effects of other chemotherapeutic agents Although VCR and doxorubicin are two other most common agents used during remission-induction, we do not know their isolated effects on coagulation system. During the past decade, agents like MTX, CTX and ARAC have been added to remission induction and/or consolidation phase of ALL therapy, especially for patients with high-risk ALL. The data on effect of these agents on coagulation system is also very scant. 3.1.3.1. Effects of other chemotherapeutic agents on haemostatic proteins. Totan et al. [76] evaluated effects of high-dose MTX (3 g/m2) on haemostatic parameters in 20 children with ALL following consolidation therapy with etoposide and ARA-C. After administration of MTX, significant decrease in PT, PTT, fibrinogen, PC, PS, AT, and marked elevation of FDP were noted on day 1 compared to

baseline. All the parameters, except FDP, began to increase on day 7 following MTX. There was no change in the level of t-PA. The exact etiology of these changes observed during the MTX therapy is unknown. Although the authors proposed MTX-induced inhibition of protein synthesis, none of their patients had severe liver dysfunction [76]. Alternatively, this could be an effect of prior chemotherapy. Since as a group, these patients started of with prolonged PT, PTT, significantly lower PC and PS values and elevation of FDP before the initiation of MTX. The values of haemostatic parameters at the time of diagnosis of ALL and during the phases of remission-induction and consolidation are unavailable for comparison. Arico et al. [77] studied clotting functions in 17 patients with ALL on maintenance chemotherapy compared to 15 healthy age- and sex-matched controls. They noted slight prolongation of PT with significantly reduced levels of factors VII, IX and a trend towards reduction in F X without any demonstrable changes in anticoagulant factors. Marra et al. [78] studied AT levels in 14 adult patients with hematologic malignancies receiving high-dose ARA-C with or without sequential L-asparaginase and noted that high-dose ARA-C alone did not influence AT levels. 3.1.3.2. Effects of other chemotherapeutic agents on cellular components. There are not many reports evaluating the effects of chemotherapeutic agents, other than asparaginase and steroids on cellular components of haemostatic system. Recent studies indicate that chemotherapeutic agents can directly and indirectly interact with platelets, endothelial cells and malignant cells [79,80]. Togna et al. [79] showed that Cisplatinum, a commonly used antineoplastic agent, induces hyper-reactivity in human platelets. In response to subthreshold, non-aggregating concentrations of agonists, Cisplatinum-treated platelets had increased aggregation and enhancement of thromboxane formation. Parades et al. [80] showed that chemotherapeutic agents commonly used in the treatment of ALL (e.g. vincristine, adriamycin, asparaginase, hydrocortisone, Ara-C, MTX, etoposide) can potentially upregulate TF on the surface of T24/83 cells leading to increased thrombin generation. This direct interaction of chemotherapeutic agents with platelets and malignant cells may possibly contribute to thrombosis, especially during initial remission-induction. Clinical and laboratory studies evaluating the effects of chemotherapeutic agents, other than asparaginase and steroids, on coagulation system and cellular components are needed.

4. Effects of other therapy-associated factors 4.1. Role of central venous line CVLs are widely used in the practice of pediatric oncology and have greatly improved the supportive care

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and quality of life in children with cancer. However, these devices are associated with unwanted complications like infections, catheter dysfunctions and catheter-related TEs. The catheter-related TEs have potential for serious complications including recurrence of thrombus (4– 19%), postphlebitic syndrome (5 – 25%), pulmonary embolism (8 – 15%), and death (2 –4%) [81,82]. Although majority of the CVL associated TEs are asymptomatic, approximately half of the symptomatic DVTs in children with ALL were in relation with CVL [4,7,19,24,53,54,83 – 86]. Glacer et al. [82], using contrast venography (the gold standard for diagnosis of upper vessel occlusion), showed that 50% of children with cancer and implantable ports had evidence of deep vein thrombosis at the site of catheter placement. Three of the twelve patients with TE had associated clinical signs or symptoms; the rest were asymptomatic. Children with ALL are more likely to have CVL-related TE compared to those with malignancies other than ALL [87,88]. PARKAA study showed over 30% incidence of asymptomatic TE in association with CVL; 83% of these events were located at the entry site of the catheter in the vein rather than the location of the catheter tip. The risk of TE was significantly increased with the CVL on the left side of body, CVL placed in the subclavian vein and when inserted by percutaneous technique rather than venesection [89]. The significance of the CVL-related TE in the development of DVT elsewhere in the body, PE or CNS TE is unknown at present time. 4.2. Role of infections Bacterial infections and sepsis is a well-known complication in children undergoing intensive induction or reinduction therapy for ALL [16]. In addition, the presence of CVL increases the risk of infections [81]. The recent laboratory and clinical evidence of close interactions of coagulation and fibrinolytic system with inflammatory pathways provide support for the infection-induced activation of coagulation. Various coagulation proteins including AT, PC, PS, TF and thrombin act as important mediators of sepsisinduced inflammatory reactions [90,91]. Thus, the infectious episodes are likely to increase the risk of TE in patients receiving therapy for ALL.

5. Conclusion Although the exact etiology is unknown, children with ALL have evidence of increased thrombin generation at diagnosis. The observation that TE in children with ALL is most commonly reported only after the initiation of antileukemic therapy indicates a possible interaction of leukemia and its therapy leading to TE. Both asparaginase and steroids are shown to induce hypercoagulable state by suppression of natural anticoagulants (especially AT and

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plasminogen) and by elevation in F VIII/vWF complex, respectively. In addition, steroid therapy causes hypofibrinolytic state by dose-dependent increase in PAI-1 levels. Furthermore, chemotherapeutic agents, especially steroids, are shown to improve platelet counts and may alter platelet functions. Combination of these effects coupled with increased thrombin generation may be responsible for the higher incidence of TE observed with concomitant administration of asparaginase and steroids. Further studies to delineate the mechanism of increased thrombin genaration in association with ALL and the effects of antileukemic therapy on haemostatic system in children with ALL are needed.

Acknowledgements We thank Dr Mohan Pai for critical review of the manuscript. This work was supported in part by a Grant-inaid NA5030 from the Heart and Stroke Foundation of Ontario, Canada. Anthony K.C. Chan is a recipient of a Research Scholarship award from the Heart and Stroke Foundation of Canada.

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