HEMOSTATIC SIDE EFFECTS OF HIGH-DOSE METHOTREXATE IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

The purpose of this study is to investigate the hemostatic side effects of HDMTX. Between 2001 and 2002, 20 children with acute lymphoblastic leukemia at the Dr. Sami Ulus Children's Hospital, Department of Pediatric Hematology and Oncology, treated according to the St. Jude ALL XIII protocol were eligible to this study. Methotrexate at a dose of 2 g/m² was infused over 24 hours. Coagulation screening studies included prothrombin time (PT), APTT, fibrinogen, fibrin degradation product (D-Dimer), factor II, factor V, factor VII, factor VIII, factor IX, factor X, PC, PS, AT-III determinations before HDMTX therapy (PreT), 1 day after (PostT^1D), and 1 week after (PostT^1W) the end of the HDMTX infusion. We found that PT and APTT were prolonged, PC, PS, and AT-III levels were decreased with a slight increase in D-Dimer 1 day after the administration of HDMTX and all of them returned to the normal levels by 7 days. In addition we found that FVII, FIX, FX were significantly decreased 1 day after therapy and normalised by 7 days.

Methotrexate (MTX) has been used in the treatment of childhood acute lymphoblastic leukemia (ALL) for over 40 years and is one of the important drugs utilized in the continuing or maintenance therapy of this disease. High-dose methotrexate (HDMTX) is synonymous with a 4–24-hour intravenous infusion of 1000–33600 mg per square meter [1–3]. After HDMTX infusion, the patient is given leucovorin (5-formyl tetrahydrofolic acid: LV), which replenishes intracellular stores of reduced folate and attenuates the toxicity secondary to HDMTX [2, 4–6]. A number of the acute toxicities are common with HDMTX infusion, including myelosuppression, vomiting, orointestinal mucositis, hepatic and renal toxicities, neurological disturbances [6–8].

Hemostatic abnormalities are found in 50% of the patients with malignancy [9–11]. Hemostatic changes in malignancy may occur with the disease process itself or due to the result of chemotherapy. Abnormal activation of the coagulation pathway, alteration of platelet activation and aggregation, decreased hepatic synthesis of coagulant and anticoagulant proteins because of hepatic infiltration of disease and/or chemotherapy (L-asparaginase), release of the plasminogen activators are all possible causes of the hemostatic abnormalities [11–13]. The most common hemostatic abnormality in patients with leukemia is hemorrhage and much rarer complication is thrombosis [14].

The purpose of this study is to investigate the hemostatic side effects of HDMTX (2 g/m² infused over 24 hours). To our knowledge, this is the first study in the literature with hemostatic side effects of HDMTX (2 g/m² infused over 24 hours) therapy in childhood acute lymphoblastic leukemia.

MATERIAL AND METHODS

Between 2001 and 2002, 20 children with acute lymphoblastic leukemia at the Dr. Sami Ulus Children's Hospital, Department of Pediatric Hematology and Oncology, treated according to the St. Jude ALL XIII protocol were eligible to this study [15]. The diagnosis and subclassification of acute leukemia were based on morphologic characteristics of the blasts, cytochemistry, and immunophenotyping analysis. Remission induction therapy consisted of prednisone, vincristine, daunomycin, asparaginase, etoposide, cytarabine. High-dose MTX plus LV rescue was given on days 44, 51, and once per 2 months during continuation therapy. Methotrexate at a dose of 2 g/m² was infused over 24 hours. Careful urinary alkalinization (urine pH > 7.5) with adequate hydration (3000 mL fluid/m²/24 hours) was administered before HDMTX infusion. Folinic acid rescue was given after starting HDMTX at 36 hour 30 mg/m² and at every 6 hour for 2 doses, then 15 mg/m² po at 48 h, and 5 mg/m² po for 3 doses at 54, 64, 72 h [15].

Routine biochemical and hematological studies were performed in all of the patients including complete blood count, serum urea nitrogen, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT). Coagulation screening studies included prothrombin time (PT), APTT, fibrinogen, fibrin degradation product (D-Dimer), factor II, factor V, factor VII, factor VIII, factor IX, factor X, PC, PS, AT-III determinations before HDMTX therapy (PreT), 1 day after (PostT^1D), and 1 week after (PostT^1W) the end of the HDMTX infusion. A 5-mL blood sample was collected in a 3.8% sodium citrate tube by venipuncture. The blood was immediately centrifuged, and plasma samples were stored at −60°C until analyzed (maximum 2 weeks). Prothrombin time, APTT, fibrinogen, and D-dimer were assayed by using Sigma Diagnostics kits (by Amax-Amelung CS-190 full automatic otoanalysor). Factor II, FV, FVII, FVIII, FIX, FX, PC, PS, AT-III were also assayed by using Sigma Diagnostics kits (by Amax-Amelung CS-190 full automatic otoanalysor. Voluntary informed consent was obtained from each patient, parent, or guardian as appropriate.

Statistical analyses were done with SPSS statistical software. The mean and standard errors were calculated for all parameters. Pretreatment values and 1 and 7 days after the treatment values were compared by multivariate analysis of variance (MANOVA). The results having significant difference were again compared to each other by using paired t or Wilcoxon tests (p <. 05 was considered statistically significant).

RESULTS

Twenty consecutive children eligible for this study were successfully enrolled. The ages, sexes, and the number of HDMTX administration for 20 children are shown Table 1.

Patient Demographics Characteristics and the Number of HDMTX Administration

	HDMTX (n = 20)		The number of HDMTX addministration
	Age	Sex
1	3.5	B	3
2	5	G	3
3	4	G	4
4	11	G	3
5	2	G	4
6	6	B	6
7	9	B	5
8	7	B	4
9	13	B	4
10	2.5	B	4
11	7	G	5
12	4.5	B	5
13	6	B	5
14	4	G	4
15	8	B	6
16	9	B	5
17	8.5	G	3
18	3.5	B	3
19	5	G	5
20	4	B	6
Median	6.12 ± 2.91	—	4.35 ± 1.04
Range	2–13	9G-11B	3–6

Mucositis was observed in 4 patients (20%). The symptoms typically began 3 day after start of the infusion and were fully developed on day 6.

Hemoglobin levels at first week after treatment (9.29 ± 1.1/g/dL) were significantly lower than pretreatment and first day after treatment (10.38 ± 1.3 g/dL, 10.09 ± 1.3 g/dL; p <. 05). Leukocyte count at the first day and first week after treatment (3215 ± 1530/mm³, 3175 ± 1076/mm³) were significantly lower than pretreatment leukocyte count (4425 ± 1874/mm³; p <. 01). Although thrombocyte count at 1 week after treatment was lower than the previous levels it remained within normal levels (p <. 01).

Creatinine levels of first day and first week after treatment were not significantly higher than pretreatment creatinine level (p >. 05). Although BUN level at first day after treatment was slightly increased (p <. 05), all the BUN values (pretreatment, first day and first week after treatment) were normal limits.

Hepatic function tests (AST-ALT) were within normal limits before the initiation of HDMTX therapy (27 ± 15 U/L, 27.8 ± 31 U/L). There were significantly increased at first day after HDMTX treatment (99.2 ± 49.8 U/L, 67.8 ± 29.9 U/L; p <. 01) and normalised at first week after treatment (45 ± 25 U/L, 40 ± 26 U/L).

Prothrombin time and APTT (11.7 ± 0.92 sec, 32.9 ± 3.34 sec) were within normal limits before initiation of HDMTX therapy and they significantly prolonged at 1 day after treatment (15.6 ± 2.12 sec, 43.8 ± 5.7 sec; p <. 01) and normalised at one week after HDMTX infusion.

The levels of FVII, FIX, FX (42.6 ± 17.8%, 49.8 ± 14.1%, 50.4 ± 18%) were significantly decreased at first day after treatment as compared with the normal limits before the initiation of HDMTX therapy (113.7 ± 23.5%, 105.4 ± 20.2%, 94.5 ± 22.9; p <. 01 in each comparison) and normalized by seven days. Although all FV levels were normal limits, its level at first day after treatment was decreased (104.1 ± 21.8, 72.6 ± 21.9; p <. 01) as compared with pretreatment levels. In contrast to other factors levels, FVIII level were significantly increased at first day after treatment than its level before the HDMTX therapy (94.4 ± 25, 55.3 ± 14; p <. 01) and it returned to normal levels at first week after therapy.

Plasma values for PC, PS, and AT-III (45.6 ± 17.8%, 50 ± 14.5%, 77.9 ± 13.9%) were found to be significantly lower at first day after treatment than before the initiation of HDMTX therapy (p <. 01 for each comparison). All these abnormal values normalized on first week after treatment.

Alterations of all parameters before and after (1 day and 1 week) HDMTX therapy are summarized in Table 2.

Alterations of All Parameters Before and After (1 Day and 1 Week) HDMTX Therapy

	HDMTX (n = 20)
	PreT	AfterT^1D	AfterT^1W	p(PreT − AfterT^1D/PreT − AfterT^1W)
Hb (g/dL)	10.38 ± 1.3	10.09 ± 1.3	9.29 ± 1.1^*	−./02
Bk (mm^3)	4425 ± 1874	3215 ± 1530^*	3175 ± 1076^*	.008−/.004
Platelet(1000 × mm^3)	262 ± 112	220 ± 99	193 ± 77^**	−/.000
Creatinine (mg/dL)	0.47 ± 0.1	0.52 ± 0.1	0.5 ± 0.18	−/−
BUN (mg/dL)	9.37 ± 4.1	13 ± 6.2^*	10.1 ± 4.9	.017/−
AST (U/L)	27 ± 15	99.2 ± 49.8^**	45 ± 25^**	.000/.002
ALT (U/L)	27.8 ± 31	67.8 ± 29.9^**	40 ± 26	.001/−
PT (sec)	11.7 ± 0.92	15.6 ± 2.12^**	12.1 ± 0.95	.000/−
APTT (sec)	32.9 ± 3.34	43.8 ± 5.7^**	36 ± 3.8	.000/−
D-Dimer (ng/mL)	117 ± 78	275 ± 182^**	197 ± 124^*	.000/.002
Fibrinogen (mg/dL)	419.7 ± 115.9	388.8 ± 138.4	377.4 ± 125.3	−/−
FII (%)	119.3 ± 14.6	104.4 ± 24.6^*	112.5 ± 13.3	.04/−
FV(%)	104.1 ± 21.8	72.6 ± 21.9^**	94.6 ± 35.1	.000/−
FVII (%)	113.7 ± 23.5	42.6 ± 17.8^**	102.1 ± 26.9	.000/−
FVIII (%)	94.4 ± 25	155.3 ± 14^**	106.4 ± 30.4	.000/−
FIX (%)	105.4 ± 20.2	49.8 ± 14.1^**	95.6 ± 20	.000/−
FX (%)	94.5 ± 22.9	50.4 ± 18^**	95.9 ± 16.1	.000/−
PC (%)	97.7 ± 22.4	45.6 ± 17.8^**	94.9 ± 24.2	.000/−
PS (%)	95.5 ± 34.6	50 ± 14.5^**	96.4 ± 28	.000/−
AT-3 (%)	117.8 ± 18.9	77.9 ± 13.9^**	118.8 ± 17.1	.000/−

^* p <. 05.

^* p <. 01.

DISCUSSION

Administration of HDMTX is one of the cornerstones of antileukemic treatment in ALL. Myelosuppression, mucositis, hepatotoxicity, nephrotoxicity, and neurological disturbances such as seizures, loss of consciousness, paresis, or leukoencephalopathy are well-known possible side effects of HDMTX treatment [6–8].

We observed mucositis in 20% (4 patients), myelosupression presenting with leukopenia and anemia in 65% (13 patients), transient hepatotoxicity in 70% (14 patients) in our study group. Erythrocyte transfusion was not required for patient in whom anemia was developed. Hepatotoxicity appeared with the elevation in AST and ALT up to twofold of basement values at first day after treatment and returned to the normal by seven days. Except for a slight increase in BUN levels, a significant nephrotoxicity did not occur in any patient, which was attributed to careful hydration and adequate urinary alkalinization. Febrile neutropenia was observed in 10% (2 patients). Infection was controlled with combined empirically antibiotherapy. These mild side effects of HDMTX therapy were expected and resolved spontaneously by seven or ten days after administration of MTX.

Hemorrhagic and thromboembolic complications has been reported to occur in 1–11% of the patients with malignancy and these complications may be due to disease process itself or may occur a result of chemotherapy [11, 14]. Hemostatic and anticoagulant system abnormalities are well defined following L-asparaginase therapy in a number of studies [12, 13]. L-asparaginase causes inhibition of plasma proteins including coagulant and anticoagulant factors [16, 17]. On the other hand, a decrease in coagulant and anticoagulant proteins are shown after the use of MTX, including combined with other chemotherapy protocols [18]. However there has been only one study in literature in which hemostatic system was investigated after HDMTX therapy alone [19]. In that study HDMTX (3 g/m²) is infused over 3 hours and they reported that PT and APTT were found to be prolonged, PC and PS levels were decreased with an increase in D-Dimer when compared to basement levels [19].

In our study, exposure time of MTX is prolonged by infusing the drug over 24 hours and its effect on hemostatic system is investigated. As in previous study of Totan et al., we also found that PT and APTT were prolonged, PC, PS and AT-III levels were decreased with a slight increase in D-Dimer 1 day after the administration of HDMTX and all of them returned to the normal levels by seven days. In addition, we found that FVII, FIX, FX were significantly decreased 1 day after therapy and normalised by 7 days. We concluded that liver cell damage might have been caused these alterations in hemostatic system since all of the decreased coagulant and anticoagulant proteins are synthesized by the liver. As presented in Table 3, there was a significant positive correlation between AST, ALT levels, and PT, APTT levels at 1 day after HDMTX therapy. Moreover, there was a negative correlation between liver function tests and FVII, FIX, FX levels in our study group. Although D-Dimer was increased 1 day after MTX therapy, fibrinogen level did not decrease as in appeared in consumption. D-Dimer removes from the circulation by liver cells, liver cell damage might have been caused this slight increase in D-Dimer rather than consumption [20, 21]. However, for the exact exclusion of consumption, we should have been studied fibrinolytic and thrombotic factors such as fibrinopeptide A-B, prothrombin fragment 1 + 2 (F 1 + 2), thrombin-antithrombin (TAT) complexes and tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), plasmin levels, and plasmin-antiplasmin (PAP) complexes.

Correlation Analysis of Parameters

	AfterT^1D (r =)
AST-PT	.75
AST-APTT	.54
ALT-PT	.56
ALT-APTT	.72
AST-FVII	−.71
ALT-FVII	−.58
ALT-FIX	−.55
ALT-FX	−.59

Neither hemorrhagic nor thromboembolic complications related to the HDMTX therapy were seen in our patient. This is attributed to opposite alterations in hemostatic system such as decreased levels of PC, PS, and some coagulant factors.

Although it did not appear in any patient in our study group, in case one encounters a hemorrhagic or thromboembolic complication in a patient after HDMTX administration, the above hemostatic system alterations due to the use of HDMTX therapy should be born in mind.