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© 2003 American Society for Clinical Oncology Homocysteine, Pharmacogenetics, and Neurotoxicity in Children With Leukemia
From the Department of Pharmaceutical Sciences, Biostatistics, Hematology-Oncology, St. Jude Children’s Research Hospital, and University of Tennessee, Memphis, TN; Texas Tech School of Pharmacy, Amarillo, TX; and Department of Human Genetics, University of Chicago, Chicago, IL. Address reprint requests to Mary V. Relling, PharmD, Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105; email: mary.relling{at}stjude.org.
Purpose: Despite its clinical success, methotrexate (MTX) therapy is associated with toxicities such as seizures, the pathogenesis of which remains unclear. It has been suggested that hyperhomocysteinemia is caused by MTX and is responsible for its neurotoxic effects. The purposes of this study were to explore whether hyperhomocysteinemia was related to MTX administration and toxicity and whether homocysteine or MTX toxicity differed by methylenetetrahydrofolate reductase (MTHFR) or reduced folate carrier (RFC) genetic polymorphisms. Patients and Methods: We studied 53 children with newly diagnosed acute lymphoblastic leukemia who were consecutively treated on a single clinical protocol that included two courses of high-dose MTX (high-dose methotrexate [HDMTX]; 2.5 or 5.0 g/m2 per day) as consolidation therapy. Results: The study participants’ median plasma homocysteine concentrations at 23 and 44 hours after HDMTX (9.00 µmol/L and 10.12 µmol/L, respectively) were greater than the concentrations immediately before HDMTX (5.77 µmol/L, P < .0001 for both comparisons). Seven days after HDMTX treatment, their plasma concentration returned to baseline. Nine patients experienced seizures, and five patients experienced thrombosis during the first 15 months of therapy, with a tendency for there to be higher plasma homocysteine in patients with seizures across all time points (P = .063) but not in patients with thrombosis (P = .59). We observed no significant differences in plasma or cerebrospinal fluid homocysteine levels or in toxicity based on the MTHFR 677C/T or RFC 80G/A genotypes. Conclusion: We conclude that homocysteine was transiently elevated after HDMTX and may be related to seizure risk in children with leukemia.
THE ANTIFOLATE methotrexate (MTX) has been widely used as an antineoplastic agent in the treatment of malignant diseases, such as acute lymphoblastic leukemia (ALL).1 Despite its clinical success, MTX can be associated with serious toxicities, such as seizures,2,3 the pathogenesis of which remains unclear. MTX and its active polyglutamate metabolites inhibit enzymes involved in folate homeostasis and induce cellular depletion of reduced folates, including 5-methyltetrahydrofolate, a carbon donor in the conversion of homocysteine to methionine.4,5 Hyperhomocysteinemia is an independent risk factor for vascular disease6 and atherothrombosis,7 probably because it increases nitric oxide synthesis.8,9 It has been suggested that hyperhomocysteinemia is caused by MTX and may be responsible for the neurotoxicity associated with MTX.10–12 The enzyme 5, 10-methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, is crucial to folate metabolism. MTHFR deficiency results in hyperhomocysteinuria.13 Patients with severe MTHFR deficiency show a wide range of symptoms, such as developmental delay, motor and gait abnormalities, seizures, psychiatric manifestations, and vascular complications.14 A common polymorphism, a C-to-T substitution at nucleotide 677 (replacing alanine with valine),15 reduces the activity of MTHFR but results in a much less severe phenotype than the rare mutations that cause severe deficiency. Approximately 10% of American whites, 20% of some Italian populations, and 1% of African-Americans are homozygous for the mutant allele.16 The 677T/T genotype has been associated with hyperhomocysteinemia,17,18 especially in patients with low folate.19,20 Several studies have noted a higher incidence of a gastrointestinal or hepatic toxicity after chronic, low-dose MTX therapy among patients with the 677T allele.21,22 The reduced folate carrier (RFC) is the gene product responsible for cellular uptake of reduced folate and MTX23 and is subject to a common polymorphism, a G-to-A substitution at position 80.24 If MTX-related toxicity is affected by MTHFR or RFC genotype, it is possible that these genotypes could be used to assist in individualizing MTX therapy. We assessed the changes in homocysteine concentration in plasma and cerebrospinal fluid (CSF) in serially, prospectively studied children with newly diagnosed ALL treated with HDMTX. The purposes of the study were to determine whether the plasma or CSF homocysteine concentration is related to MTX administration and the occurrence of MTX toxicity and whether the MTHFR or RFC genotype is related to plasma or CSF homocysteine levels and MTX-related toxicity.
Children with newly diagnosed ALL were treated on St. Jude Children’s Research Hospital (Memphis, TN) protocol Total XIV between 1998 and 1999 after informed consent had been obtained from the parent, guardian, or patient (as appropriate). Measures of homocysteine were incorporated into this protocol, and all patients agreed to participate in this aspect of the study. The treatment is outlined in Table 1  . All patients received remission induction therapy, including up-front HDMTX therapy with leucovorin and triple intrathecal treatment. Consolidation consisted of HDMTX (5 g/m2 or 2.5 g/m2 over 24 hours for those patients assigned to the standard/high- or low-risk arms of the study, respectively), along with intrathecal treatment. HDMTX was given weekly with leucovorin rescue (15 mg/m2 intravenously for standard/high-risk patients or 10 mg/m2 for low-risk patients), which started 44 hours after the start of MTX infusion and was given every 6 hours for a total of five doses, with increased leucovorin for that minority of patients who had plasma MTX concentrations greater than 1 µM at 44 hours. After consolidation therapy, patients received continuation treatment for 120 weeks.
Neurotoxicity was assessed using the National Cancer Institute common toxicity criteria version 2.0 (available at http://ctep.cancer.gov/reporting/ctc.html). All patients who had seizures (regardless of grade) and all patients who had other grade 3 or 4 neurotoxicity were eligible for consideration in this analysis. Other than one patient with severe somnolence related to bacterial meningitis (who was not considered), neurotoxicity consisted of seizures. Except for the diagnostic sample, orders for plasma and CSF homocysteine measures were timed relative to the HDMTX treatment. Blood (3 to 4 mL) was collected in heparinized tubes at diagnosis, immediately before consolidation 1 (7 weeks after the up-front HDMTX plus leucovorin administration), immediately before consolidation 2 (7 days after the prior HDMTX plus leucovorin administration), 23 and 44 hours after the start of consolidation 2 (all before the second course’s leucovorin treatment), and immediately before week 31 of continuation therapy (4 weeks after the last MTX administration). CSF was collected into polypropylene tubes at diagnosis, immediately before consolidation 1, immediately before consolidation 2, and immediately before week 31 of continuation therapy. The samples were stored at -80°C, shipped to the analyst on dry ice, stored at -80°C, and thawed on ice at the time of assay. Homocysteine was measured by a modified high-performance liquid chromatography method.11,25 The assay conditions for the CSF samples were optimized for the lower concentrations of homocysteine in these samples. The limit of detection for CSF was 75 fmol on column with these modifications. The overall coefficient for variation for the assay of homocysteine in CSF was 5%. The plasma MTX concentration was measured by fluorescence polarization immunoassay (TDx/TDxFLx systems; Abbott Laboratories, Abbott Park, IL). DNA was extracted from blood cells, and the MTHFR 677C/T and RFC 80G/A genotypes were assessed using modifications of previously described polymerase chain reaction–based methods.15,24 For the MTHFR 677C/T polymorphism, a different forward primer was used for the PCR than what has previously been described.15 The sequence of the forward primer used was 5'-CAGTCCCTGTGGTCTCTTCAT-3'. A 329-bp amplification product that contains the polymorphic site was obtained using standard PCR conditions and AmpliTaq polymerase enzyme (Applied Biosystems, Foster City, CA). Digestion of the amplified product with the Hinfl restriction enzyme gave a 175-bp and a 154-bp product in the presence of the 677T allele, whereas no digestion occurred in the presence of the 677C allele. For genotyping of the RFC 80G/A polymorphism, amplification of the polymorphic region was performed using primers 5'-AGAAGCAGGTGCCCGTGGAA-3' (forward) and 5'-TGCGCCATGAAGCCGTAGAAG-3' (reverse), which produce a 94-bp fragment. PCR was performed in a 10-µL volume using 100 nmol/L for each primer and Hot Star Taq polymerase (Qiagen, Valencia, CA). Amplification products were sequenced in forward and reverse directions, analyzed on an ABI 3700 automated sequencer (Applied Biosystems), and genotyped using PolyPhred, version 4.05.26 Genotype distributions were found to be consistent with Hardy Weinberg equilibrium.
Statistical Analysis
Of the 53 patients who were enrolled onto the protocol, 23 were male, 38 were white, and 15 were nonwhite. The median age was 6 years (range, 0 to 18 years). The number of patients who received HDMTX (and who were, therefore, eligible for assessment of homocysteine) and the number of patients on study at the time points were as follows: 50 of 50 patients received consolidation 1; 47 of the 50 patients received consolidation 2 (one patient refused further therapy, and two had HDMTX withheld because of toxicity—one patient had seizures, and one had nonneurologic toxicity); and 39 of 45 patients received HDMTX at week 31 (two patients refused therapy, two had seizures, and two had other nonneurologic toxicity). All CSF and plasma samples obtained from patients were assayed for homocysteine if available.
Plasma and CSF Homocysteine Concentrations Before and After HDMTX Therapy
Plasma homocysteine concentration between patients who received 2.5 g/m2 of MTX versus 5.0 g/m2 did not differ (repeated measure analysis of variance [ANOVA] analysis based on values 7 days after consolidation 1 and at 23 hours and 44 hours after consolidation 2, P = .518). However, there was a weak positive correlation (r = 0.38, P = .0102) between the steady-state plasma MTX concentration (at 23 hours) after consolidation 2 and plasma homocysteine concentration at 44 hours (Fig 2 ).
Concurrent plasma and CSF homocysteine were measured at diagnosis, immediately before consolidation 1, immediately before consolidation 2, and at week 31. The only correlation between plasma and CSF homocysteine levels was found at week 31 of continuation therapy (r = 0.50, P = .0353). CSF homocysteine concentrations did not differ significantly among time points (Fig 3 ). CSF homocysteine concentration measured 7 days after HDMTX consolidation did not differ (P = .079) between patients who received 2.5 g/m2 of MTX (n = 19; median concentration, 0.204 µmol/L; range, 0.039 to 1.14 µmol/L) and patients who received 5.0 g/m2 of MTX (n = 26; median concentration, 0.108 µmol/L; range, 0 to 0.852 µmol/L).
Homocysteine Concentration and MTX-Induced Toxicity Neurotoxicity consisted of seizures. Nine patients experienced seizures; two of these patients experienced concurrent sagittal sinus thromboses. Three patients had seizures during induction treatment, and six patients had seizures during continuation treatment. None of the seizures were followed by long-term neurologic deficits. Six patients received additional HDMTX after their first seizure; four of these patients had no further seizures, whereas two patients did have a recurrent seizure, after which HDMTX was not given again. When the two patients whose seizures were concurrent with CNS thrombosis were included in the seizure group, the plasma homocysteine concentrations of patients who experienced seizures tended to be higher (P = .063, repeated measures ANOVA) than the concentrations of patients who did not have seizures (Fig 4 ), particularly when measured at a long interval from the last HDMTX plus leucovorin treatment (ie, immediately before consolidation 1 HDMTX, P = .0008). Findings were similar if these two patients were excluded from the analysis (P = .073, ANOVA). CSF homocysteine levels were not different between the patients who had seizures and those who did not (P = .68 and .65 for the comparisons in which the two patients who have concurrent thrombosis were included and excluded, respectively; repeated measures ANOVA). Five patients experienced thrombosis (three patients during induction and two during reinduction). We found no relationship between plasma or CSF homocysteine levels and the development of thrombosis (P = .59 and P = .55, respectively; repeated measures ANOVA).
MTHFR and RFC Genotypes The plasma and CSF homocysteine concentrations did not significantly differ by MTHFR 677C/T genotype (P = .99 and .29, respectively; repeated measures ANOVA) or by RFC 80G/A genotype (P = .36 and P = .69, respectively; Fig 5 ), although at diagnosis, plasma homocysteine tended to be higher among those heterozygous or homozygous for the MTHFR 677T allele. There was no evidence for a difference in the occurrence of seizures or of thromboses among the MTHFR genotypes (P = .31 for seizure and P = .47 for thrombosis, Table 3) or RFC genotypes (P = .77 for seizure and P = .70 for thrombosis, Table 4). However, the number of patients who had the MTHFR T/T genotype was small (n = 2), and therefore, the impact of this genotype was difficult to define. When patients were grouped by their combined MTHFR and RFC genotypes (MTHFR 677T alleles combined with RFC 80G alleles), there was also no trend for an association with the plasma or CSF homocysteine or with toxicity (data not shown).
Hyperhomocysteinemia has been associated with neurologic defects. Patients with severe MTHFR deficiency, which is accompanied by hyperhomocysteinuria, have severe neurologic deficits, seizure disorders, and mental retardation.14 Neurotoxicity, including seizures, encephalopathy, ataxia, or hemiparesis, has been linked to antifolate therapy, particularly in patients who receive systemic and intrathecal MTX therapy with little or no systemic leucovorin rescue.3,29 Because acute elevations in homocysteine have been reported after MTX therapy,30–32 it is reasonable to hypothesize that elevated homocysteine caused by MTX could be a biomarker for or etiologic in MTX-associated neurotoxicity among children with ALL. We did find a trend for overall higher plasma homocysteine levels among patients with seizures (P = .063), especially when measured several weeks after leucovorin rescue (P = .0008).
Homocysteine is converted to methionine by a reaction that uses 5-methyltetrahydrofolate (the active metabolite of leucovorin) as the methyl donor. MTX decreases intracellular pools of 5-methyltetrahydrofolate33 and can lead to an increase in homocysteine.10 After intravenous administration of 1.0 to 13.6 g of MTX to 7 adults over a 2- to 4-hour period, Refsum et al30 observed a modest increase in serum homocysteine that was attenuated with subsequent treatment. These findings were confirmed when 12 children with ALL received 8.0 g of MTX,31 when 16 patients with ALL were given a 24-hour infusion of 33.6 g,32 and when five patients received a 4-hour infusion of 8 g of MTX to treat osteogenic sarcoma.32 Each of these studies showed an acute increase in plasma homocysteine concentration within 24 hours after HDMTX treatment. Our study of a larger number of uniformly treated patients receiving contemporary ALL treatment supports an effect of HDMTX on plasma homocysteine and further indicates that the increase in homocysteine may be greater in those patients who developed neurotoxicity (Fig 4
We detected no significant difference in the plasma homocysteine levels at 1, 2, or 7 days after the 2.5-g/m2 versus 5.0-g/m2 dose of MTX with leucovorin rescue. Because leucovorin causes a rapid decrease in homocysteine concentrations in cell lines34 and in patients30,32 and because plasma homocysteine increased for several days after unrescued low-dose MTX,35 it is possible that our use of leucovorin after HDMTX may have confounded any dose-related effect of MTX on hyperhomocysteinemia, which might require several days or weeks of unrescued folate status to fully manifest itself. Moreover, interpatient variability in plasma MTX clearance resulted in some overlap of MTX exposure between the 2.5 g/m2 and 5 g/m2 doses, and in fact, examining concentration rather than dose did indicate a correlation between steady-state MTX exposure and homocysteine level (Fig 2
There are few published reports regarding CSF homocysteine concentration and MTX treatment. In the present study, in which CSF homocysteine was compared at identical times after therapy in patients who did and did not experience neurotoxicity, we found no higher CSF homocysteine in the patients with neurotoxicity. We acknowledge that we had no CSF (or plasma) for analysis in one patient at consolidation 2 and in two patients at week 31 because prior neurotoxicity precluded delivery of further HDMTX, and this may have complicated our chances of observing higher CSF homocysteine in patients with neurotoxicity. We cannot rule out that CSF homocysteine might have been higher at the time of seizure compared with the identical time after MTX therapy in children who did not develop neurotoxicity because it would not have been feasible to sample CSF at identical times in the cases and controls. Quinn et al11 hypothesized that CSF homocysteine concentration might be associated with MTX-related neurotoxicity; however, they did not have the opportunity to compare CSF concentrations at identical times after therapy in patients who did versus patients who did not exhibit neurotoxicity. In this study, we found no relationship between CSF homocysteine and MTX-related toxicity. Moreover, the CSF homocysteine concentration was not elevated 7 days after HDMTX plus leucovorin rescue in our prospective, serially studied cohort (Fig 3 Interestingly, plasma homocysteine before any antileukemic therapy was greater than at any time point after chemotherapy (as previously reported31) and was higher in patients who had standard/high-risk ALL than in patients with low-risk ALL (P = .0102). Plasma homocysteine concentration may be elevated in patients with a higher tumor burden and possible folate deficiency; it was also elevated in patients with a hyperproliferative disease such as psoriasis.35 Thus, the higher baseline homocysteine level before therapy (but not after therapy) among patients who had standard/high-risk ALL may have reflected their untreated disease burden, but by the end of a 6-week remission induction therapy, such baseline differences between risk groups were not present.
Accumulating data indicate that individuals who have the MTHFR T/T genotype have higher homocysteine, especially in those who have low folate status.17,19,20 Only two patients in the present study had a T/T MTHFR genotype, and although they tended to have higher plasma homocysteine, the small number of patients with this genotype limited our statistical power. At diagnosis (before the first HDMTX), the plasma homocysteine in patients heterozygous or homozygous for the 677T polymorphism tended to be higher than in patients with two MTHFR 677C alleles (Fig 5 In this analysis, we did not demonstrate a relationship between seizures or thromboses and MTHFR or RFC genotypes, which was consistent with a poor correlation of homocysteine with these genotypes. Associations between the MTHFR 677T genotype and gastrointestinal toxicity or hepatotoxicity have been demonstrated in patients receiving low doses of MTX without leucovorin rescue.21,22 It is possible that, in a setting in which leucovorin rescue is given, the effect of homocysteine on the risk of MTX toxicity (in this study, neurotoxicity) is attenuated. There are conflicting reports on associations between MTHFR genotype and the risk of thrombosis,38,39 and our data in this relatively small group of patients do not support such an association. In conclusion, our results showed that plasma homocysteine concentrations are elevated acutely after MTX administration and may be related to toxicity. In the setting of leucovorin rescue, neither MTHFR nor RFC genotype was related to homocysteine concentration or MTX-related neurotoxicity. Whether the acute increase in homocysteine can prove an informative biomarker for neurotoxicity requires additional testing with alternative regimens of MTX.
We thank Pam McGill and Nancy Duran for technical assistance; Wen Jian Yang and Nancy Kornegay for database management; our clinical staff; and the patients and their families for participating.
Supported by grant nos. CA 51001, CA 78224, and CA21765 from the National Cancer Institute and the National Institutes of Health/National Institute of General Medical Sciences Pharmacogenetics Research Network and Database grant nos. U01GM61393 and U01GM61374 from the National Institutes of Health, Bethesda, MD; by a Center of Excellence grant from the State of Tennessee; and by American Lebanese Syrian Associated Charities. C-H.P. is an American Cancer Society F.M. Kirby Clinical Research Professor.
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33. Fiskerstrand T, Ueland PM, Refsum H: Folate depletion induced by methotrexate affects methionine synthase activity and its susceptibility to inactivation by nitrous oxide. J Pharmacol Exp Ther 282:1305–1311, 1997 34. Ueland PM, Refsum H, Male R, et al: Disposition of endogenous homocysteine by mouse fibroblast C3H/10T1/2 Cl 8 and the chemically transformed C3H/10T1/2 MCA Cl 16 cells following methotrexate exposure. J Natl Cancer Inst 77:283–289, 1986[Medline] 35. Refsum H, Helland S, Ueland PM: Fasting plasma homocysteine as a sensitive parameter of antifolate effect: A study of psoriasis patients receiving low-dose methotrexate treatment. Clin Pharmacol Ther 46:510–520, 1989[Medline] 36. Thyss A, Milano G, Etienne MC, et al: Evidence for CSF accumulation of 5-methyltetrahydrofolate during repeated courses of methotrexate plus folinic acid rescue. Br J Cancer 59:627–630, 1989[Medline] 37. Kamen BA, Vietti T: Evidence for CSF accumulation of 5-methyltetrahydrofolate during repeated courses of methotrexate plus folinic acid rescue. Br J Cancer 60:799, 1989[Medline]
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39. Kluijtmans LA, Boers GH, Verbruggen B, et al: Homozygous cystathionine beta-synthase deficiency, combined with factor V Leiden or thermolabile methylenetetrahydrofolate reductase in the risk of venous thrombosis. Blood 91:2015–2018, 1998 Submitted July 9, 2002; accepted May 23, 2003. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 or Fisher’s exact test. 
) and did not have seizures (
). The numbers of patients are 8, 8, 6, 4, 4, and 3 in the seizure group and 36, 41, 41, 37, 40, and 32 in the nonseizure group at diagnosis, before consolidation 1, before consolidation 2, 23 hours and 44 hours after consolidation 2, and before high-dose methotrexate (week 31), respectively. Abbreviations: HDMTX, high-dose methotrexate; LV, leucovorin; ITMHA, intrathecal methotrexate, hydrocortisone, and cytarabine.
