Treatment of Cancer-Childhood Leukemia Cooperative Group. N .... Supported by The Children's Research Center of Michigan, Art ... Karmanos Cancer Institute.
Correspondence
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likely due to deterioration of the samples over years. Results were similar when using a 5 × 10−3 threshold and lower thresholds can of course be used in the same way, provided that the sensitivity is sufficient. This technique has been successfully exported to several laboratories and has now been currently used for routine MRD detection in more than 500 patients. The rapid recognition of patients with a very bad prognosis is a major goal, because these patients display a very high risk of early relapse and alternative therapeutics have probably to be applied as soon as possible to be efficient. So far, the technique has enabled results to be known within 2 to 15 days of sample reception in virtually all patients displaying a TCR or IGH rearrangement, giving evidence of its feasibility. C Guidal1 E Vilmer2 B Grandchamp1,3 H Cave´1
1
Laboratoire de Biochimie Ge´ne´tique, Hoˆpital Robert Debre´, Paris, France; Service d’He´matologie, Hoˆpital Robert Debre´, Paris, France; and 3INSERM U409, Faculte´ de Me´decine Xavier Bichat, Paris, France 2
References 1 Cave´ H, van der Werff ten Bosch J, Suciu S, Guidal C, Waterkeyn C, Otten J, Bakkus M, Thielemans K, Grandchamp B, Vilmer E. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer-Childhood Leukemia Cooperative Group. N Engl J Med 1998; 339: 591–598.
2 van Dongen JJ, Seriu T, Panzer-Grumayer ER, Biondi A, PongersWillemse MJ, Corral L, Stolz F, Schrappe M, Masera G, Kamps WA, Gadner H, van Wering ER, Ludwig WD, Basso G, de Bruijn MA, Cazzaniga G, Hettinger K, van der Does-van den Berg A, Hop WC, Riehm H, Bartram CR. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998; 28: 1731–1738 3 Elsworth AM, Evans PAS, Morgan GJ, Kinsey SE, Shiach CR. Quantitative PCR of the immunoglobulin heavy chain gene using genomic DNA. Br J Haematol 1996; 95: 700–703. 4 Owen RG, Goulden NJ, Oackhill A, Shiach C, Evans PA, Potter MN. Comparison of fluorescent consensus IgH PCR and allele-specific oligonucleotide probing in the detection of minimal residual disease in childhood ALL. Br J Haematol 1997; 97: 457–459. 5 Cave´ H, Guidal C, Rohrlich P, Delfau MH, Broyard A, Lescoeur B, Rahimy C, Fenneteau O, Monplaisir N, d’Auriol L, Vilmer E, Grandchamp B. Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of ␦ and ␥ T-cell receptor genes. Blood 1994; 83: 1892–1902. 6 Trainor KJ, Brisco MJ, Wan JH, Neoh S, Grist S, Morley AA. Gene rearrangement in B- and T- lymphoproliferative disease detected by the polymerase chain reaction. Blood 1991; 78: 192–196. 7 Szczepanski T, Langerak AW, Wolvers-Tettero IL, Ossenkoppele GJ, Verhoef G, Stul M, Petersen EJ, de Bruijn MA, van’t Veer MB, van Dongen JJ. Immunoglobulin and T cell receptor gene rearrangement patterns in acute lymphoblastic leukemia are less mature in adults than in children: implications for selection of PCR targets for detection of minimal residual disease. Leukemia 1998; 12: 1081– 1088
Polymorphisms in methylenetetrahydrofolate reductase and methotrexate sensitivity in childhood acute lymphoblastic leukemia Leukemia (2002) 16, 764–765. DOI: 10.1038/sj/leu/2402428 TO THE EDITOR In view of the importance of methotrexate (MTX) therapy in the treatment of childhood ALL particularly during the consolidation and maintenance phases of treatment, identifying parameters associated with MTX sensitivity and resistance have direct relevance to the treatment of patients with ALL. Pharmacologic and biologic parameters which correlate with clinical MTX sensitivity and resistance in the treatment of ALL include: (1) altered intracellular MTX transport via the reduced folate carrier (RFC); (2) generation of long-chain MTXpolyglutamates catalyzed by folypolyglutamate synthase (FPGS); (3) levels of dihydrofolate reductase (DHFR), the primary target of MTX inhibition; and (4) the relationship of MTX sensitivity and leukemia cytogenetics including hyperdiploidy.1–6 It is conceivable that additional factors may also be determinants of MTX response. The main mechanism of action of MTX as an anti-folate agent, results in the depletion of metabolically active pools of tetrahydrofolate cofactors with a suppression of folate-dependent synthesis of DNA, RNA and protein and thus, it is conceivable that alterations in reduced folate pools may lead to altered MTX sensitivity. Recently, polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) (gene localized to 1p36.3), an enzyme which catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the main circulating form of reduce folates, have been identified. Reduced activity of the MTHFR C677T allele (with the resultant substitution of valine for alanine), in both the heterozygous and homozygous state has been linked to hyperhomocysteinemia due to folate pool imbalances.7 Correspondence: JW Taub, Children’s Hospital of Michigan, 3901 Beaubien Blvd, Detroit, MI, 4820, USA; Fax: 313 745–5237 Received 24 September 2001; accepted 17 December 2001 Leukemia
In this pilot study, we determined if there was a relationship between the MTHFR C677T genotype and in vitro MTX sensitivity in pediatric ALL patients. Cryopreserved lymphoblasts were available from six patients with ALL treated at Children’s Hospital of Michigan (CHM) who had suffered from MTX-related neurotoxicity (eg seizures) or hepatotoxicity (eg persistent elevation in transaminase levels) and the MTHFR gene status had been analyzed to assess if there was a relationship with MTX toxicity. Increased treatment-related toxicity has been reported in breast cancer patients homozygous for the MTHFR C677T allele treated with combined cyclophosphamide, MTX, fluorouracil therapy and bone marrow transplant patients who received MTX for graft-versus-host disease prophylaxis.8,9 MTHFR genotype for the C677T allele was determined by PCR amplification of DNA extracted from peripheral blood lymphocytes obtained from the patients in remission. Following PCR amplification of the MTHFR gene from genomic DNA, the PCR products were digested with the restriction enzyme HinfI with the DNA fragments size separated on a agarose gel. MTX in vitro sensitivity was determined by an in situ thymidylate synthase inhibition assay with (1) short-term 3 h exposure to MTX followed by an 18 h drug-free period and (2) continuous 21 h exposure to MTX. This assay measures the conversion of 3H-dUMP to dTMP and 3H2O with 5-3H-2⬘-deoxycytidine as a precursor for dUMP.1 In this group of six ALL patients, the two patients homozygous for the MTHFR C677T allele (T/T), exhibited greater MTX sensitivity compared to patients with the wild-type (C/C) or heterozygous (T/C) genotypes and were classified as high risk by the NCI leukemia classification (Table 1). Of note, none of the patients studied had cytogenetic evidence of hyperdiploidy, a group which is known to have a favorable response to MTX. One of the two samples with the C/C genotype demonstrated greater MTX sensitivity compared to the samples with the heterozygous (T/C) genotype; this sample may have exhibited other parameters which contribute to MTX sensitivity including increased RFC expression and/or low DHFR levels. These preliminary results suggest that the relationship of MTX sensitivity and
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Table 1
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Relationship of in vitro methotrexate sensitivity and MTHFR gene polymorphism
Patient No.
Age
WBC (/l)
MTHFR (C677T)
MTX TSI50 short (M)
MTX TSI50 long (M)
1 2 3 4 5 6
5 11 22 m 19 m 3 14
57,900 300,000 33,200 43,000 62,000 13,200
T/T T/T C/T C/T C/C C/C
0.18 0.25 1.43 3.46 4.70 0.98
0.048 0.099 0.545 0.264 0.615 0.183
Cytogenetics
46,XX [9]/49,XX,+X,del(6)(q21q25),+15,+21 [11] 45,X,t(X;9)(p21;p13),t(5;12)(p15;p13),−7 [10] 46,XY [6]/46,XY,del(20)(p12) [9] 46,XY [3]/46,XY,t(9;17)(p22;q25) [14] 46,XY [7]/47,XY,add(17)(p11),add(18)(q11),+19 [10] 46,XY [6]/46,XY,add(14)(q24) [10]
TSI50 is the concentration of MTX that inhibits 50% of the activity of thymidylate synthase as compared to the controls. Short, 3 h exposure followed by an 18 h drug-free incubation; long, continuous 21 h exposure to MTX. MTHFR genotype should be tested in a large-scale study and may provide additional insights into parameters accounting for MTX responsiveness clinically. It is possible that additional polymorphisms involving MTHFR (A1298C) or other genes (cystathionine--synthase, methionine synthase) which interact with the reduced folate pathway, may be associated with altered MTX cytotoxicity.
Acknowledgements Supported by The Children’s Research Center of Michigan, Art Gagnon Memorial Fund, BPCT Golf Charity, and Leukemia Research, Life, Inc. (Detroit, MI). 1 JW Taub1 Children’s Hospital of Michigan 1 Karmanos Cancer Institute LH Matherly Y Ravindranath1 Wayne State University School of Medicine Detroit, MI, USA G-JL Kaspers2 2 University Hospital MG Rots2 Vrije Universiteit, Amsterdam CH van Zantwijk2 The Netherlands
References 1 Rots MG, Pieters R, Kaspers GJL, van Zantwijk CH, Noordhuis P, Mauritz R, Veerman AJP, Jansen G, Peters GJ. Differential methotrexate resistance in childhood T-versus common preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay. Blood 1999; 93: 1067–1074.
2 Matherly LH, Taub JW. Molecular and cellular correlates of methotrexate response in childhood acute lymphoblastic leukemia. Leuk Lymphoma 1999; 35:1–20. 3 Rots MG, Pieters R, Peters GJ, Noordhuis P, van Zantwijk CH, Henze G, Janka-Schaub GE, Veerman AJP, Jansen G. Methotrexate resistance in relapsed childhood acute lymphoblastic leukemia. Br J Haematol 2000; 109: 629–634. 4 Matherly LH, Taub JW, Wong SC, Ekizian R, Buck S, Simpson P, Amylon M, Pullen J, Camitta B, Ravindranath Y. Increased frequency of expression of elevated dihydrofolate reductase in T-cell versus B-precursor acute lymphoblastic leukemia in children. Blood 1997; 90: 578–589. 5 Matherly LH, Taub JW, Ravindranath Y, Proefke SA, Wong SC, Gimotty P, Buck S, Wright JE, Rosowsky A. Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphoblastic leukemia. Blood 1995; 85: 500–509. 6 Zhang L, Taub JW, Williamson M, Wong SC, Hukku B, Pullen J, Ravindranath Y, Matherly LH. Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy. Clin Cancer Res 1998; 4: 2169–2177. 7 Bailey LB, Gregory JF. Polymorphisms of methylenetetrahydrofolate reductase and other enzymes: metabolic significance, risks and impact on folate requirement. J Nutr 1999; 129: 919–922. 8 Toffoli G, Veronesi A, Boiocchi M, Crivellari D. MTHFR gene polymorphism and severe toxicity during adjuvant treatment of early breast cancer with cyclophosphamide, methotrexate, and fluorouracil (CMF). Ann Oncol 2000; 11: 373–374. 9 Ulrich CM, Yasui Y, Storb R, Schubert MM, Wagner JL, Bigler J, Ariail KS, Keener CL, Li S, Liu H, Farin FM, Potter JD. Pharmacogenetics of methotrexate: toxicity among marrow transplantation patients varies with the methylenetetrahydrofolate reductase C677T polymorphism. Blood 2001; 98: 231–234.
Leukemia
