title: Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia.
abstract: BACKGROUND
  Methotrexate (MTX) eradicates leukemic cells by disrupting de novo nucleotide biosynthesis and DNA replication, resulting in cell death. Since its introduction in 1947, MTX-containing chemotherapeutic regimens have proven instrumental in achieving curative effects in acute lymphoblastic leukemia (ALL). However, drug resistance phenomena pose major obstacles to efficacious ALL chemotherapy. Moreover, clinically relevant molecular mechanisms underlying chemoresistance remain largely obscure. Several alterations in MTX metabolism, leading to impaired accumulation of this cytotoxic agent in tumor cells, have been classified as determinants of MTX resistance. However, the relation between MTX resistance and long-term clinical outcome of ALL has not been shown previously.
  
  
  METHODS
  We have collected clinical data for 235 childhood ALL patients, for whom samples taken at the time of diagnosis were also broadly characterized with respect to MTX resistance. This included measurement of concentrations of MTX polyglutamates in leukemic cells, mRNA expression of enzymes involved in MTX metabolism (FPGS, FPGH, RFC, DHFR, and TS), MTX sensitivity as determined by the TS inhibition assay, and FPGS activity.
  
  
  RESULTS
  Herein we demonstrate that higher accumulation of long-chain polyglutamates of MTX is strongly associated with better overall (10-year OS: 90.6 vs 64.1%, P = 0.008) and event-free survival (10-year EFS: 81.2 vs 57.6%, P = 0.029) of ALL patients. In addition, we assessed both the association of several MTX resistance-related parameters determined in vitro with treatment outcome as well as clinical characteristics of pediatric ALL patients treated with MTX-containing combination chemotherapy. High MTX sensitivity was associated with DNA hyperdiploid ALL (P < 0.001), which was linked with increased MTX accumulation (P = 0.03) and elevated reduced folate carrier (RFC) expression (P = 0.049) in this subset of ALL patients. TEL-AML1 fusion was associated with increased MTX resistance (P = 0.023). Moreover, a low accumulation of MTX polyglutamates was observed in MLL-rearranged and TEL-AML1 rearranged ALL (P < 0.05).
  
  
  CONCLUSIONS
  These findings emphasize the central role of MTX in ALL treatment thereby expanding our understanding of the molecular basis of clinical differences in treatment response between ALL individuals. In particular, the identification of patients that are potentially resistant to MTX at diagnosis may allow for tailoring novel treatment strategies to individual leukemia patients.
authors.affiliation: Department of Pediatric Oncology/Hematology, VUmc Cancer Center Amsterdam, VU University Medical Center, Room CCA 4.28, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands.
authors.forename: Anna
authors.initials: A
authors.lastname: Wojtuszkiewicz
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.affiliation: Department of Pediatric Oncology/Hematology, VUmc Cancer Center Amsterdam, VU University Medical Center, Room CCA 4.28, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: van Woerden
authors.affiliation: Department of Pediatric Oncology/Hematology, VUmc Cancer Center Amsterdam, VU University Medical Center, Room CCA 4.28, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands.
authors.forename: Boas
authors.initials: B
authors.lastname: Dubbelman
authors.affiliation: Department of Pediatric Hematology/Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Escherich
authors.affiliation: Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark.|Department of Pediatrics and Adolescent Medicine, University Hospital Rigshospitalet, Copenhagen, Denmark.
authors.forename: Kjeld
authors.initials: K
authors.lastname: Schmiegelow
authors.affiliation: Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Sonneveld
authors.affiliation: Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands.|Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: Pieters
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Peter M
authors.initials: PM
authors.lastname: van de Ven
authors.affiliation: Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.affiliation: Department of Pediatric Oncology/Hematology, VUmc Cancer Center Amsterdam, VU University Medical Center, Room CCA 4.28, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands.
authors.forename: Gertjan J L
authors.initials: GJ
authors.lastname: Kaspers
authors.affiliation: Department of Pediatric Oncology/Hematology, VUmc Cancer Center Amsterdam, VU University Medical Center, Room CCA 4.28, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands. j.cloos@vumc.nl.|Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands. j.cloos@vumc.nl.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
mesh_terms: D000293:Adolescent; D002648:Child; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000627:therapeutic use; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000627:therapeutic use; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy*; D016896:Treatment Outcome
chemical_list: D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate