Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia.
BACKGROUND
Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with combination chemotherapy containing methotrexate (MTX), drug resistance contributes to treatment failure for a substantial fraction of patients. The primary transporter for folates and MTX is the reduced folate carrier (RFC). Impaired drug transport is a documented mechanism of MTX resistance in patients with ALL; however, to the authors' knowledge it is not known whether inactivating RFC mutations are a contributing factor.


METHODS
The authors devised a genomic polymerase chain reaction-single strand conformational polymorphism assay followed by sequencing and screened the entire RFC coding region for sequence alterations in DNA from 246 leukemia specimens from patients with diverse ethnic variation, 24 at the time of recurrence and the rest at the time of diagnosis. This cohort was comprised of 203 B-precursor ALL specimens (82.5%), 32 T-lineage ALL specimens (13%), and 11 acute myeloblastic leukemia specimens (4.5%).


RESULTS
Of 246 DNA samples, only 3 diagnosis B-precursor ALL specimens (1.2%) were found to harbor alterations in the RFC gene, including heterozygous single nucleotide changes resulting in D56H and D522N substitutions in the first extracellular loop and the C-terminus of this transporter, respectively. The third sample had a sequence alteration in exon 3 that could not be identified because of the lack of availability of DNA.


CONCLUSIONS
Whereas inactivating RFC mutations are a frequent mechanism of MTX resistance in human leukemia cell lines and in patients with osteosarcoma, they are not common and do not appear to play any significant role in intrinsic or acquired resistance to MTX in childhood leukemia. This is the first study of RFC mutations in multiple pediatric leukemia specimens.
Kaufman
Yotam
Y
Department of Biology, The Technion, Haifa, Israel.
Drori
Stavit
S
Cole
Peter D
PD
Kamen
Barton A
BA
Sirota
Jenny
J
Ifergan
Ilan
I
Arush
Myriam Weyl Ben
MW
Elhasid
Ronit
R
Sahar
Dvora
D
Kaspers
Gert Jan L
GJ
Jansen
Gerrit
G
Matherly
Larry H
LH
Rechavi
Gideon
G
Toren
Amos
A
Assaraf
Yehuda G
YG
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000494:pharmacology*; D002648:Child; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm; D006801:Humans; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology*; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000235:genetics*; D058978:Reduced Folate Carrier Protein; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008727:Methotrexate