Kaufman Yotam, Drori Stavit, Cole Peter D, Kamen Barton A, Sirota Jenny, Ifergan Ilan, Arush Myriam Weyl Ben, Elhasid Ronit, Sahar Dvora, Kaspers Gert Jan L, Jansen Gerrit, Matherly Larry H, Rechavi Gideon, Toren Amos, Assaraf Yehuda G
Department of Biology, The Technion, Haifa, Israel.
Cancer. 2004 Feb 15;100(4):773-82. doi: 10.1002/cncr.20018.
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.
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%).
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.
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.
尽管大多数急性淋巴细胞白血病（ALL）患儿通过含甲氨蝶呤（MTX）的联合化疗得以治愈，但耐药性导致相当一部分患者治疗失败。叶酸和MTX的主要转运体是还原型叶酸载体（RFC）。药物转运受损是ALL患者MTX耐药的一种已被证实的机制；然而，据作者所知，尚不清楚失活的RFC突变是否为一个促成因素。
作者设计了一种基因组聚合酶链反应 - 单链构象多态性分析方法，随后进行测序，并对来自246份白血病标本（来自不同种族的患者）的DNA中的整个RFC编码区进行序列改变筛查，其中24份在复发时采集，其余在诊断时采集。该队列包括203份B前体ALL标本（82.5%）、32份T系ALL标本（13%）和11份急性髓细胞白血病标本（4.5%）。
在246份DNA样本中，仅3份诊断为B前体ALL的标本（1.2%）被发现RFC基因存在改变，包括杂合单核苷酸变化，分别导致该转运体第一个细胞外环和C末端出现D56H和D522N替代。第三个样本外显子3存在序列改变，但由于DNA无法获取而未能识别。
尽管失活的RFC突变是人类白血病细胞系和骨肉瘤患者中MTX耐药的常见机制，但在儿童白血病中它们并不常见，似乎在MTX的固有或获得性耐药中不起任何重要作用。这是对多个儿童白血病标本中RFC突变的首次研究。
