Prognostic Role of the Reduced Folate Carrier, the Major Membrane Transporter for Methotrexate, in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group

Abstract

Purpose:The value of measuring expression of individual genes relevant to particular chemotherapy drugs and encoding metabolizing enzymes, transporters, or drug targets, as predictors of treatment response and outcome in pediatric acute lymphoblastic leukemia (ALL), remains controversial. Experimental Design: In a case-control population of 91 pediatric B-precursor ALL patients [42 relapsed within 4 years (cases) and 49 did not relapse (controls)], we used real-time reverse transcription-PCR to measure transcript levels for 20 genes relevant to chemotherapy with the five major drugs used to treat this disease, including asparaginase, 6-mercaptopurine, methotrexate, prednisone, and vincristine. Results were confirmed in a separate case-control population of 26 patients. Results: Only the human reduced folate carrier (hRFC) gene, encoding the major membrane transporter for methotrexate, showed a significant difference in median transcript levels between the 42 cases and the 49 controls (P = 0.0278, Wilcoxon test). Using cutoffs for hRFC expression levels (based on Akaike information criterion), there were statistically significant associations between hRFC transcripts and treatment relapse (P = 0.0052). hRFC-B, corresponding to the major hRFC transcript form in ALL, was also measured by real-time reverse transcription-PCR and was prognostic. The association between treatment relapse and hRFC levels was validated in a separate study population of 14 cases and 12 controls from an earlier case-control study (P = 0.0221). Conclusions: Our results strongly suggest the prognostic importance of hRFC gene expression to treatment outcomes in pediatric ALL.They validate our previous studies of hRFC transcriptional regulation in pediatric ALL and provide further compelling evidence for the critical role for methotrexate in the successful treatment of this disease.

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

“…cDNAs were synthesized using random hexamers, RNase inhibitor, and MuLV reverse transcriptase and purified with the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA). Quantitative real-time RT-PCR was performed on a Roche LightCycler 1.2 (Roche Diagnostics, Indianapolis, IN) with gene-specific primers and FastStart DNA Master SYBR Green I Reaction Mix (Roche Diagnostics) (Ge et al, 2007). Primers are listed in Supplemental Table 3S.…”

Human reduced folate carrier: translation of basic biology to cancer etiology and therapy

“…This figure is from Hou et al [126] on chromosome 12 to chromosome 21 at the AML1 locus, the hRFC locus is translocated (B. Hukku and L. H. Matherly, unpublished). While accumulations of MTX polyglutamates were reported to be low in t(12;21) ALLs [140], no consistent hRFC expression pattern has yet to emerge in t(12;21) B-precursor ALL patients versus non t (12;21) ALLs [136,141].…”

“…Of course, upon cloning hRFC in 1995, it became possible to directly measure levels of gene expression in primary patient specimens by Northern blotting [131] and by quantitative RT-PCR [129,134] including real time RT-PCR [76,86,135,136]. A key assumption behind these expression-based assays is that relative transport by hRFC is reasonably proportional to levels of hRFC transcripts, a concept that has not always been tested.…”

“…In ALL, low levels of hRFC were likewise associated with a poorer prognosis [131,135]. In a very recent case-control study of 91 pediatric B-precursor ALL patients with real time RT-PCR to measure transcript levels of 20 genes (encoding proteins involved with transport, metabolism, therapeutic targets, or apoptosis signaling and considered to be associated with success or failure of chemotherapy with the major drugs used to treat ALL), only hRFC was significantly different between cases (failed within 4 years) and controls (did not fail) (increased median of ∼2-fold in cases over controls; p<0.03) [136].…”

Chapter 5 Structure and Function of the Reduced Folate Carrier

“…In 42 primary osteosarcoma samples from patients who experienced poor responses to chemotherapy including MTX, 65% showed low level RFC expression (Guo et al, 1999). Similarly, in primary acute lymphoblastic leukemia, low levels of RFC were associated with a poor prognosis (Gorlick et al, 1997;Levy et al, 2003;Ge et al, 2007).…”