Abstract 1624: Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia
Authors: Wojtuszkiewicz Anna , Peters Godefridus J. , van Woerden Nicole L. , Dubbelman Boas , Escherich Gabrielle , Schmiegelow Kjeld , Sonneveld Edwin , Pieters Rob , van de Ven Peter M. , Jansen Gerrit , Assaraf Yehuda G. , Kaspers Gertjan J L , Cloos Jacqueline 
Published: August 2015

Abstract
Methotrexate (MTX) is an essential component of contemporary ALL treatment regimens. This antifolate eradicates leukemic cells by disrupting de novo biosynthesis of nucleotides, leading to inhibition of DNA replication and consequent cell death. Over the past decades, MTX-containing chemotherapy has proven to be crucial in achieving curation in ALL. However, drug resistance continues to be a major obstacle to curative ALL treatment. A number of alterations in MTX metabolism, resulting in impaired accumulation of this antifolate in cancer cells, have been identified as determinants of MTX resistance. However, it remains unclear which of these molecular mechanisms have clinical relevance. Therefore, the current study aimed at determining the relation between MTX resistance and long-term clinical outcome of ALL.
Towards this end, we collected the available clinical data of 235 childhood ALL patients, including clinical data with a follow-up time of 10 years. For these patients, samples obtained at the time of diagnosis were also characterized with respect to MTX resistance. This included determination of MTX polyglutamate levels in leukemic cells, mRNA expression of enzymes involved in (anti)folate metabolism and transport (i.e. FPGS, FPGH, DHFR, TS and RFC); MTX sensitivity was determined by the thymidylate synthase inhibition assay (TSIA) and in vitro FPGS activity assay was performed.
High levels of long-chain MTX polyglutamates was strongly associated with favorable long-term event-free survival (EFS, p = 0.029) and overall survival (OS, p = 0.008) of ALL patients (10-year EFS 81% and OS 91%, compared to EFS 58% and OS 64% in patients with low levels of this active metabolite). Similar relations were observed for the total accumulation of MTX polyglutamates and FPGS activity, which were associated with overall survival (p = 0.018 and p = 0.039, respectively). In the multivariate Cox regression model, including clinical variables, the level of long chain MTX polyglutamates showed a trend towards an association with event-free survival (p = 0.073) but not with overall survival (p = 0.465). In addition, we assessed the association between MTX resistance-related variables and the treatment outcome in patients with different cytogenetic alterations. High MTX sensitivity was associated with hyperdiploid ALL (P&lt;0.001), which was also associated with increased MTX accumulation (p = 0.03) and elevated RFC expression (p = 0.049). Moreover, cells characterized by TEL-AML1 fusion displayed elevated MTX resistance (p = 0.023) compared to samples devoid of this aberration, while MLL-rearrangements were associated with low accumulation of cellular MTX polyglutamates (p = 0.012). These findings emphasize the central role which MTX lays in ALL treatment thereby highlighting the necessity for further exploration of the molecular mechanisms underlying MTX resistance in ALL patients.
Financial support by Kika (Children cancer-free)
Citation Format: Anna Wojtuszkiewicz, Godefridus J. Peters, Nicole L. van Woerden, Boas Dubbelman, Gabrielle Escherich, Kjeld Schmiegelow, Edwin Sonneveld, Rob Pieters, Peter M. van de Ven, Gerrit Jansen, Yehuda G. Assaraf, Gertjan J L Kaspers, Jacqueline Cloos. Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1624. doi:10.1158/1538-7445.AM2015-1624