Imaging, Diagnosis, Prognosis DNA Variants in Region for Noncoding Interfering Transcript of Dihydrofolate Reductase Gene and Outcome in Childhood Acute Lymphoblastic Leukemia Fidaa Al-Shakfa, 1,3 Stéphanie Dulucq, 1 Ivan Brukner, 1 Iva Milacic, 1 Marc Ansari, 1 Patrick Beaulieu, 1 Albert Moghrabi, 1,2 Caroline Laverdière, 1,2 Stephen E. Sallan, 4,5 Lewis B. Silverman, 4,5 Donna Neuberg, 4 Jeffery L. Kutok, 6 Daniel Sinnett, 1,2 and Maja Krajinovic 1,2,3 Abstract Purpose: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A -317 and C -1610 alleles. Haplotype *1 was also associated higher DHFR expression. Experimental Design: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript. Results: Six polymorphisms were identified, of which five were single nucleotide poly- morphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter poly- morphisms revealed diversification of haplotype *1 into five subtypes ( *1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G 308 A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels. Conclusions: The study provides a new insight into DHFR regulatory variations predis- posing to an event in ALL patients. (Clin Cancer Res 2009;15(22):6931–8) Methotrexate, a folic acid antagonist, is an important compo- nentofthetreatmentofacutelymphoblasticleukemia(ALL).A major mechanism of methotrexate action involves competitive inhibition of the dihydrofolate reductase (DHFR; refs. 1, 2). In both experimental and clinical settings, altered levels of DHFR are found in relapsed patients and in cells manifesting metho- trexate-resistant phenotype (3–5), suggesting that DHFR can play an important role in development of methotrexate resis- tance. Changes in the level of DHFR expression and conse- quently in the sensitivity to methotrexate can also be due to genetic polymorphisms, particularly those located in the regu- latoryelements.Werecentlyanalyzedpolymorphismsin DHFR gene located in the region 2 kb upstream of the first transcrip- tion initiation site (6). Association of these polymorphisms with treatment responses in children with ALL showed that a reduction in event-free survival (EFS) was associated with A -317 and C -1610 alleles and with the haplotype *1, harboring these alleles. The haplotype *1 conferred higher transcriptional activityasshownbyreportergeneassayandquantitativemRNA analysis (6). In addition to this regulatory region that precedes the first or minor transcript initiation site, human DHFR gene has al- so a downstream major promoter controlling the transcrip- tion of productive mRNA at the major transcript initiation Authors' Affiliations: 1 Research Center, CHU Sainte-Justine; Departments of 2 Pediatrics and 3 Pharmacology, University of Montreal, Montreal, Quebec, Canada and 4 Department of Pediatric Oncology, Biostatistics and Computational Biology, Dana-Farber Cancer Institute; 5 Division of Hematology/Oncology, Children's Hospital; and 6 Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts Received 3/13/09; revised 7/31/09; accepted 8/7/09; published OnlineFirst 10/27/09. Grant support: Canadian Institutes of Health Research, Leukemia Lympho- ma Society of Canada, Research Center of CHU Sainte Justine, Charles Bru- neau Foundation, and Centre d'excellence en Oncologie pédiatrique et en soins palliatifs. Dana-Farber Cancer Institute ALL treatment protocols are supported by the National Cancer Institute/NIH grant P01 CA 68484. M. Krajinovic and D. Sinnett are scholars of the Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment ofpagecharges.Thisarticlemustthereforebeherebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: MajaKrajinovic,ResearchCenter,CHUSainte-Justine, 3175 chemin de la Côte-Ste-Catherine, Montreal, Quebec, Canada H3T 1C5. Phone: 514-345-4931, ext. 6259; Fax: 514-345-4731; E-mail: maja. krajinovic@umontreal.ca. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0641 6931 Clin Cancer Res 2009;15(22) November 15, 2009 www.aacrjournals.org