RAS mutation is associated with hyperdiploidy and parental characteristics in pediatric acute lymphoblastic leukemia JL Wiemels 1 , Y Zhang 1 , J Chang 2 , S Zheng 3 , C Metayer 2 , L Zhang 2 , MT Smith 2 , X Ma 4 , S Selvin 2 , PA Buffler 2 and JK Wiencke 3 1 Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, and UCSF Comprehensive Cancer Center, University of California, San Francisco, CA, USA; 2 School of Public Health, University of California, Berkeley, CA, USA; 3 Department of Neurological Surgery, University of California San Francisco, CA, USA; and 4 Department of Epidemiology and Public Health, Yale University, New Haven, CT, USA We explored the relationship of RAS gene mutations with epidemiologic and cytogenetic factors in a case series of children with leukemia. Diagnostic bone marrow samples from 191 incident leukemia cases from the Northern California Childhood Leukemia Study were typed for NRAS and KRAS codon 12 and 13 mutations. A total of 38 cases (20%) harbored RAS mutations. Among the 142 B-cell acute lymphoblastic leukemia (ALL) cases, RAS mutations were more common among Hispanic children (P ¼ 0.11) or children born to mothers o30 years (P ¼ 0.007). Those with hyperdiploidy at diagnosis (450 chromosomes) had the highest rates of RAS mutation (P ¼ 0.02). A multivariable model confirmed the significant associations between RAS mutation and both maternal age and hyperdiploidy. Interestingly, smoking of the father in the 3 months prior to pregnancy was reported less frequently among hyperdiploid leukemia patients than among those without hyperdiploidy (P ¼ 0.02). The data suggest that RAS and high hyperdiploidy may be cooperative genetic events to produce the leukemia subtype; and furthermore, that maternal age and paternal preconception smoking or other factors associated with these parameters are critical in the etiology of subtypes of childhood leukemia. Leukemia (2005) 19, 415–419. doi:10.1038/sj.leu.2403641 Published online 27 January 2005 Keywords: childhood leukemia; RAS; hyperdiploidy; smoking; maternal age Introduction The etiology of childhood leukemia is uncertain but may be different for distinct molecular subtypes of leukemia. A dominant-acting oncogene, RAS, is mutated in a large percen- tage of human tumors, in particular those with putative chemical causes such as lung and colon cancers, 1 and in chemically induced rodent tumors. 2 In leukemia, RAS mutations are historically correlated to pediatric and adult myeloid and less so to lymphoblastic subtypes. 3 RAS is not associated with prognostic outcome of leukemia in studies of childhood AML 4 and ALL, 5 but has been associated in etiology studies with occupational chemical exposures in adult AML. 6,7 The RAS genes are part of the small GTPase family and consist of three separate genes, NRAS, KRAS2, and HRAS. HRAS is rarely mutated in hematologic tumors and is expressed at a low level compared to the other two isoforms in leukemia and the hematopoietic cells from which they derive, 8 and hence is not further considered here. The three RAS genes code for proteins that are nearly identical except for the C-terminus, and recent work has shown that these differences lead to discrete subcellular locations of RAS proteins, and distinct interacting proteins including nucleotide exchange and GTPase-activating proteins (reviewed in Hingorani and Tuveson 9 and Ehrhardt et al 10 ). The RAS proteins activate several downstream pathways to promote proliferation, differentiation, survival, and apoptosis depending on cellular conditions. We assessed RAS mutation status in cases derived from the Northern California Childhood Leukemia Study (NCCLS) with the hypothesis that the RAS-mutation positive subgroup would be associated with exposures to chemicals, specifically carcino- gens from parental cigarette smoke. We also considered the relationship of RAS mutation with our most prevalent cytoge- netic subgroup – high hyperdiploid leukemia (those with 450 chromosomes) – and patient demographic characteristics. RAS mutations were unexpectedly linked to hyperdiploidy; and among hyperdiploid patients, a negative association with paternal smoking at the time of pregnancy was apparent. Materials and methods Study population Subjects were derived from the Northern California Childhood Leukemia Study (NCCLS). Included in this study were 191 incident cases of childhood leukemia (0–14 years), who were enrolled in from 1995 to 2000 and had cryopreserved pretreatment bone marrow aspirates obtained from the clinical center that first diagnosed the case. A detailed description of this approximately population-based study design can be found elsewhere, 11 as well as the subset used for this analysis. 12 Parental demographic characteristics and smoking information was provided by the case mother (97.5%) or father (2.5%) through in-person interviews in the home of the parents. Cytogenetics Patient diagnostic cytogenetics were subjected to a standardized review, supported by Fluorescence In Situ Hybridization (FISH) to help categorize TEL-AML1 translocations and cryptic high hyperdiploidy (hereafter referred to as ‘hyperdiploidy’). Hyper- diploidy is defined here as the presence of 50 or more chromosomes in diagnostic karyotypes (overt hyperdiploidy) or the concurrent present of more than two chromosomes 21 and X (identified by centromeric FISH) among cases where the karyotype failed or was unavailable (cryptic hyperdiploidy). The presence of extra chromosomes 21 and X distinguish 97% of hyperdiploid leukemia 13 and has been used elsewhere to indicate hyperdiploidy. 14 Received 28 September 2004; accepted 29 November 2004; Published online 27 January 2005 Correspondence: Dr J Wiemels, Laboratory for Molecular Epidemiol- ogy, University of California San Francisco, 500 Parnassus Ave, MU-420W, Box 0560, San Francisco, CA 94143, USA; Fax: þ 1 415 476-6014; E-mail: wiemels@itsa.ucsf.edu Leukemia (2005) 19, 415–419 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu