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Leukemia (2005) 19, 415419 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu RAS mutation is associated with hyperdiploidy and parental characteristics in pediatric acute lymphoblastic leukemia JL Wiemels1, Y Zhang1, J Chang2, S Zheng3, C Metayer2, L Zhang2, MT Smith2, X Ma4, S Selvin2, PA Buffler2 and JK Wiencke3 1Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, and UCSF Comprehensive Cancer Center, University of California, San Francisco, CA, USA; 2School of Public Health, University of California, Berkeley, CA, USA; 3Department of Neurological Surgery, University of California San Francisco, CA, USA; and 4Department 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 0.11) or children born to mothers o30 years (P 0.007). Those with hyperdiploidy at diagnosis (450 chromosomes) had the highest rates of RAS mutation (P 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 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, 415419. 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 percentage 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 AML4 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 Correspondence: Dr J Wiemels, Laboratory for Molecular Epidemiology, University of California San Francisco, 500 Parnassus Ave, MU-420W, Box 0560, San Francisco, CA 94143, USA; Fax: 1 415 476-6014; E-mail: wiemels@itsa.ucsf.edu Received 28 September 2004; accepted 29 November 2004; Published online 27 January 2005 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 Tuveson9 and Ehrhardt et al10). 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 carcinogens from parental cigarette smoke. We also considered the relationship of RAS mutation with our most prevalent cytogenetic 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 (014 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'). Hyperdiploidy 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 leukemia13 and has been used elsewhere to indicate hyperdiploidy.14 RAS and hyperdiploidy in childhood ALL JL Wiemels et al 416 RAS characterization to other racial/ethnic groups (Table 1). In our case series, 36% of fathers smoked in 3 months prior to pregnancy, and 12% of Screening for RAS mutations was performed by laboratory mothers smoked during pregnancy. personnel on coded samples. Laboratory personnel at UC San RAS mutations were present in 20% (38/191) of all leukemias Francisco did not have access to epidemiologic variables or including 17 KRAS, 18 NRAS, and three with both K and NRAS, cytogenetic status, information which was kept at UC Berkeley. and were similarly prevalent in AML and ALL (21 vs 16%, DNA was isolated using the QIAamp DNA Blood Mini Kit P 0.49). The presence of RAS was similar in B- and T-lineage (Qiagen) from 5 ml bone marrow sample. leukemias (20 and 29%, respectively). Subsequent analyses A Restriction Endonuclease-Mediated Selective (REMS)-PCR were confined to the B-cell leukemia subgroup since it was the Screen was first used to identify RAS mutations, essentially as described.15 REMS-PCR is a two-step nest PCR, the second PCR largest homogeneous group. A higher proportion of RAS mutant bone marrows were observed among the hyperdiploid B-cell being subjected to a selective restriction endonuclease digestion ALL group compared to other B-cell subtypes (P 0.02, Table 1). of the normal wild-type sequence to allow for detection of the Maternal age was inversely associated with RAS mutation mutant. Primers and specific methods are available from the (P 0.007, Table 1). authors by request. In order to detect associations between variables while In order to confirm the results obtained by REMS-PCR, adjusting for other covariates, a multivariable log-linear analysis oligonucleotides were synthesized specific for each mutation, was performed with ALL cases (Table 2). This model includes nonradioactively labeled, and used on dot blots of the first round PCR reaction essentially as previously described.16 Finally, all the seven variables of interest as main effects as well as in interaction with each other, in categorization of 157 ALL cases. mutations were confirmed by Sanger DNA sequencing (ABI 377, In the multivariable model, RAS mutation remained significantly Foster City, CA, USA). associated with maternal age (P 0.01) and hyperdiploidy (P 0.03). In addition, paternal smoking 3 months prior to pregnancy was significantly inversely associated with hyperdi- Statistical analysis ploidy among ALL cases (7/42, or 17% among hyperdiploids vs Pearson's w2 tests or Fisher's exact tests (when 25% of the cells had expected counts o5) were used to compare the distribution 29/63, or 46% among nonhyperdiploids, P 0.002 in univariable analysis, and P 0.02 in the multivariable model log-linear model, Table 2). of RAS mutation by childhood leukemia subtypes, child's demographics, and parental characteristics. Multivariable ana- lysis was performed using log-linear regression to assess the Discussion associations between seven variables (presence of RAS muta- tion, presence of hyperdiploidy, paternal smoking 3 months This is the first study to consider RAS mutation along with before pregnancy, maternal smoking during pregnancy, mater- cytogenetic subtypes of pediatric ALL and epidemiologically nal age, child's race/ethnicity, and income) (see Table 2). The derived variables in a series of leukemias. We report the variable `income' was included in this model since it has been association of mutations within the RAS oncogene (N and K loci) found to be associated with leukemia risk in previous analyses.11,17 Although log-linear model and logistic model with mother's age at time of the child's birth, and hyperdiploidy (Table 1). We also report an unexpected inverse association are generally similar, log-linear model is more appropriate for between paternal smoking and hyperdiploidy. These associa- this analysis because it does not require an outcome variable. tions are not likely to be explained by bias, since patient families Log-linear model was chosen for the analysis because it is not were not aware of their child's RAS mutation status. Likewise, clear whether the hyperdiploidy is an antecedent event to the laboratory personnel were blinded to the subjects' cytogenetic RAS mutation. Application of log-linear model is an extension of the Pearson's w2-test used to assess association in a two- status and epidemiologic information. Lastly, all subjects were cases, and all subjects would share any interview or response dimensional (row by column) contingency table and it allows bias introduced by case status. evaluation of associations in a multidimensional contingency Childhood leukemia like other cancers is thought to be a table. A log-linear model consists of two parts, the additive part multistep process in which two or more mutations occur at with the main effect terms and the part with the measures of association terms (interaction terms). The statistical significance different periods in development of the child as well as the ontological development of the blood cell.1820 Hyperdiploidy of each interaction term was assessed by a P-value generated is the gain of a number of extra chromosomes (522 more than from the log-likelihood ratio test comparing the sub-model without the measure of association (interactive) term to the full the diploid 46) which is thought to occur in a single catastrophic mitosis,21 and in some cases appears to be a prenatal event.22,23 model including the measures of association. This process The association of RAS mutations with maternal age suggests identifies the degree of pairwise associations between the RAS that RAS mutation may also be a prenatal event, and indicates mutation and hyperdiploidy and five other variables that make that an examination of archived neonatal blood samples for RAS up the analysis (Table 2). mutations in children with RAS-mutation positive leukemia could be informative. A recent animal model suggests that RAS Results may operate as an initiating or a second event in a two-hit disease;24,25 our data suggest that hyperdiploidy may represent a complementary genetic event in leukemias with RAS mutation. A total of 157 cases were diagnosed with acute lymphoblastic leukemia (ALL), 32 with acute myeloid leukemia (AML), and Recent evidence that FLT3 mutations may also be such a complementary event in hyperdiploid leukemia26,27 is compa- two with chronic myeloid leukemia (CML). The mean and tible with the current results, as FLT3 signals in part through the median ages of the cases were 6.1 and 5.0 years, respectively RAS pathway. Furthermore, mutations in another RAS-pathway (range: 0.214.9 years). In all, 49% of cases were non-Hispanic gene, PTPN11, are genetically restricted to leukemias that do (NH) White, 33% were Hispanic, and the remaining belonged not have RAS mutations, and additionally are found in the TEL- Leukemia RAS and hyperdiploidy in childhood ALL JL Wiemels et al Table 1 RAS mutations in pediatric B-cell acute lymphoblastic leukemia, and relationships to patient and parental characteristics in the NCCLS study RAS mutation P-valuea Childhood leukemia (n 191)b Childhood leukemia phenotypesc ALL (n 157) AML (n 32) Yes (%) 38 (20) 33 (21) 5 (16) No (%) 153 (80) 124 (79) 27 (84) F 0.49 ALL subtypes B lineage (n 142) T lineage (n 14) 29 (20) 113 (80) 4 (29) 10 (71) 0.48 B-lineage ALL cytogenetic subtypesd Nonhyperdiploid B cell (n 86) 12 (14) Hyperdiploid B cell (n 56) 17 (30) 74 (86) 39 (70) 0.02 Genderd Female (n 69) Male (n 73) 10 (14) 19 (26) 59 (86) 54 (74) 0.09 Race/ethnicityd Hispanic (n 48) Non-Hispanic White (n 65) Other (n 23) 13 (27) 8 (12) 6 (26) 35 (73) 57 (88) 17 (74) 0.11 Father's aged o30 (n 58) X30 (n 75) 15 (26) 12 (16) 43 (74) 63 (84) 0.16e Mother's aged o30 (n 73) X30 (n 61) 21 (29) 6 (10) 52 (71) 55 (90) 0.007e Incomed o$30 000 (n 43) $30 000$75 000 (n 56) 4$75 000 (n 35) 11 (26) 11 (20) 5 (14) 32 (74) 45 (80) 30 (86) 0.46f Father smoked 3 months prior to pregnancyd Yes 4 (12) No 16 (25) 29 (88) 47 (75) 0.13 Mother smoked 3 months prior to pregnancyd Yes 4 (19) No 23 (20) 17 (81) 90 (80) 0.89 Mother smoked during pregnancyd Yes 2 (15) No 25 (21) 11 (85) 96 (79) 0.65 aP-value derived from w2-test. bThree cases had both KRAS and NRAS mutations. cThe two CML cases included in the study did not have RAS mutations. dThese included 152 ALL cases for whom the diagnosing hospital or the UC Berkeley performed FISH screening for t(12;21) and hyperdiploidy AND for whom the t(12;21) and hyperdiploidy informa- tion was captured from the hospital clinical cytogenetics report. eP-value for trend when father's age treated as a continuous variable 0.40; P-value for trend for mother's age 0.02. fP-value for trend when treated as a six-level ordinal variable 0.20. AML1-negative common ALL subgroup, particularly those with hyerpdiploidy,28 the same subgroup we have found an excess of RAS mutations. Like PTPN11, FLT3 mutations were shown to be genetically restricted to pediatric (myeloid) leukemias without RAS mutations.29 Future work will need to determine whether mutations in RAS and FLT3 are exclusive to each other in childhood ALL. We did not detect a higher prevalence of parental smoking among RAS-mutation positive cases compared to RAS-mutation negative cases or case subtypes (Table 1), perhaps due to limited power. Previous studies suggest that RAS mutations may be associated with chemical exposures. Two epidemiologic studies have linked RAS mutation in adult myeloid leukemia with `high risk' occupations for leukemogenesis.6,7 Another casecase pediatric leukemia study (like the current one) suggested a role for parental hydrocarbon exposures including some specific for the father for leukemias with RAS mutations compared to those without.30 In addition, mutagenic chemicals from maternal smoking cross the placenta enhancing the plausibility of an effect of parental smoking on pediatric leukemia risk.31 Our analysis showed a significant association between paternal smoking and hyperdiploid leukemia, in the inverse direction, when compared to other leukemia subtypes (see Results). Because parental smoking was not significantly associated with RAS mutation-positive leukemia overall (Table 1), this suggests that another molecular subtype of leukemia may be positively associated with paternal smoking. Future studies should strive to assess the role of parental smoking and other hydrocarbons in pediatric leukemia among the various key tumor genetics subtypes (eg TEL-AML1, MLL, hyperdiploidy, and RAS). It should be noted that the current study did not include population-based controls and therefore population risks were not assessed. The unexpected significant inverse association with preconception paternal smoking and hyperdiploid leukemia is unprecedented. This relationship was, however, not seen for maternal smoking, possibly since the prevalence of maternal smoking at the time of pregnancy was far lower (37% paternal, 12% maternal). No risk factors have been associated with hyperdiploidy apart from age of the child in past studies. The apparent lower prevalence of preconception paternal smoking among hyperdiploid cases needs to be further assessed by examining leukemia risk in a casecontrol analysis. We cannot exclude the possibility that smoking associated mutagens may be toxic to hyperdiploid clones, which have been shown to be especially sensitive to therapeutic chemical agents albeit those associated with poisoning the folate metabolic pathway.32,33 RAS mutation was more than twice as frequent among Hispanics (28%) compared to non-Hispanic whites (13%). Also, maternal age was clearly associated with incidence of RAS mutation (Tables 1 and 2). However, when both of these factors were included in the same model, ethnicity was not a significant factor but maternal age remained significant (Table 2), suggesting that ethnic identity may be confounded with maternal age which is the true associated factor. Maternal age at child's birth is not an established risk factor in leukemia, but a slightly increased risk in children born from older mothers has been observed.34 The association with maternal age in the current study, that is, higher prevalence of RAS-mutant positive leukemias in children born of younger mothers, may be a reflection of an increased prevalence of other genetic subtypes in children born of older mothers. Furthermore, maternal age may be confounded with another causal variable, which should be considered in future studies. The current study raises many questions and highlights the potential interactive role of RAS mutation, maternal age, ethnicity, cytogenetics, and parental smoking. The significant associations found here should guide the design of future etiology studies, which have at their heart a goal of teasing out the causal pathway to childhood leukemia, and emphasize the 417 Leukemia RAS and hyperdiploidy in childhood ALL JL Wiemels et al 418 Table 2 Multivariable log-linear model and selective associations: B-cell acute lymphoblastic leukemia cases in the NCCLS (n 152) Variables included in the model:a Var1 FSMpre; Var2 MSMpreg; Var3 MomAgeo30; Var4 Hyperdip; Var5 RAS; Var6 Race; Var7 Income Full log-linear model:a Logcount a b1Var1 . . . b7Var7 c1Var1 Var2 c2Var1 Var3 . . . c6Var1 Var7 d1Var2 Var3 d2Var2 Var4 . . . d5Var2 Var7 e1Var3 Var4 e2Var3 Var5 . . . e4Var3 Var7 f1Var4 Var5 f2Var4 Var6 f3Var4 Var7 g1Var5 Var6 g2Var5 Var7 h1Var6 Var7 Measure of association terms DFb w2-valuec P-valued FSMprenRAS MSMpregnRAS FSMprenHyperdip MSMpregnHyperdip MomAgeo30nRAS HyperdipnRAS RASnRace 1 0.35 1 0.10 1 5.89 1 0.52 1 4.47 1 6.43 2 0.39 0.55 0.75 0.02 0.47 0.03 0.01 0.82 aDetails: FSMpre paternal smoking during preconception (two categories, one parameter); MSMpreg maternal smoking during pregnancy (two categories, one parameter); MomAgeo30 maternal age at birth o30 years (two categories, one parameter); Hyperdip hyperdiploidy (two categories, one parameter); Ras any Ras mutation (two categories, one parameter); Race child's race/ethnicity (three categories, two parameters); Income annual household income (three categories, two parameters). bDF degrees of freedom. cw2 value derived from log-likelihood ratio test comparing the submodel without the measure of the association term with the full model including the measure of association term. dP-value associated with the w2 value derived from log-likelihood ratio test. critical role of tumor genetic subgroups in epidemiologic study of pediatric leukemia. 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