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NEOPLASIA CYP1A1*2B (Val) allele is overrepresented in a subgroup of acute myeloid leukemia patients with poor-risk karyotype associated with NRAS mutation, but not associated with FLT3 internal tandem duplication David T. Bowen, Marion E. Frew, Sara Rollinson, Philippa L. Roddam, Ann Dring, Martyn T. Smith, Stephen E. Langabeer, and Gareth J. Morgan The etiology of acute myeloid leukemia (AML) is largely unknown. Biologic and epidemiologic data implicate exogenous toxicants, including cytotoxic drugs, benzene, radiation, and cigarette smoking. Allelic variation in genes encoding enzymes such as NADP(H) quinone oxidoreductase (NQO1) and glutathione Stransferase T1 (GSTT1) that metabolize environmental toxicants predispose to subtypes of AML, including therapy-related AML. We assayed NRAS oncogene mutation and FLT3 internal tandem duplication in 447 AML patients with an abnormal karyotype treated in Medical Research Council (MRC) AML clinical trials. Functional allelic variant frequencies in Introduction genes encoding carcinogen-metabolizing enzymes GSTT1, GSTM1, CYP1A1, CYP2D6, CYP2C19, SULT1A1, and NQO1 were previously determined for this cohort. FLT3 internal tandem duplication (ITD) frequency was 17%, and NRAS mutation 12% for the entire cohort. The 2 mutations were found together in only 4 patients. No association was found between enzyme allelic variant frequencies and the presence of FLT3 ITD for the entire cohort or within cytogenetic subgroups. CYP1A1*2B (Val) high-inducibility variant allele was overrepresented in patients with NRAS mutation compared with no mutation, for (1) the entire AML cohort (n 8/53 vs 26/371; odds ratio [OR] 2.36; 95% confidence interval [CI] 1.01-5.53) and (2) the poor-risk karyotype group (n 6/14 vs 4/89; OR 15.94; 95% CI 3.71-68.52) comprising patients with partial/complete deletion of chromosome 5 or 7, or abnormalities of chromosome 3. The CYP1A1*2B allele may predispose to the development of these subgroups of AML by augmented phase 1 metabolism to highly reactive intermediates of CYP1A1 substrates, including polycyclic aromatic hydrocarbons, or by generation of oxidative stress as a metabolic by-product. (Blood. 2003;101:2770-2774) 2003 by The American Society of Hematology The etiology of acute myeloid leukemia (AML) is unknown for the majority of cases. The identification of biologically welldefined subgroups on the basis of cytogenetic analysis has improved prognostic power and led to risk-directed therapeutic strategies. It is increasingly clear that the etiologic mechanisms underlying the "good-risk" translocational karyotypes t(15:17), inv(16), and t(8:21), which predominate in younger patients, will differ from the largely deletional abnormalities seen in "poor-risk" disease, which are more common in older patients. The karyotypic similarities between therapy-related AML (tAML) and poor-risk de novo AML1 suggest that an environmental etiology is more likely in the latter group than in the de novo good-risk group. Questionnaire-based case-control studies have suggested associations between exposure to specific environmental toxicants and the development of AML, but cannot define mechanisms of leukemogenesis.2,3 The cytochrome P450 enzymes are expressed predominantly in the liver and function to detoxify many environmental toxicants (xenobiotics). These phase 1 metabolic enzymes transfer electrons onto substrate toxicants to create highly reactive intermediates, which are then available for detoxification by a variety of phase 2 enzymes including glutathione S-transferases and sulfotransferases. Several carcinogen-metabolizing genes with functional allelic variants are now known to predispose to t-AML4,5 and also to de novo AML.6-8 Furthermore, this predisposition may be greater or less in subgroups of AML defined by specific genomic damage at the karyotypic level. The paradigm for an AML-predisposition gene encoding a carcinogen-metabolizing enzyme is NADP(H) quinone oxidoreductase (NQO1). The C609T polymorphic variant, which confers reduced phase 2 metabolism, is associated with a predisposition to therapy-related AML4 and selected cytogenetic subgroups of de novo AML.6 Reduced phase 2 metabolism has the potential to result in an accumulation of reactive intermediates, which in turn oxidize DNA and protein, leading to DNA mutation and/or cell death. Phase 1 enzymes with functionally relevant allelic variants are also predisposition genes for AML and include cytochrome P450 2C19 (CYP2C19) and CYP2D6.8 The proposed mechanism of predisposition for these 2 genes is a consequence of reduced phase 1 metabolism, presumably allowing the accumulation of nonmetabolized toxic carcinogens. From the Department of Molecular and Cellular Pathology, University of Dundee, Dundee, United Kingdom; Academic Unit of Hematology and Oncology, University of Leeds, Leeds, United Kingdom; School of Public Health, University of California, Berkeley, CA; Department of Hematology, University College London, London, United Kingdom. Submitted January 25, 2002; accepted November 13, 2002. Prepublished online as Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-01-0228. Supported by Leukemia Research Fund grant no. 98/35. The AML DNA bank has been supported by the Medical Research Council, the Leukemia Research Fund, and the Kay Kendall Leukemia Fund. Reprints: David T. Bowen, Molecular and Cellular Pathology, Ninewells Hospital, Dundee, DD1 9SY United Kingdom; e-mail: d.t.bowen@dundee.ac.uk. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ``advertisement'' in accordance with 18 U.S.C. section 1734. 2003 by The American Society of Hematology 2770 BLOOD, 1 APRIL 2003 VOLUME 101, NUMBER 7 BLOOD, 1 APRIL 2003 VOLUME 101, NUMBER 7 CYP1A1*2B (Val) ALLELE IN AML 2771 We selected functional polymorphic variants in specific carcinogen-metabolizing enzymes for study. Enzymes were selected on the basis of an established role in the metabolism of known leukemogenic compounds (eg, benzene, cytotoxic chemotherapeutic drugs). We then selected only patients with well-characterized karyotypic abnormalities, to include abnormalities known to occur following genotoxic insult (eg, chromosome 5 and 7 abnormalities) and also good-prognosis translocations for which an environmental etiology may be less likely. Finally, we assayed the 2 most common molecular abnormalities in AML, namely, FLT3 internal tandem duplication (ITD) and NRAS mutation; each of which is likely to arise from a different mechanism. We demonstrate that the *2B high-inducibility variant of CYP1A1 is overrepresented in a subgroup of poor-risk AML patients with NRAS mutation and may represent a predisposition allele for these specific forms of genomic damage in AML. Patients, materials and methods Patients We studied 447 patients with de novo or secondary AML (defined as secondary to chemotherapy, or following an antecedent hematologic disorder), and who were entered into the Medical Research Council (MRC) AML 10 (younger than 60 years, n 109); AML 11 (older than 55 years, n 115), and AML 12 (younger than 60 years, n 223) clinical trials. This patient cohort overlaps with cohorts in previously published studies.6 Median age was 46 years (range, 15-79 years). DNA was extracted from presentation bone marrow by dodecyltrimethylammonium bromide lysis9 and stored at University College London (United Kingdom). Cytogenetic classification Most cytogenetic studies were done in local laboratories, all of them participants in a central quality control scheme (United Kingdom National External Quality Assessment Scheme). Where this was not available, cytogenetic analyses were done at a central facility in University College London. Standard methods of bone marrow culture were used; 20 metaphases were fully analyzed and reported in accordance with the International System for Human Cytogenetic nomenclature (ISCN) guidelines.10 A hierarchical cytogenetic classification was used to assign all patients to a cytogenetic subgroup as previously described.11 For patients with more than one chromosomal abnormality, the subgroup assigned was determined by the most important abnormality in the following order (with the most important listed first): established translocation, established deletion, established trisomy, nonestablished translocation, nonestablished deletion, nonestablished trisomy.11 Data in this study are now presented for cytogenetic risk groups on the basis of the MRC hierarchical classification12 and defined here as (1) good-risk, t(15;17), t(8;21), inv(16); (2) poor-risk, partial or complete deletion of chromosomes 5 and 7 (5q/7q), abnormalities of 3q; and (3) intermediate-risk, which includes 8, 11q23 abnormalities, 11, 17p, 21, 20q, 12p (but excludes normal karyotype). Carcinogen-metabolizing enzyme gene allelic variant analysis Assays for allelic variants at the following loci have been previously described: GSTM1 (null), GSTT1 (null),7 CYP2D6 (poor/extensive metabolizer), CYP2C19*2 (681G3A; aberrant splice), CYP1A1*2B allele (2455A3G, Ile3Val),8 and NQO1 (609C3T; Pro3Ser).6 In addition, a polymorphism in SULT1A1 (*2, 213G3 A; Arg3His) was assayed by allelic discrimination by means of the polymerase chain reaction (PCR) based TaqMan technology (PE Applied Biosystems, Foster City, CA). Primer and probe sequences for this assay were as follows: forward primer, 5-GGTTGAGGAGTTGGCTCTGC-3 (300 nM); reverse primer, 5ACGTGTGCTGAACCATGAAGT-3 (300 nM) (annealing temperature, 62C); wild-type (WT) probe, 5-AGTTTGTGGGGCGCTCCCTG-3 (100 nM) (Tet labeled); variant probe 5-AGTTTGTGGGGCACTCCCTGC-3 (200 nM) (Fam labeled). FLT3 internal tandem duplication assay Genomic DNA was amplified from exons 12 and 13 of the FLT3 gene.13 This produced a wild-type fragment of 328 bases plus a highermolecular weight fragment containing internal tandem duplicated sequences (when present) of varying length as previously described.13,14 Bands with higher molecular weight than wild type were cut from the gel and directly sequenced by means of a fluorescent primer adapted chain-termination method15 on an ABI 3100 sequencer (PE Applied Biosystems) to determine the start site and size of each duplication. NRAS mutational analysis Mutation screen. Separate assays were developed for mutation detection at "hot spots" in codons 12/13 (exon 1) and codon 61 (exon 2). Oligonucleotide primers amplifying short fragments (241 base pair [bp], exon 1; 201 bp, exon 2) were designed for PCR as follows: N12/13 assay: forward, 5-GACTGAGTACAAACTGGTGG-3; reverse, 5-TGCATAACTGAATGTATACCC-3. N61 assay: forward, 5-CAAGTGGTTATAGATGGTGAAACC-3; reverse, 5-AAGATCATCCTTTCAGAGAAAATAAT-3. PCR products were then subjected to denaturing heteroduplex high-pressure liquid chromatography (HPLC) analysis (dHPLC) (Transgenomic WAVE, Crewe, United Kingdom). PCR conditions were as follows: (1) Exon 1, HotStarTaq (Qiagen, Valencia, CA), 0.625 U. Primers (12.5 pmol), N12/13 forward, N12/13 reverse. Denaturing, 95C for 15 minutes and 94C for 30 seconds; annealing, 55.5C for 1 minute; extension, 72C for 1 minute for 35 cycles; final cycle at 72C for 10 minutes. (2) Exon 2, HotStarTaq (Qiagen), 0.625 U. Primers (12.5 pmol), N61F forward, N61R reverse. Denaturing, 95C for 15 minutes; 94C for 30 seconds; annealing, 55.5C for 1 minute; extension, 72C for 1 minute for 35 cycles; final cycle at 72C for 10 minutes. Heteroduplexes were then generated by means of a thermal cycler as follows: 95C for 5 minutes; 95C, reducing at 1C per 22 seconds, for 70 cycles. Then, 10 L heteroduplexed PCR product per well was loaded from 96-well plates and analyzed by dHPLC under the following conditions: flow, 0.9 mL/min, 47% to 52% buffer (B) in 0.1 minutes, to 60% B in 4 minutes at 61C. Representative dHPLC plots are shown in Figure 1. Mutation confirmation. Samples exhibiting an abnormal dHPLC profile were confirmed as mutant by direct sequencing and/or PCR-restriction fragment length polymorphism mutation-sensitive digestion analysis for codons 12 and 13.16,17 If these assays were insufficiently sensitive to confirm mutation, PCR products were cloned (Original TA Cloning Kit; Invitrogen, Groningen, the Netherlands) and sequenced.15 The sensitivity of the dHPLC is such that 15% mutant DNA can be confidently detected in a sample containing wild-type and mutant sequence (M.E.F., manuscript in preparation). Statistical analysis CYP1A1 allellic variants were analyzed as dichotomous variables (Ile/Val plus Val/Val, versus Ile/Ile). Odds ratios (ORs) and 95% confidence intervals were calculated from 2 2 tables for mutant versus nonmutant (NRAS or FLT3) and Ile/Val plus Val/Val, versus Ile/Ile. Logistic regression was used to test these associations within the entire cohort and also between the 3 cytogenetic risk groups. Power calculations were prospectively computed to inform sample size for the case-control study reported elsewhere6 and were based upon expected enzyme variant polymorphism frequencies. These prospective calculations were not done for NRAS or FLT3 ITD frequency within the patient cohort alone. Retrospective power calculations were performed with the assumption of an expected CYP1A1*2B allele variant frequency (Ile/Val plus Val/Val) of 11%,8 an FLT3 ITD mutant frequency of 25%,14 and an NRAS mutant frequency of 15%. The sample size of 427 patients was sufficient to provide 80% power at a P value less than .05 to detect an OR of 2.5 for the difference in CYP1A1*2B variant frequency between FLT3 ITD mutant versus no ITD, and an OR of 2.9 for NRAS mutation versus no NRAS mutation. 2772 BOWEN et al BLOOD, 1 APRIL 2003 VOLUME 101, NUMBER 7 Figure 1. Denaturing HPLC profiles from representative AML patient DNA. (A) Denaturing HPLC profile from DNA containing wild-type codon 12/13 NRAS sequence only. (B) Denaturing HPLC profile from DNA containing wild-type plus mutant sequences. In this profile, wild-type homoduplexes are retained on the dHPLC column for longer (6.36 minutes) than mutant/wild-type heteroduplexes (5.97 minutes). Results FLT3 ITD frequency was 17% (77 of 447) and NRAS mutation was 12% (53 of 443) for the entire cytogenetically selected AML cohort. The 2 mutations were found together in only 4 patients, confirming previous observations.18 Presence of FLT3 ITD is not associated with variant allele frequency in carcinogen-metabolizing enzymes No differences in carcinogen-metabolizing enzyme variant allele frequency were found for GSTT1, GSTM1, CYP2D6, CYP2C19, CYP1A1, SULT1A1, or NQO1, either for the presence versus absence of FLT3 ITD (data for CYP1A1 variant alleles shown in Table 1) or in relation to start site or duplication size (data not shown). Table 1. CYP1A1 allele frequency in AML patients (all cases and subgroups) with or without FLT3 internal tandem duplication CYP1A1 allele frequency FLT3 ITDs no. (%) ITDs no. (%) OR 95% CI All cases* Ile/Ile Ile/Val or Val/Val Good-risk karyotypes t(15;17), t(8;21), inv(16) Ile/Ile Ile/Val or Val/Val Intermediate karyotypes Ile/Ile Ile/Val or Val/Val Poor-risk karyotypes 5q/7q or 3q Ile/Ile Ile/Val or Val/Val 67 (93) 5 (7) 48 (94) 3 (6) 14 (100) 0 (0) 5 (71) 2 (19) 326 (92) 0.84 0.31-2.25 29 (8) 154 (91) 0.64 0.18-2.31 15 (9) 77 (94) 5 (6) 0.49 0.02-9.3 95 (91) 4.22 0.71-25.0 9 (9) Ile/Ile, homozygous for Ile allele common among whites. Ile/Val or Val/Val, heterozygous or homozygous, respectively, for the *2B Val allele. CI indicates confidence interval. *n 427. CYP1A1*2B allele is overrepresented in AML with NRAS mutation Patients with NRAS-mutant AML had a higher frequency of the CYP1A1*2B allele (combined Ile/Val plus Val/Val) compared with nonmutant AML cases (z 2.0; P .045) (Table 2). When the cohort was subdivided by cytogenetic risk group, this association was confined to the poor-risk karyotype group (Table 2), which was in turn statistically significantly different (logistic regression) from the good-risk group alone (z 2.78; P .005); the intermediaterisk group alone (z 2.0; P .045); and the combined good- plus intermediate-risk groups (z 3.026; P .005). The poor-risk group comprised patients with abnormalities of chromosomes 5 and/or 7 (5q/7q) and also patients with abnormalities of 3q. The frequency of the *2B allele in patients with 5q/7q abnormalities was 4 of 7 for patients with NRAS mutation versus 4 of 79 for patients without the mutation. Corresponding frequencies Table 2. CYP1A1 allele frequency in AML patients (all cases and subgroups) with or without NRAS mutation CYP1A1 allele frequency Mutant NRAS,no. (%) No mutant NRAS, no. (%) OR 95% CI All cases* Ile/Ile Ile/Val or Val/Val Good-risk karyotypes t(15;17), t(8;21), inv(16) Ile/Ile Ile/Val or Val/Val Intermediate karyotypes Ile/Ile Ile/Val or Val/Val Poor-risk karyotypes 5q/7q or 3q Ile/Ile Ile/Val or Val/Val 45 (84.9) 8 (15.1) 24 (96) 1 (4) 13 (93) 1 (7) 8 (57) 6 (43) 345 (93) 26 (7) 2.36 1.01-5.53 190 (88) 17 (12) 70 (93) 5 (7) 0.46 0.06-3.66 1.08 0.12-9.99 85 (96) 4 (4) 15.94 3.71-68.52 See Table 1 for abbreviations. *n 424. BLOOD, 1 APRIL 2003 VOLUME 101, NUMBER 7 CYP1A1*2B (Val) ALLELE IN AML 2773 for the 3q group were 2 of 7 for patients with the NRAS mutation versus 0 of 10 for those without. No difference in variant allele frequency was found for GSTT1, GSTM1, CYP2D6, CYP2C19, SULT1A1, or NQO1 when patient groups with and without the NRAS AML mutation were compared. The CYP1A1*2B allele is not associated with a specific NRAS signature mutation The NRAS mutation spectrum for patients with the CYP1A1*2B allele is presented in Table 3. Codon 12 mutations were most frequent; 6 of 7 of those mutations were at hot-spot sites, compared with 24 of 45 mutations at codon 12 among patients lacking the *2B allele (not significant). The other base change at codon 33 is a novel NRAS mutation in a patient with trisomy 8. Discussion We have previously observed that the allele frequency for the CYP1A1*2B variant is no different in AML patients than in controls8 and cannot therefore be considered a predisposition allele for the development of all types of AML. We now show that the CYP1A1*2B allele may indeed predispose patients to develop a subgroup of AML characterized by specific genomic damage, namely, NRAS mutation and poor-risk karyotype. CYP1A1 is a phase 1 detoxification enzyme, expressed predominantly in extrahepatic tissue and with a wide variety of substrates, including the carcinogenic polycyclic aromatic hydrocarbons (PAHs). Baseline cellular expression of CYP1A1 is usually low, but high inducibility is mediated through the binding of inducers such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and PAH to a cellular protein, the aryl hydrocarbon receptor (AhR). AhR is in turn bound to heat-shock protein 90 (Hsp90) and releases this before translocation to the nucleus to associate with another protein, the Ah receptor nuclear translocator (Arnt). This complex binds an enhancer sequence activating transcriptional induction of CYP1A1 mRNA.19 Thus, phase 1 detoxification capacity of CYP1A1 is influenced at 3 levels: baseline expression level, expression induction, and enzymatic activity. Several polymorphic allelic variants have now been described within the CYP1A1 gene (reviewed in Wormhoudt et al20). The most common variant allele, designated *2A, introduces an Msp1 restriction site in the 3 end of the gene. The next most common, and potentially functionally more significant, variant is a single nucleotide polymorphism in exon 7 (A3G) producing an Iso3Val substitution at amino acid 462 (allelic variant designated *2B). These alleles are often, but not always, in linkage disequilibrium.20 While the data linking the *2A allele to high inducibility of Table 3. Mutation spectrum for patients with NRAS-mutant AML and who have the CYP1A1*2B (Val) allele Karyotype CYP1A1 allele Mutation: codon-base substitution/ amino acid change 5q/7q 5q/7q 5q/7q 5q/7q 3q 3q 8 t(15;17) Ile/Val Ile/Val Ile/Val Val/Val Ile/Val Val/Val Ile/Val Ile/Val 12.2 G 3 A/Gly 3 Asp 12.2 G 3 A/Gly 3 Asp 61.1 C 3 A/Gln 3 Lys 12.2 G 3 A/Gly 3 Asp 12.1 G 3 A/Gly 3 Ser 12.2 G 3 C/Gly 3 Ala 33.3 T 3 G/Asp 3 Glu 12.1 G 3 A/Gly 3 Ser CYP1A1 in human systems remain inconclusive,21,22 the *2B allele does appear to increase induced gene expression and enzymatic function in lymphocytes from healthy donors with a significant trend through heterozygotes to homozygotes for the variant (Val) allele.22 In the Crofts et al22 study, CYP1A1 expression was particularly high among smokers with at least one *2B allele but was not elevated for smokers with at least one *2A allele. Thus, augmented phase 1 metabolism resulting from high inducibility and increased expression of CYP1A1 upon exposure to environmental inducers and substrates will lead to accumulation of toxic metabolic intermediates should phase 2 metabolic pathways be saturated. The high concentration of PAH in cigarette smoke has led to extensive studies of CYP1A1 allelic variant frequencies in smokingassociated cancers. Although most studies are small, the *2B allele may be overrepresented in smokers developing lung,23 breast,24 and colorectal25 cancers. Several studies provide plausible epidemiologic support for the association between AML and substrates and inducers of CYP1A1. A cohort of subjects exposed accidentally to the potent CYP1A1 inducer TCDD have shown a 3.8-fold increased risk of myeloid leukemia with a latency of 15 years.26 Exposure to cigarette smoke is a weak but reasonably consistent risk factor for AML in large case-control studies.27,28 Smoking history data were unfortunately not available for most subjects in our study. NRAS mutations in our patients with the CYP1A1*2B allele show no signature mutation, and this is perhaps not surprising given the diversity of putative environmental carcinogens in the etiology of AML. It is of note, however, that in those patients reported here and in our extended cohort of AML samples (data not shown), most NRAS mutations are transitions, as has previously been reported in AML and other hematologic malignancies.29 Thus, although PAHs are the most attractive candidate carcinogenic substrate of CYP1A1 in the etiology of AML, on the basis of the mutation spectrum in our study, it is likely that the other substrates are involved. This may indeed be the case in other tumors, including lung cancer. Although a signature p53 mutation spectrum is evident in cohorts of smokers with lung cancer,30 a recent study found a higher prevalence of p53 mutation in lung cancers from heavy smokers (compared with nonsmokers) with variant CYP1A1 alleles (*2A/*2B), but the usual signature mutation spectrum was not found in this subgroup.31 This suggests that the mutation signature may be less evident in patients with variant CYP1A1 alleles. Also in this study, smokers with a variant CYP1A1 allele had an increased frequency of KRAS mutation but the authors do not comment upon the mutation spectrum. Cigarette smoke contains a multitude of carcinogenic compounds in addition to both TCDD and PAHs. Exposure to combinations of CYP1A1 inducers and substrates may therefore exacerbate the potential for genomic damage on the background of inheritance of the high-inducibility *2B allele. A final alternative hypothesis is that reactive oxygen species known to be generated as a byproduct of CYP1A1mediated carcinogen metabolism32,33 may oxidize DNA. Oxidative DNA damage may generate a more diverse mutation spectrum, including transitions (eg, 5OH-cytosine G3A) and transversions (8OH-guanine G3T). It remains unclear why the association between the CYP1A1*2B variant allele and NRAS mutation should be confined to the poor-risk karyotype groups. In the context of therapy-related AML, however, point mutations in critical regulatory genes are more common within the "deletional" karyotypes, namely, p53 mutation 2774 BOWEN et al BLOOD, 1 APRIL 2003 VOLUME 101, NUMBER 7 associated with chromosome 5 deletion,34 and RAS mutation associated with chromosome 7 deletion.1 These data suggest that genomic instability leads to AML through different pathways,1 and in the context of de novo AML, CYP1A1 variant allele status may represent one determinant of this instability. It is not surprising that FLT3 ITD is not associated with the CYP1A1*2B allele within these cytogenetic subgroups, as the ITD most likely arises from a mechanism different from that of point mutation, perhaps aberrant recombination or defective double-strand DNA repair. In conclusion, our data suggest that the variant CYP1A1*2B allele may predispose to the development of a subgroup of AML patients characterized by poor-risk karyotypes and NRAS mutation. Epidemiologic data also suggest that substrates and inducers of CYP1A1 represent candidates for environmental carcinogens with potential to cause AML, but alternative mechanisms such as CYP1A1-induced oxidative stress are also proposed. Acknowledgments We wish to thank Mrs Helen Walker for her assistance in characterizing the DNA bank samples and Dr Simon Ogston for statistical advice. We also wish to thank Professors Alan Burnett (Chairman, MRC Adult Leukemia Working Party) and David Linch for initiating, administering, and providing access to the DNA bank. Finally, we wish to thank David Baty and Dot Mechan for assistance with dHPLC assays, and Professor Roland Wolf for access to the ABI 7700 TaqMan. References 1. Pedersen-Bjergaard J, Andersen MK, Christiansen DH, Nerlov C. Genetic pathways in therapy-related myelodysplasia and acute myeloid leukemia. Blood. 2002;99:1909-1912. 2. Taylor JA, Sandler DP, Bloomfield CD, et al. Ras oncogene activation and occupational exposures in acute myeloid leukemia. J Natl Cancer Inst. 1992;84:1626-1632. 3. Crane MM, Keating MJ. Exposure histories in acute nonlymphocytic leukemia patients with a prior preleukemic condition. Cancer. 1991;67: 2211-2214. 4. Larson RA, Wang Y, Banerjee M, et al. Prevalence of the inactivating 609C--T polymorphism in the NAD(P)H:quinone oxidoreductase (NQO1) gene in patients with primary and therapy-related myeloid leukemia. Blood. 1999;94:803-807. 5. Allan JM, Wild CP, Rollinson S, et al. Polymorphism in glutathione S-transferase P1 is associated with susceptibility to chemotherapy-induced leukemia. Proc Natl Acad Sci U S A. 2001;1159211597. 6. Smith MT, Wang Y, Kane E, et al. Low NAD(P)H: quinone oxidoreductase 1 activity is associated with increased risk of acute leukemia in adults. Blood. 2001;97:1422-1426. 7. Rollinson S, Roddam P, Kane E, et al. Polymorphic variation within the glutathione S-transferase genes and risk of adult acute leukaemia. Carcinogenesis. 2000;21:43-47. 8. Roddam PL, Rollinson S, Kane E, et al. Poor metabolizers at the cytochrome P450 2D6 and 2C19 loci are at increased risk of developing adult acute leukaemia. Pharmacogenetics. 2000;10: 605-615. 9. Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques. 1991;11:298-300, 302. 10. Mitelman F. ISCN: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland: Karger; 1995. 11. Moorman AV, Roman E, Willett EV, et al. Karyotype and age in acute myeloid leukemia: are they linked? Cancer Genet Cytogenet. 2001;126:155161. 12. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukemia Working Parties. Blood. 1998;92:2322-2333. 13. Yokota S, Kiyoi H, Nakao M, et al. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies: a study on a large series of patients and cell lines. Leukemia. 1997;11:16051609. 14. Kottaridis PD, Gale RE, Frew ME, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood. 2001;98:1752-1759. 15. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74:5463-5467. 16. Horiike S, Misawa S, Nakai H, et al. 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