FLT3-TKD mutation in childhood acute myeloid leukemia - Nature

4 downloads 0 Views 92KB Size Report
Children's Hospital, Linkou, Taiwan; and 4Biostatistics Department, Childhood Cancer Foundation, Taipei, Taiwan. Mutations of receptor tyrosine kinases are ...
Leukemia (2003) 17, 883–886 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

FLT3-TKD mutation in childhood acute myeloid leukemia D-C Liang1, L-Y Shih2, I-J Hung3, C-P Yang3, S-H Chen1, T-H Jaing3, H-C Liu1, L-Y Wang1 and W-H Chang4 1

Division of Pediatric Hematology-Oncology, Mackay Memorial Hospital, Taipei, Taiwan; 2Division of Hematology-Oncology, Chang Gung Memorial Hospital and Chang Gung University, Taipei, Taiwan; 3Division of Hematology-Oncology, Chang Gung Children’s Hospital, Linkou, Taiwan; and 4Biostatistics Department, Childhood Cancer Foundation, Taipei, Taiwan

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. An internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence of the FLT3 gene (FLT3-ITD) is found in 20–25% of adult acute myeloid leukemia (AML) and at a lower frequency in childhood AML. FLT3-ITD is associated with leukocytosis and a poor prognosis, especially in patients with normal karyotype. Recently, there have been three reports on point mutations at codon 835 of the FLT3 gene (D835 mutations) in adult AML. These mutations are located in the activation loop of the second tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD). The clinical and prognostic relevance of the TKD mutations is less clear. To the best of our knowledge, there has been no report to describe FLT3-TKD mutations in childhood AML. In this pediatric series, FLT3-TKD mutations occurred in three of 91 patients (3.3%), an incidence significantly lower than that of FLT3-ITD (14 of 91 patients, 15.4%) in the same cohort of patients. None of them had both FLT3-TKD and FLT3-ITD mutations. Sequence analysis showed one each of D835 Y, D835 V, and D835 H. Of the three patients carrying FLT3-TKD, two had AML-M3 with one each of L- and V-type PML-RARa, and another one had AML-M2 with AML1-ETO. None of our patients with FLT3-TKD had leukocytosis at diagnosis. At bone marrow relapse, one of the four patients examined acquired FLT3-ITD mutation and none gained FLT3-TKD mutation. Leukemia (2003) 17, 883–886. doi:10.1038/sj.leu.2402928 Keywords: childhood acute myeloid leukemia; FLT3-ITD; FLT3-TKD; D835 mutation

that FLT3-ITD itself did not have prognostic significance but the mutant to wild-type allelic ratio did.10–12,19 Although the presence of FLT3-ITD did not influence the outcome of APL patients,7 the alterations of FLT3 in APL was associated with more aggressive clinical features.22 Thus, mutationally activated FLT3 receptors represent a promising target for AML therapy. Inhibitors of FLT3 tyrosine kinase activity are therefore being actively pursued, and several inhibitors of FLT3 have been identified as potential candidates for treatment of leukemia carrying FLT3-activating mutations.23–27 More recently, point mutations in codon 835 of the FLT3 gene have been described in adult AML.28 These mutations are located in the activation loop of the second TKD of FLT3 (FLT3TKD). Like FLT3-ITD, TKD mutations of FLT3 also induce constitutive tyrosine phosphorylation and enhance cell proliferation.28 There have only been three studies on FLT3-TKD mutations in adult AML.11,28,29 The FLT3-TKD mutations occurred in approximately 7% in adults AML.11,28,29 To the best of our knowledge, there has been no report on FLT3-TKD mutations in children with AML. In this study, we reported the FLT3-TKD mutations in childhood AML, and analyzed the clinical characteristics of the AML cases with FLT3-TKD mutations.

Materials and methods Introduction The FLT3 gene belongs to the receptor tyrosine kinase class III family, which plays a central role in hematopoiesis.1 This gene locates at chromosome 13q12 and is predominantly expressed in hematopoietic stem cells.2 FLT3 is also expressed in human leukemia or lymphoma cell lines.3,4 Recently, an internal tandem duplication (ITD) of the FLT3 gene (FLT3-ITD) was found in 20–25% of adult acute myeloid leukemia (AML).5–12 FLT3-ITD was also found to be associated with leukocytosis in acute promyelocytic leukemia (APL),7 the AML transformation of myelodysplastic syndrome,13 and atypical cases of therapyrelated myelodysplasia or AML.14 The ITD mutation of FLT3 occurs at the juxtamembrane domain through the first tyrosine kinase domain (TKD).1 The altered FLT3 gene is always transcribed in-frame.5,6 In childhood AML, there have been several studies on FLT3-ITD;15–19 the incidences of FLT3-ITD varied from 5.3 to 16.5%, which were lower than those of adult AML.6–12 FLT3-ITD has been reported to be associated with a poor outcome in adult and childhood AML,8,9,15–18,20,21 whereas some studies observed Correspondence: Dr L-Y Shih, Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, 199 Tung Hwa North Road, Taipei, Taiwan; Tel: +886-3-3281200 ext. 2524; Fax: +886 3 3286697; E-mail: [email protected] Received 7 January 2003; accepted 11 February 2003

Patients In all, 91 children (age p18 years) with de novo AML diagnosed at Mackay Memorial Hospital and Chang Gung Children’s Hospital were studied at initial diagnosis. Informed consent was obtained from the guardians. The morphologic subtypes were classified according to the FAB Cooperative Group.30–32 Three patients were associated with Down’s syndrome. Immunophenotyping was performed by flow cytometry using monoclonal antibodies against HLA-DR, CD13, CD14, CD15, CD33, CD41, CD61, CD34, CD2, CD3, CD5, CD7, and CD19. One patient had acute mixed-lineage leukemia with myeloid and lymphoid components. The mononuclear cells from diagnostic marrow samples were enriched by Ficoll–Hypaque (1.077 gm/ ml; Pharmacia Biotech, Uppsala, Sweden) density-gradient centrifugation and cryopreserved in 10% dimethyl sulfoxide and 20% fetal bovine serum in liquid nitrogen until test. The treatment protocols were previously described.19 Before 1997, all AML patients were treated with the nation-wide Taiwan Pediatric Oncology Group (TPOG) AML 901 protocol, consisting of induction therapy with epidoxorubicin, cytosine arabinoside (Ara-C), and 6-thioguanine, consolidation therapy with etoposide and cyclophosphamide, and then maintenance therapy with five courses of epidoxorubicin, Ara-C and 6-thioguanine, followed by etoposide and cyclophosphamide and then by 6-mercaptopurine and methotrexate. After 1997, those with APL were treated with TPOG-APL-97 protocol with

FLT3-TKD in childhood AML D-C Liang et al

884 analyzed. The karyotypes were interpreted according to the International System for Human Cytogenetic Nomenclature.35 The detection of common fusion transcripts by reverse transcriptase (RT) PCR assays for AML1-ETO, PML-RARa, CBFb-MYH11, and 11q23 abnormalities including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-ELL, followed by Southern blot analysis, was performed as previously described.36

all-trans retinoic acid first, followed by idarubicin and Ara-C if the white blood cell (WBC) count was increased. The postremission therapy consisted of six courses of idarubicin and Ara-C. Since 2001, a new protocol TPOG-APL-2001, which was modified from the PETHEMA protocol,33 has been used. The non-APL patients were randomized to receive either the TPOG-AML-97A protocol consisting of induction therapy with idarubicin and Ara-C, postremission therapy with four courses of high-dose Ara-C and etoposide alternated with high-dose Ara-C and mitoxantrone, and then four courses of idarubicin and AraC; or the TPOG-AML-97B protocol, which was modified from MRC AML10 protocol.34 The clinicohematologic characteristics including age, gender, WBC count, FAB classification, karyotype, specific fusion transcripts at diagnosis, event-free survival (EFS), and overall survival were analyzed.

Statistical methods The comparison between groups was examined by Fisher’s exact test. Induction failure was defined as failure to achieve remission after induction therapy and early death before achieving remission. For the calculation of EFS, all deaths and relapses occurring after the start of chemotherapy were classified as events, and induction failure was assigned a time of zero. The univariate analysis between variables and survival or EFS was carried out using the log-rank test. A statistically significant difference was defined as a P-value o0.05.

DNA PCR assay for FLT3-ITD Genomic DNAs were extracted from frozen bone marrow mononuclear cells collected at diagnosis by using a DNA extraction kit (Puregene Gentra System, Minneapolis, MN, USA) according to the manufacturer’s instruction. The PCR assay for FLT3-ITD, genescan analysis to determine the allelic distribution, and sequencing of the duplicated fragments of FLT3-ITD were previously described.19

Results

FLT3-TKD A mutation of FLT3-TKD was present in three of 91 pediatric patients with AML (3.3%), an incidence significantly (P ¼ 0.015) lower than that of FLT3-ITD (14 of 91 patients, 15.4%). None of our patients had both ITD and TKD mutations. The clinicohematologic features of the three patients with FLT3-TKD are shown in Table 1. None of the three children had leukocytosis; their WBC counts at diagnosis were 6400, 5500, and 1600/ml, respectively. FLT3-TKD was found in two patients with APL, one with L-type PML-RARa and the other with V-type PML-RARa; the third patient had AML-M2 with AML1-ETO. In this series, FLT3-TKD occurred in one of 22 patients (4.5%) with AML-M2, and two of 12 patients (16.7%) with AML-M3, and it was not found in the remaining four M0, 11 M1, 16 M4, 13 M5, two M6, and 11 M7 patients. Sequence analysis showed that all mutations occurred at codon 835 of FLT3. In the patient with AML1-ETO, the second nucleotide A of D835 was substituted with T, resulting in an Asp to Val amino-acid change (D835 V). The substitution for the first nucleotide G of D835 with C, resulting in an Asp to His change (D835 H) was found in the APL patient with V-type PML-RARa. The substitution for the first nucleotide G of D835 with T, resulting in an Asp to Tyr change (D835 Y) was found in the other APL patient with L-type PML-RARa. Of the three patients carrying FLT3-TKD, the one with M2 subtype died during remission induction therapy, and the other two patients with M3 subtype had an EFS of 12+ and 23+ months, respectively. With a median follow-up of 45 months,

Detection of FLT3-TKD mutations For detection of the FLT3-TKD mutations at codons 835 and 836, genomic DNA was amplified by PCR using the forward primer of 50 -CCGCCAGGAACGTGCTTG-30 and the reverse primer of 50 -GCAGCCTCACATTGCCCC-30 .28 A 25 ml of amplified products were digested with 10 U EcoRV (New England Biolab, Inc., Beverly, MA, USA) for 1 h at 371C and then run onto a 8% polyacrylamide gel. Undigested band was cut out from the gel, purified and directly sequenced in both directions with the BigDye Terminator Cycle Sequencing Ready Reaction kit, which contained AmpliTaq DNA polymerase FS (Applied Biosystems), on an automated DNA sequencing system according to the manufacturer’s instructions. PCR products with faint aberrant bands were individually cut from the gel, purified with a MinElute gel extraction kit (Qiagen, Hilden, Germany), cloned into the pCR II-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA), and then subjected to sequencing.

Cytogenetic analysis and detection of common fusion transcripts Cytogenetic analysis was performed using a direct method or short-term unstimulated cultures. G-banded metaphases were

Table 1 Patient

1 2 3

Clinicohematologic features in three children with FLT3-TKD Age (years)

Sex

WBC (/ml)

Percentage of blasts in BM

FAB subtype

Cytogenetic abnormality

FLT3-TKD mutation

OS (months)

EFS (months)

13 3 6

M F F

6400 5500 1600

83 84 98

M2 M3 M3

45X, -Y, t(8;21) 46XX, t(15;17) 46XX, t(15;17)

Asp835 Val Asp835 His Asp835 Tyr

1.5 24.5+ 13+

0 23+ 12+

OS, overall survival; EFS, event-free survival. Leukemia

FLT3-TKD in childhood AML D-C Liang et al

885 the 3-year EFS in AML patients without FLT3-TKD was 51.1% (95% confidence interval, 39.6–65.9%), and in those with FLT3ITD and those without FLT3-ITD, it was 50.0% (95% confidence interval, 25.0–100%), and 51.5% (95% confidence interval, 39.4–67.4%), respectively. The small number of patients with FLT3-TKD precluded a meaningful comparison of treatment outcome between patients with and without this abnormality. Patients with a WBC count X50 000/ml had a shorter overall survival (P ¼ 0.019), whereas age (P ¼ 0.253) and FAB subtype (P ¼ 0.149) did not influence overall survival. Similarly, a WBC count of X50 000/ml predicted poor EFS (P ¼ 0.028), whereas age (P ¼ 0.199) and FAB subtype (P ¼ 0.794) did not. Four patients had paired bone marrow samples at both diagnosis and relapse available for FLT3 mutation analysis. These four patients had neither FLT3-TKD mutations nor FLT3-ITD mutations at initial diagnosis. At relapse, one of them acquired FLT3-ITD mutation with a mutant to wildtype ratio of 0.66. None of them had FLT3-TKD mutations at relapse.

Discussion We found that 3.3% of childhood AML had FLT3-TKD and 15.4% had FLT3-ITD mutations. None of our patients harbored both ITD and TKD mutations. Taken together, a total of 18.7% of childhood AML possessed an FLT3-activating mutation. Similar to the previous reports on adult AML that demonstrating a lower frequency of FLT3-TKD (7%) as compared with 20–25% of FLT3-ITD,11,28,29 the present study on a same cohort of pediatric patients with AML revealed that the incidence of FLT3-TKD was also significantly lower than that of FLT3-ITD. Furthermore, our data also showed that the frequency of FLT3TKD in childhood AML was lower than that of adult AML. D835 Y is the most common FLT3-TKD mutation in adult AML, the others include D835 H, D835 V, D835 E, and D835 N, etc.11,28,29 D835 Y, D835 H, and D835 V occurred in one each of our patients. We have recently demonstrated that adult patients with AML might acquire FLT3-ITD at the time of relapse.37 Likewise, one of the four children with relapsed AML who did not have ITD mutation at diagnosis, gained FLT3-ITD at relapse, whereas no acquisition of TKD mutation was found in the four relapsed samples. Thiede et al 11 found that FLT3-TKD mutations in adult AML were associated with leukocytosis, high percentage of marrow blasts, and M5 subtype, however, no correlation was observed between FLT3-TKD mutations and WBC count or other clinicohematologic characteristics by other group.28 None of our three patients with FLT3-TKD had leukocytosis, the small number of patients harboring FLT3-KTD mutations precluded a meaningful analysis of the correlation between TKD mutation and clinicohematologic features. However, it is of interest to note that all three patients with TKD mutations in the present study had specific chromosomal translocations, which was contradictory to the findings of more prevalence of normal karyotypes in adult AML.11,28 As patients carrying TKD mutations were rare in the reported adult series and our pediatric series, the clinical relevance of FLT3-TKD requires additional studies on more patients with this mutation. The prognostic impact of FLT3-TKD mutation has been evaluated in adult AML, but no consistent results were found;28,29 FLT3-TKD mutation was reported to be associated with a poor disease-free survival in the unselected adult AML,28 whereas others failed to find FLT3-TKD mutation to be of prognostic significance.29 The

number of patients carrying FLT3-TKD was small in our pediatric series, it is unable to draw a definitive conclusion.

Acknowledgements This work is supported in part by Grants 91-01 from the Childhood Cancer Foundation of ROC and NSC91-2314-B-182032 from the National Science Council, Taiwan. We thank Ms Meng-Chu Chou and Ms Siew-Hoon Teoh for their technical assistance.

References 1 Rosnet O, Schiff C, Pebusque MJ, Marchetto S, Tonnelle C, Toiron Y et al. Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells. Blood 1993; 82: 1110–1119. 2 Rosnet O, Stephenson D, Mattei MG, Marchetto S, Shibuya M, Chapman VM et al. Close physical linkage of the FLT1 and FLT3 genes on chromosome 13 in man and chromosome 5 in mouse. Oncogene 1993; 8: 173–179. 3 Meierhoff G, Dehmel U, Gruss HJ, Rosnet O, Birnhaum D, Quentmeier H et al. Expression of FLT3 receptor and FLT3-ligand in human leukemia–lymphoma cell lines. Leukemia 1995; 9: 1368–1372. 4 Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. Leukemia 1996; 10: 588–599. 5 Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K et al. Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911–1918. 6 Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T 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: 1605–1609. 7 Kiyoi H, Naoe T, Yokota S, Nakao M, Minami S, Kuriyama K et al. Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia 1997; 11: 1447–1452. 8 Rombouts WJC, Blokland I, Lowenberg B, Ploemacher RE. Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the flt3 gene. Leukemia 2000; 14: 675–683. 9 Abu-Duhier FM, Goodeve AC, Wilson GA, Gari MA, Peake IR, Rees DC et al. FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group. Br J Haematol 2000; 111: 190–195. 10 Whitman SP, Archer KJ, Feng L, Baldus C, Becknell B, Carlson BD et al. Absence of the wildtype allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a Cancer and Leukemia Group B study. Cancer Res 2001; 61: 7233–7239. 11 Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002; 99: 4326–4335. 12 Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002; 100: 59–66. 13 Horiike S, Yokota S, Nakao M, Iwai T, Sasai Y, Kaneko H et al. Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia. Leukemia 1997; 11: 1442–1446. 14 Christiansen DH, Pedersen-Bjergaard J. Internal tandem duplications of the FLT3 and MLL genes are mainly observed in atypical cases of therapy-related acute myeloid leukemia with a normal karyotype and are unrelated to type of previous therapy. Leukemia 2001; 15: 1848–1851. 15 Kondo M, Horibe K, Takahashi Y, Matsumoto K, Fukuda M, Inaba J et al. Prognostic value of internal tandem duplication of the FLT3 Leukemia

FLT3-TKD in childhood AML D-C Liang et al

886 16

17

18

19 20

21

22

23 24 25 26

Leukemia

gene in childhood acute myelogenous leukemia. Med Pediatr Oncol 1999; 33: 525–529. Iwai T, Yokota S, Nakao M, Okamoto T, Taniwaki M, Onodera N et al. Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia. Leukemia 1999; 13: 38–43. Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H et al. Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children. Br J Haematol 1999; 105: 155–162. Meshinchi S, Woods WG, Stirewalt DL, Sweetser DA, Buckley JD, Tjoa TK et al. Prevalence and prognostic significance of FLT3 internal tandem duplication in pediatric acute myeloid leukemia. Blood 2001; 97: 89–94. Liang DC, Shih LY, Hung IJ, Yang CP, Chen SH, Jaing TH et al. Clinical relevance of internal tandem duplication of the FLT3 gene in childhood acute myeloid leukemia. Cancer 2002; 94: 3292–3298. Rombouts WJ, Lowenberg B, van Putten WL, Ploemacher RE. Improved prognostic significance of cytokine-induced proliferation in vitro in patients with de novo acute myeloid leukemia of intermediate risk: impact of internal tandem duplications in the Flt3 gene. Leukemia 2001; 15: 1046–1053. Boissel N, Cayuela JM, Preudhomme C, Thomas X, Grardel N, Fund X et al. Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy. Leukemia 2002; 16: 1699–1704. Noguera NI, Breccia M, Divona M, Diverio D, Costa V, De Santis S et al. Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol. Leukemia 2002; 16: 2185–2189. Tse KF, Novelli E, Civin CI, Bohmer FD, Small D. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 2001; 15: 1001–1010. Teller S, Kramer D, Bohmer SA, Tse KF, Small D, Mahboobi S et al. Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3. Leukemia 2002; 16: 1528–1534. Minami Y, Kiyoi H, Yamamoto Y, Yamamoto K, Ueda R, Saito H et al. Selective apoptosis of tandemly duplicated FLT3-transformed leukemia cells by Hsp90 inhibitors. Leukemia 2002; 16: 1535–1540. Tse KF, Allebach J, Levis M, Smith BD, Bohmer FD, Small D. Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor. Leukemia 2002; 16: 2027–2036.

27 Levis M, Allebach J, Tse KF, Zheng R, Baldwin BR, Smith BD et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002; 99: 3885–3891. 28 Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97: 2434–2439. 29 Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol 2001; 113: 983–988. 30 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French–American–British Cooperative Group. Ann Intern Med 1985; 103: 620–625. 31 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR et al. Criteria for the diagnosis of acute leukemia of megakaryocyte lineage (M7). Ann Intern Med 1985; 103: 460–462. 32 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DAG, Gralnick HR et al. Proposal for the recognition of minimally differentiated acute myeloid leukemia (AML-M0). Br J Haematol 1991; 78: 325–329. 33 Sanz MA, Martin G, Rayon C, Esteve J, Gonzalez M, DiazMediavilla J et al. A modified AIDA protocol with anthracyclinebased consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. PETHEMA group. Blood 1999; 94: 3015–3021. 34 Hann IM, Stevens RF, Goldstone AH, Rees JKH, Wheatley K, Gray RG et al. Randomized comparison of DAT versus ADE as induction chemotherapy in children and younger adults with acute myeloid leukemia. Results of the Medical Research Council’s 10th AML Trial (MRC AML 10). The Adult and Childhood Leukaemia Working Parties of the Medical Research Council. Blood 1997; 89: 2311–2318. 35 Mitelman F (ed). An International System for Human Cytogenetic Nomenclature. Basel: Karger, 1995. 36 Liang DC, Shih LY, Yang CP, Hung IJ, Chen SH, Liu HC. Molecular analysis of fusion transcripts in childhood acute myeloid leukemia in Taiwan. Med Pediatr Oncol 2001; 37: 555–556. 37 Shih LY, Huang CF, Wu JH, Lin TL, Dunn P, Wang PN et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002; 100: 2387–2392.