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FLT3 mutations in childhood acute lymphoblastic leukemia at first relapse
Leukemia (2005) 19, 467–468. doi:10.1038/sj.leu.2403655 Published online 27 January 2005 TO THE EDITOR
The FMS-like tyrosine kinase 3 (FLT3) plays an important role in the pathogenesis of hematopoietic malignancies.1 Constitutive activation of FLT3, resulting in cell proliferation and survival, occurs either through coexpression of FLT3 ligand and autocrine signalling or through mutations of the FLT3 gene, which lead to autophosphorylation and activation of the FLT3 receptor. FLT3 activating mutations, internal tandem duplications (ITD) and kinase domain (KD) mutations were initially discovered in acute myeloid leukemias (AML) and are associated with poor prognosis in both adult and pediatric AML.2 Recently, it was shown that FLT3 mutations are common in two subtypes of childhood acute lymphoblastic leukemia (ALL).3,4 In infant ALL with translocations involving the mixed lineage leukemia (MLL) gene and in hyperdiploid ALL, FLT3 mutations are detected in 18.2%3 and in 21.5%3–25%4, respectively. While the presence of FLT3 mutations in infant ALL with MLL rearrangements was found to be associated with an inferior prognosis, the presence of the mutations in hyperdiploid ALL did not affect clinical outcome.3 To substantiate these important findings, we investigated whether the frequency of FLT3 mutations is increased in relapsed childhood ALL, and if FLT3 mutations are of prognostic relevance. We analyzed 134 diagnostic leukemic samples from patients with first relapse of ALL with bone marrow (BM) involvement enrolled within two consecutive years, 2000 and 2001, into the multicenter relapse trials of the Berlin–Frankfurt– Muenster Study group (ALL-REZ BFM).5 Although leukemic samples were not available from 62 patients within the indicated time period, statistical analysis of patient characteristics of the studied (n ¼ 134) vs the not available samples (n ¼ 62) did not disclose any significant differences. Only the percentage of samples with TEL/AML1 fusion transcripts was slightly higher in the studied group (P ¼ 0.018). Informed consent for treatment and accompanying scientific studies was obtained from the parents or the guardians. FLT3 length mutations in the juxtamembrane domain were identified by Genescan analysis, and RFLP-mediated PCR was used for detection of activation loop mutations in the KD, as published elsewhere.6 All samples with detectable mutations were sequenced (different control DNA were a gift from S Schnittger, University Hospital Grosshadern, Munich, Germany). We found FLT3 mutations in leukemic cell samples of three patients: one I836M (49% mutant allele), one D835Y (5%) and one ITD of 30 bp (65.6%). The FLT3 length mutation was detected in a patient with MLL rearrangement (MLL/AF4). At initial ALL diagnosis, when the patient was an infant, the leukemic cells were MLL/AF4 positive but FLT-ITD negative, Correspondence: Dr S Wellmann, Department of Pediatric Oncology and Hematology, Charite, Medical University of Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany; Fax: þ 49 30 450 566946; E-mail:
[email protected] Received 24 September 2004; accepted 7 December 2004; Published online 27 January 2005
suggesting the occurrence of an FLT-ITD positive subclone. All other included patients with an MLL aberration at relapse (n ¼ 4) depicted no FLT3 mutations and were older than 1 year at initial diagnosis. Both FLT3 activation loop mutations were neither associated with known cytogenetics nor with hyperdiploidy. While FLT3-D835Y could also not be detected in the corresponding initial ALL, no leukemic sample was available to study the clonal evolution of the FLT3-I836M mutation. All three patients suffered a subsequent relapse or died after achieving complete remission, while in the entire study group 77/134 patients and in the not analyzed group 41/62 had an adverse event (P ¼ 0.126). As the blast count in only 12/134 analyzed BM samples was below 70% (but above 10%), it is unlikely that mutations were missed. Taken together, our results indicate that FLT3 mutations emerged at ALL relapse (2/3) and occur with a lower frequency than Taketani et al3 and Armstrong et al4 found in their analyses regarding ALL at first presentation in patients older than 1 year, 6/112 and 10/71, respectively. As we found no FLT3 mutations in hyperdiploid ALL, our data are in agreement with the observation of Taketani et al3 that these patients have good clinical outcomes and that the mutations may not affect the growth advantage of hyperdiploid ALL cells. In conclusion, albeit FLT3 mutations do not accumulate in relapsed ALL, recent findings indicate that even wild-type FLT3 remains an interesting therapeutic target in ALL. Constitutively activated wild-type FLT3 was found in some B-cell precursor ALL cell lines,7 and childhood ALL cells with high levels of FLT3 expression were killed by inhibition of FLT3 signalling.8
Acknowledgements This work was supported by a grant from the Deutsche Jose´ Carreras Leuka¨mie-Stiftung (Project DJCLS-R03/16).
S Wellmann1 E Moderegger1 A Zelmer1 M Bettkober1 A von Stackelberg1 G Henze1 K Seeger1
1
Department of Pediatric Oncology and Hematology, Charite, Medical University of Berlin, Berlin, Germany
References 1 Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 2003; 3: 650–665. 2 Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia 2003; 17: 1738–1752. 3 Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R et al. FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood 2004; 103: 1085–1088. 4 Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA et al. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood 2004; 103: 3544–3546. 5 Henze G, Fengler R, Hartmann R, Kornhuber B, Janka-Schaub G, Niethammer D et al. Six-year experience with a comprehensive approach to the treatment of recurrent childhood acute lymphoLeukemia
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468 blastic leukemia (ALL-REZ BFM 85). A relapse study of the BFM group. Blood 1991; 78: 1166–1172. 6 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.
7 Zheng R, Levis M, Piloto O, Brown P, Baldwin BR, Gorin NC et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood 2004; 103: 267–274. 8 Brown P, Levis M, Shurtleff S, Campana D, Downing J, Small D. FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression. Blood 2004, September 16 [E-pub ahead of print].
NUP214-ABL1 amplification in t(5;14)/HOX11L2-positive ALL present with several forms and may have a prognostic significance
Leukemia (2005) 19, 468–470. doi:10.1038/sj.leu.2403654 Published online 27 January 2005 TO THE EDITOR
HOX11L2 expression, mostly resulting from the chromosomal cryptic translocation t(5;14)(q35;q32), is observed in more than 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL) and in about 13% of adult T-ALL.1,2 The prognostic significance of the genetic abnormality in patients with this subtype of ALL is still controversial, claimed as unfavorable for some groups3,4 or regarded as neutral for others.5,6 Recently, episomal amplification encompassing the ABL1 gene has been found in 2.3 and 4.3% of childhood and adult T-ALL.7 The incidence of ABL1 amplification rose to 5.8% of T-ALL by Graux et al,8 who showed that a new fusion gene, NUP214-ABL1, was included in the episomal amplicon. Moreover, the latter authors showed that the NUP214-ABL1 fusion was mainly present in T-ALL expressing HOX11 or HOX11L2. We hypothesized that the expression of the fusion gene might account for the discrepancies observed in the evolution of individual HOX11L2-positive patients with T-ALL. We report the finding of fluorescence in situ hybridization (FISH) and real-time PCR in a child with T-ALL with a peculiar form of amplification and rapidly fatal evolution.
Case report GD, a 9-year-old boy was admitted to the hematological unit of the Armand Trousseau hospital (Paris) with a suspicion of leukemia because of fever, erythematous angina, enlarged cervical lymph nodes and hyperleukocytosis. Clinical and radiological examination displayed mediastinal enlargement, hepatosplenomegaly and voluminous mesenteric lymph nodes. Hematologic data confirmed the diagnosis of acute leukemia: in peripheral blood, 302 109/l leukocytes with 93% lymphoblasts, 116 109/l platelets and hemoglobin 117 g/l. Immunophenotyping revealed an immature T-cell leukemia scoring positive for c-CD3, CD2, CD4, CD5, CD7 and CD8 antigens. All the myeloid antigens tested (CD13, CD14, CD15, CD33, Correspondence: Dr R Berger, EMI 0210 INSERM, Tour Pasteur, Hoˆpital Necker, 149 rue de Se`vres, Paris 75015, France; Fax: þ 33 0 1 4219 27 40; E-mail:
[email protected] Received 29 November 2004; accepted 6 December 2004; Published online 27 January 2005 Leukemia
CD117, CD35 and c-MPO) were negative, with the exception of CD33. The child immediately received induction therapy including prednisolone prephase, vincristine 1.5 mg/m2 weekly 4, cyclophosphamide 1 g/m2 day 8, asparaginase 6000 IU 8, daunorubicin 40 mg/m2 days 8, 9, 10 and 15 and two intrathecal injections of methotrexate and aracytine. At day 7, he demonstrated corticoid resistance, and at day 21, bone marrow was aplastic with persistence of 83% leukemic cells. No remission could be obtained and the child died 30 days after the diagnosis because of massive intracranial hemorrhage. Cytogenetic studies were performed, prior to any treatment, on blood cells after 24 h in vitro culture with R-banding technique. The karyotype was 47,XY, þ 8[26]/46,XY[1]. No structural aberration was detected.
FISH studies Two series of FISH studies were performed according to usual techniques.9 The first allowed the detection of a t(5;14) translocation with dual-color FISH with whole chromosome 14 painting probe and YAC 885A6, as described previously.1 The chromosomal breakpoints were more precisely located with BAC probes, on chromosome 5 between BACs 45L16 remaining to chromosome 5 and 546B8 translocated to 14q32 (without split), and on chromosome 14 to BAC 2576L4 giving split signals on normal chromosome 14, der(14) and der(5). In the second step, amplification of ABL1 was investigated with the commercial LSI BCR-ABL1 ES dual probe covering 300 kb of ABL1 from intron 5 toward telomere (Abbott, Rungis). Surprisingly, a strong signal was present on the long arm of chromosome 2 in addition to the normal signals on both 9q34 copies. No extrachromosomal hybridization signal could be observed. It was concluded that amplification of ABL1 sequences were located to 2q23–24. Hybridization with whole chromosome 9 painting (WCP) probe also showed a signal on 2q, consistently less strong that the signal obtained with ABL probes alone. The difference of the two signals was evidenced by dual-color FISH with ABL and chromosome 9 WCP probe showing that the signals generated by the ABL probe were larger than those of the WCP probe. To ascertain the involvement of NUP214, dual-color FISH studies were performed with BACs RP11 235J21 (ABL1) and RP11235F20 and RP11-106L9 (NUP214). Colocalization of ABL1 and NUP214 probes to both chromosomes 9 as well as to chromosome 2q confirmed the amplification of DNA sequences of the two genes (Figure 1).
