Originally published as JCO Early Release 10.1200/JCO.2005.01.6253 on January 17 2006

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BAALC Expression and FLT3 Internal Tandem Duplication Mutations in Acute Myeloid Leukemia Patients With Normal Cytogenetics: Prognostic Implications

From the Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Medizinische Klinik III, Berlin; Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Medizinische Klinik I; Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Deutsche Studieninitiative Leukämie Statistical Group, Dresden, Germany; and The Ohio State University, Comprehensive Cancer Center, Columbus, OH

Address reprint requests to Claudia D. Baldus, MD, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Department of Hematology, Oncology and Transfusion Medicine, Hindenburgdamm 30, 12203 Berlin, Germany; e-mail: claudia.baldus{at}charite.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
PURPOSE: Evaluate the impact of BAALC (brain and acute leukemia, cytoplasmic), a gene whose expression has been associated with adverse outcome in acute myeloid leukemia (AML) with normal cytogenetics, and FLT3 internal tandem duplication (ITD) mutations as independent prognostic factors in a larger study.

PATIENTS AND METHODS: BAALC expression was determined by real-time reverse transcriptase polymerase chain reaction in pretreatment blood samples of 307 adults 60 years of age with AML with normal cytogenetics. Patients were dichotomized at BAALC's median expression into low and high expressers. The FLT3 ITD mutant:wild-type ratio was determined by fragment analysis.

RESULTS: Compared with low-BAALC patients, high-BAALC patients had a higher rate of primary resistant leukemia (16% v 6%; P = .006). High BAALC expression was associated with a higher cumulative incidence of relapse (CIR; P = .018) and an inferior overall survival (OS; 3-year OS, 36% v 54%; P = .001). On multivariable analysis, high BAALC was independently predictive of resistant disease (P = .019), and high BAALC as well as a high FLT3 mutant:wild-type ratio were confirmed as the only factors predicting a high CIR (BAALC, P = .03; FLT3, P = .01) and inferior OS (BAALC, P = .001; FLT3, P = .012).

CONCLUSION: This study strengthens BAALC expression as one of the most important prognostic factors in AML patients with normal cytogenetics. BAALC expression and FLT3 mutation status should assist in tailoring induction and postremission therapies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease. Currently, cytogenetic findings provide the most important prognostic information.^1,2 Approximately half of adult AML patients lack clonal chromosome aberrations at diagnosis, and although this group has an intermediate prognosi,s only 40% are long-term survivors. The identification of molecular markers that precisely differentiate a patient's risk could improve treatment outcome by the use of sophisticated risk adaptive treatment strategies.^3,4

Genes involved in signal transduction have been the focus of molecular analyses. Mutations in the fms-like tyrosine kinase 3 (FLT3) receptor, most commonly an internal tandem duplication (ITD), are frequent in AML.^5-8 Several studies have shown that overrepresentation of the ITD relative to the wild-type FLT3 allele is especially predictive of an adverse outcome.^9-11

BAALC (brain and acute leukemia, cytoplasmic) is a gene implicated in normal hematopoiesis and leukemia.¹² The gene, located at chromosome 8q22.3, encodes a protein of yet-unknown function. In hematopoiesis, BAALC reflects a stage-specific marker and is aberrantly expressed in a subset of acute leukemias.¹³ High mRNA expression levels of BAALC have been shown to be an adverse risk factor in newly diagnosed AML patients with normal cytogenetics. The first study to show this included 86 de novo AML patients younger than 60 years with a more favorable FLT3 mutation status treated on Cancer and Leukemia Group B (CALGB) protocol 9621.¹⁴

Independent confirmation is required to validate the initial results so that BAALC expression may be exploited for risk-adapted treatment stratification of AML patients with normal cytogenetics. Here we present the results of a Deutsche Studieninitiative Leukämie (DSIL) study comprising 307 adult AML patients 60 years with normal cytogenetics treated on the AML'96 protocol. In contrast to the CALGB study, our study includes patients diagnosed with secondary AML (sAML), and patients with a high-risk FLT3 mutation status, thus allowing a more comprehensive characterization of the patient's risk profile.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Patients
We studied 307 patients treated on AML'96.¹⁵ Two hundred seventy-eight had de novo AML, and 29 had sAML, including 24 patients with a prior myelodysplastic syndrome (MDS) and five with therapy-related AML (tAML).

Treatment
The treatment schema is summarized in Supplementary Figure A1 (online only). Patients received two cycles of induction therapy. Postinduction therapy was stratified for adults 60 years according to cytogenetic risk group. For patients with de novo AML and normal cytogenetics, those having a human leukocyte antigen–identical sibling donor were referred for allogeneic stem-cell transplantation (SCT). Those without a sibling donor were randomly assigned to receive intermediate dose cytarabine (ara-C; 1,000 mg/m² every 12 hours, days 1 through 6; I-MAC) or high-dose ara-C (3,000 mg/m² every 12 hours, days 1 through 6) plus mitoxantrone (10 mg/m² days 4 to 6; H-MAC) and both of these were followed by autologous peripheral blood (PB) SCT. Patients not eligible for allogeneic or autologous SCT were randomly assigned to receive I-MAC or H-MAC followed by one consolidation cycle. Patients with sAML who were younger than 45 years were referred for unrelated-donor SCT. The Technical University Dresden ethics board approved this study. All patients gave written informed consent according to the Declaration of Helsinki.

Patient Samples
Of 473 adult AML patients 60 years with normal cytogenetics enrolled onto AML'96, 307 had diagnostic PB samples stored in the DSIL tissue bank. There was no significant difference in percentage of PB blasts between the 307 available samples and the remaining 166 (P = .67). Pathologic diagnoses were reviewed centrally and classified according to the French-American-British (FAB) schema. Pretreatment cytogenetic analyses of bone marrow (BM) or PB were performed in institutional cytogenetics laboratories, and karyotypes were reviewed centrally. Patients were classified as having normal cytogenetics on the basis of analysis of BM or PB metaphases; in most cases 20 metaphases were assessable.¹⁶

When possible, patients had a centrally determined pretreatment BM FLT3 and MLL genotype.⁸ Of the 307 patients analyzed for BAALC expression, 268 had sufficient material for FLT3 mutation studies and 179 for analysis of the MLL partial tandem duplication (PTD).

FLT3 and MLL Studies
The FLT3 genotype was determined as previously described.¹¹ Polymerase chain reaction (PCR) for exons 14 and 15 was performed on genomic DNA using published primer molecules. For Genescan analysis of the FLT3 mutant:wild-type ratio (FLT3 ratio) PCR primer FLT3 14F was labeled with 6-FAM (TIB MOLBIOL, Berlin, Germany). Analysis of the MLL gene PTD has been published.⁸ Reverse transcriptase (RT-) PCR assays were used to amplify the mutant MLL transcript. Confirmation was performed by DNA sequencing and for selected samples by Southern Blot analysis.

BAALC Determination
Total RNA was isolated using the RNAzol B kit (Biozol, Munich, Germany), and complementary DNA (cDNA) synthesized using 1 µg of total RNA. Comparative real-time RT-PCR assays were performed as described with slight modifications.¹⁴ Each sample was analyzed in duplicate. Glucose-phosphate isomerase (GPI) and BAALC were coamplified in the same tube using 1 µL cDNA, 1x master mix (IQ Mix; BioRad, Munich, Germany), GPI probe (5'-HEX-TTCAGCTTGACCCTCAACACCAAC-TAMRA) with GPI forward (5'TCTTCGATGCCAACAAGGAC) and reverse (5'GCATCACGTCCTCCGTCAC) primers, and BAALC probe (5'-FAM-CTCTTTTAGCCTCTGTGGTCTGAAGGCCAT-TAMRA) with BAALC forward (5'GCCCTCTGACCCAGAAACAG) and reverse (5'CTTTTGCAGGCATTCTCTTAGCA) primers. Reactions were performed using the Rotor Gene Real-Time PCR 3000 Machine (Corbett Research, Uppsala, Sweden). The comparative cycle threshold (C_T) method was used to determine the relative expression levels of BAALC, and the cycle number difference (C_T = GPI – BAALC) was calculated for each replicate. Relative BAALC expression values were calculated using the mean of C_T from the two replicates, that is µ(C_T) = (C_T)/2, and expressed as 2^µ(CT). For samples without detectable BAALC amplification within 60 cycles, BAALC expression values were set at 0 (n = 74). A calibrator (RNA from the cell line KG1a) included in each run was used for standardization between runs. Positive and negative controls were included in all assays.

Statistical Analysis
Baseline clinical features across groups were compared using the ² or a two-sided Fisher's exact test for categoric and the nonparametric Mann-Whitney U test for continuous variables. A P value less than .05 was considered significant.

Achievement of complete remission (CR) was assessed after completion of the second induction cycle and required granulocytes 1,500/µL, platelets 100,000/µL, no PB blasts, BM cellularity more than 20% with maturation of all cell lines, no Auer rods, less than 5% BM blasts, and no extramedullary leukemia. Primary resistance to chemotherapy was defined as more than 25% blasts in the BM, lack of regeneration of normal hematopoiesis, persistence of PB blasts, or extramedullary leukemia after induction. Relapse was defined as the reappearance of circulating blasts, more than 10% BM blasts, more than 20% blasts and promyelocytes in two consecutive BM aspirates within 14 days, or development of extramedullary leukemia.

Overall survival (OS) including all 307 patients was calculated using the Kaplan-Meier method, and the log-rank test was used to compare differences between survival curves.¹⁷ OS was measured from the protocol on-study date until the date of death regardless of cause, censoring for patients alive at last follow-up. Cumulative incidence of relapse (CIR) was defined for only those patients achieving a CR (n = 208; Supplementary Figure A2, online only). It was measured from the CR date until date of relapse, death or date last known alive, where death during CR was considered a competing risk. Median follow-up for patients alive was 29 months (range, one to 85 months). Estimates of CIR were calculated, and the differences among groups determined by Gray's k-sample test.¹⁸

BAALC expression values failed to satisfy the proportional hazards assumption; therefore, BAALC was dichotomized at its median with patients classified as low BAALC if they had expression values within the lower 50% and as high BAALC if they had BAALC expression values within the upper 50%. When patients were grouped into quartiles according to BAALC expression, differences between the first and second (OS, P = .8), and the third and fourth quartile (OS, P = 1.0) were not statistically significant. In contrast, comparing the first or second to the third or fourth quartiles, Kaplan-Meier analyses detected significantly inferior outcomes of the latter, supporting the use of the median for categorizing BAALC expression into low and high risk (Supplementary Figure A3, online only).

Logistical regression models were constructed for CR and primary resistance, Cox proportional hazards models for OS, and a multivariable model using Gray's method was constructed for CIR. All covariates whose univariate models reflected a P value less than .2 from the likelihood ratio test were considered for inclusion in the model. Stepwise forward selection was performed and the model-building process was stopped once the addition of variables was no longer significant at = .05. Calculations were performed using SPSS software package, version 12 (SPSS Inc, Chicago, IL), and CIR was calculated using Gray's algorithm (http://biowww.dfci.harvard.edu/gray/) and S-PLUS software, version 6.2 (Insightful AG, Reinach, Switzerland).

	RESULTS

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BAALC Expression and Clinical Features
There were no significant differences between patients with low and high BAALC expression in pretreatment age, sex, WBC, percentage of BM blasts, or history of MDS/tAML (Table 1). High-BAALC patients presented with a higher percentage of blood blasts (P = .004). High BAALC expression was associated with the more immature FAB subtypes M0/M1 (P = .001; Table 1), whereas the monocytic differentiated FAB M5b correlated with low BAALC expression (P = .001). Gingival hyperplasia was more frequent in low-BAALC patients (P = .003).

BAALC Expression and Achievement of CR
High BAALC expressers less often achieved CR than low BAALC expressers (62% v 73%; P = .038) and more often had primary resistant disease (16% v 6%; P = .006; Table 2). On multivariable analysis, neither BAALC expression nor the FLT3 ratio were independent factors for achievement of CR, but BAALC predicted primary resistance with an odds ratio of 2.8 for high-BAALC patients (P = .019; Table 3).

View larger version (19K): [in this window] [in a new window]   Fig 1. Kaplan and Meier analysis of (A) cumulative incidence of relapse and (B) overall survival showing a significantly inferior outcome for patients with high BAALC expression compared with patients with low BAALC expression.

BAALC and FLT3 Subgroups
We evaluated the impact of BAALC in conjunction with FLT3 mutation status. In multivariable analysis, the FLT3 ITD mutation status without quantitative determination for the mutated and wild-type allele was an independent prognostic factor predicting a higher CIR (P = .012), but not inferior OS. When the FLT3 mutation status was quantified by Genescan analysis, patients with a high FLT3 ratio (> 0.8) had an inferior outcome with a higher CIR (P < .0001) and inferior OS (P < .0001) compared with patients with FLT3 wild type or a low FLT3 ratio ( 0.8). Therefore, we defined patients with either FLT3 wild type or a low FLT3 ratio ( 0.8) as low-risk FLT3, and patients with a high FLT3 ratio as high-risk FLT3.

On the basis of BAALC expression and FLT3 risk status, we identified four subgroups: (I) BAALC low/FLT3 low risk (n = 125), (II) BAALC high/FLT3 low risk (n = 110), (III) BAALC low/FLT3 high risk (n = 12), and (IV) BAALC high/FLT3 high risk (n = 21). Low-risk FLT3 mutation status and low BAALC expression identified patients with the best outcome. An escalating risk for high CIR and inferior OS was noted for the groups as follows: low BAALC expression with low-risk FLT3, high BAALC expression with low-risk FLT3; low BAALC expression with high-risk FLT3; high BAALC expression with high-risk FLT3 (Table 4; Fig 2).

View larger version (16K): [in this window] [in a new window]   Fig 2. Outcome with respect to BAALC expression and FLT3 mutation status. Overall survival: group I, BAALC low/FLT3 low risk; group II, BAALC high/FLT3 low risk; group III, BAALC low/FLT3 high risk; group IV, BAALC high/FLT3 high risk. P value represents overall P values across the four groups.

For high BAALC patients undergoing allogeneic SCT (n = 26), a low CIR (3-year CIR, 16%) was observed compared with high-BAALC patients receiving autologous SCT (n = 22; 52%; P = .007; Fig 3A). In addition, high-BAALC patients undergoing allogeneic SCT had a similarly low CIR as low-BAALC (n = 22) patients (3-year CIR, 16% v 14%; P = .86; Fig 3B).

View larger version (17K): [in this window] [in a new window]   Fig 3. Consolidation treatment and BAALC expression. (A) Cumulative incidence of relapse (CIR) for patients with high BAALC expression receiving autologous (auto) stem-cell transplantation (SCT) or allogeneic (allo) SCT as consolidation therapy within the Deutsche Studieninitiative Leukämie AML'96 protocol. (B) CIR for patients with high and low BAALC expression receiving allo SCT as consolidation therapy.

	DISCUSSION

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To compare the two studies, we applied the same approach, utilizing pretreatment blood samples for the expression analyses. BAALC expression can be detected in total normal marrow,¹³ but not in normal blood. The higher BAALC expression levels in marrow from healthy donors are a result of the presence of normal CD34⁺ progenitor cells that physiologically express BAALC. Blood was studied in AML to avoid contaminating cells with physiologic BAALC expression. Another group, however, recently reported a strong correlation between BAALC expression levels in blood and marrow samples from AML patients.¹⁹

Although high BAALC expression was associated with a higher percentage of circulating blasts, and both variables were associated with outcome in the univariate analysis, in multivariable analyses only BAALC expression was identified as a significant independent prognostic factor.

Various molecular markers and global gene expression profiling have been investigated to improve risk profile characterization of AML patients with normal cytogenetics.^3,20-23 To date the most common and clinically important molecular defects are FLT3 ITD mutations; these are detected in 25% to 30% of cytogenetically normal AML and associated with inferior outcome.^9,24 Clinical trials are now evaluating the efficacy of targeting FLT3 by specific inhibitors.^25,26 We have demonstrated that high BAALC is significantly associated with a higher FLT3 ratio. For most AML patients (ie, those with a low risk FLT3 mutation status) BAALC expression allowed further discrimination of high- and low-risk patients. Importantly, in multivariable analysis high BAALC and a high FLT3 ratio were independently predictive of inferior OS and CIR. That both factors independently influence the clinical course suggests different molecular pathways may be involved.

Determination of BAALC expression in conjunction with FLT3 risk status appeared to identify four groups characterized by an increasingly adverse prognosis: BAALC low/FLT3 low risk, BAALC high/FLT3 low risk, BAALC low/FLT3 high risk, and BAALC high/FLT3 high risk. Indeed, high-level BAALC expression in conjunction with high-risk FLT3 mutation status identified patients with a predicted CIR at 20 months of 75% and 100% probability of dying by 3 years. Whereas high BAALC expression clearly identifies a new poor prognostic subgroup of patients within the FLT3 low risk group, future larger studies are required to clearly define the impact of BAALC expression within the FLT3 high-risk group.

We found a significantly higher rate of primary resistant leukemia for patients with high compared with low BAALC expression. The novel finding that high BAALC expression is associated with primary refractory disease in multivariable analysis underscores its importance for the choice of future remission-induction chemotherapy for this patient subgroup. We found high BAALC expression to be an independent factor predicting CIR, confirming its previously reported impact on disease-free survival.

In this study high-BAALC patients undergoing allogeneic transplantation had a low CIR, suggesting that these patients may benefit from this therapy. Allogeneic SCT conferred a similar low CIR for high and low BAALC patients treated on the AML'96 protocol. When additional patients who received autologous or allogeneic SCT in first CR off protocol were included (to increase the sample size), these findings were confirmed.

In summary, we conclude that high BAALC expression in conjunction with FLT3 mutation status successfully identifies high-risk patients within the heterogeneous group of cytogenetically normal AML patients. High BAALC expression is established as one of the most important independent risk factors associated with resistant disease, a high CIR, and inferior survival. Modulation of induction therapy and intensification of postremission therapy may be critical to improve outcome for these high-risk patients. Our preliminary results and exploratory analyses suggest that patients with high BAALC expression may benefit from consolidation with allogeneic SCT. However, a prospective randomized trial is necessary to confirm this. Moreover, further studies are needed to determine the yet-unknown function of BAALC protein in normal hematopoiesis and leukemia.

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
The following participants entered patients onto the trial: D. Huhn, O. Knigge (Universitätsklinikum Charité, Berlin); E. Späth-Schwalbe, S. Hesse-Amojo (Krankenhaus Spandau, Berlin); O. Rick, W. Siegert (Charité Campus Mitte, Berlin); R. Kolloch, U. Krümpelmann (Krankenanstalten Gilead, Bielefeld); K.-H. Pflüger, T. Wolff (Evang. Diakonissenanstalt Bremen); H.-H. Heidtmann (St Joseph-Hospital, Bremerhaven); F. Marquard (Allgemeines Krankenhaus, Celle); M. Hähnel, F. Fiedler, R. Herbst (Krankenhaus Küchwald, Chemnitz); M. Gramatzki, G. Helm (Universitätsklinikum, Erlangen); J.-G. Saal (Malteser Krankenhaus, Flensburg); H.-G. Höffkes, M. Arland (Städtisches Klinikum, Fulda); E. Faßhauer (St Elisabeth-Krankenhaus, Halle); N. Schmitz, P. Dreger (Allgemeines Krankenhaus St Georg, Hamburg); H. Schmidt, K. Buhrmann (Kreiskrankenhaus, Hameln); H. Dürk (St Marien-Hospital, Hamm); M. Burk (Klinikum Stadt, Hanau); A.-D. Ho, U. Mahlknecht (Universitätsklinikum, Heidelberg); A. Bartholomäus (St Bernward Krankenhaus, Hildesheim); A.A. Fauser (Klinik f. Hämatologie/Onkologie und KMT, Idar-Oberstein); H. Link, F.-G. Hagmann (Westpfalzklinikum, Kaiserslautern); G. Köchling (Kreiskrankenhaus, Leer); K.-P. Schalk (St Vincent-Krankenhaus, Limburg/Lahn); S. Fetscher (Städtisches Krankenhaus Süd, Lübeck); T. Wagner (Universitätsklinikum, Lübeck); A. Neubauer (Universitätsklinikum, Marburg); H. Bodenstein, J. Tischler (Klinikum Minden, Minden); H. Pohlmann, N. Brack (Städtisches Krankenhaus München Harlaching, München); M. Wilhelm, H. Wandt, K. Schäfer-Eckart, (Städtisches Klinikum, Nürnberg); B. Seeber (Klinikum Offenbach, Offenbach); F. Hirsch (Kreiskrankenhaus, Offenburg); T. Geer, H. Heißmeyer (Diakonie-Krankenhaus, Schwäbisch-Hall); J. Labenz (Ev. Jung-Stilling-Krankenhaus, Siegen); J. Kaesberger (Diakonissen-Krankenhaus, Stuttgart); W.E. Aulitzky, L. Leimer (Robert-Bosch-Krankenhaus, Stuttgart); M.R. Clemens, R. Mahlberg (Mutterhaus der Borromaerinnen, Trier); R. Schwerdtfeger (Deutsche Klinik für Diagnostik, Wiesbaden); R. Engberding, R. Winter (Stadtkrankenhaus, Wolfsburg); M. Sandmann (Klinikum St Antonius, Wuppertal); F. Weissinger, H. Rückle-Lanz (Universitätsklinikum, Würzburg).

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 

Conception and design: Claudia D. Baldus, Christian Thiede, Gerhard Ehninger Administrative support: Eckhard Thiel, Gerhard Ehninger Provision of study materials or patients: Christian Thiede, Gerhard Ehninger Collection and assembly of data: Claudia D. Baldus, Christian Thiede, Silke Soucek Data analysis and interpretation: Claudia D. Baldus, Christian Thiede, Silke Soucek, Clara D. Bloomfield Manuscript writing: Claudia D. Baldus, Christian Thiede, Clara D. Bloomfield Final approval of manuscript: Claudia D. Baldus, Christian Thiede, Clara D. Bloomfield, Eckhard Thiel, Gerhard Ehninger

 

	GLOSSARY

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
BAALC (brain and acute leukemia, cytoplasmic): The BAALC gene, located on chromosome 8q22.3, encodes a protein with no homology to any known proteins or functional domains. Expression of this gene was found mainly in neuroectoderm-derived tissues and hematopoietic precursors.

CIR (cumulative incidence of relapse): The use of competing risk analyses are indicated in the presence of competing events (such as death and relapse), and the Gray's test is a recommended method to estimate the CIR.

Gene expression profiling: Identifying the expression of a set of genes in a biologic sample (eg, blood, tissue) using microarray technology.

Mutations in the fms-like tyrosine kinase 3 (FLT3) receptor: The FLT3 gene, located at chromosome band 13q12,encodes a membrane-bound protein member of the class III tyrosine kinase recep-tor family. Receptors belonging to the tyrosine kinase family (eg, EGFR, PDGFR) are activated through the auto- or transphosphorylation of tyrosine residues in the cytoplasmic region of the receptors in an ATP-dependent manner. An internal tandem duplication of FLT3 is one of the most common mutations in normal karyotype acute myeloid leukemia, occurring in approximately 28% to 38% of these patients.

Risk-adapted treatment stratification: Intensive treatments such as allogeneic stem-cell transplantation are potentially curative in acute myeloid leukemia (AML) but remain to be associated with high treatment-related mortality. Molecular markers will likely be valuable to stratify karyotypically normal AML patients within risk-adapted therapies.

	Acknowledgment

 
We are grateful to Amy S. Ruppert for her statistical support. We thank Marita Hartwig, Ulrike Löwel, Marika Karger, and Peggy Grassmel for excellent technical assistance. We also thank the participants of the DSIL study group listed in the Appendix (online only) who entered their patients onto the trial.

	NOTES

 
Supported by Leukemia Clinical Research Foundation (C.D.Bloomfield) and supported by grants from the Deutsche Krebshilfe and the Bundesministerium f. Bildung u. Forschung (Kompetenznetzwerk "Akute und Chronische Leukämien"; G.E.).

C.D. Baldus and C. Thiede contributed equally to this work.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted February 15, 2005; accepted October 5, 2005.

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