J Clin Oncol 22: by American Society of Clinical Oncology INTRODUCTION

VOLUME 22 NUMBER 18 SEPTEMBER 15 2004 JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T Individual Patient Data Based Meta-Analysis of Patients Aged 16 to 60 Years With Core Binding Factor Acute Myeloid Leukemia: A Survey of the German Acute Myeloid Leukemia Intergroup R.F. Schlenk, A. Benner, J. Krauter, T. Büchner, C. Sauerland, G. Ehninger, M. Schaich, B. Mohr, D. Niederwieser, R. Krahl, R. Pasold, K. Döhner, A. Ganser, H. Döhner, and G. Heil From the Department of Internal Medicine III, University of Ulm, Ulm; Central Unit of Biostatistics, German Cancer Research Center Heidelberg, Heidelberg; Department of Hematology/ Oncology, University of Hannover, Hannover; Department of Internal Medicine A and Department of Medical Informatics and Bioinformatics, University of Münster, Münster; Department of Internal Medicine I, University of Dresden, Dresden; Department of Hematology/Oncology, University of Leipzig, Leipzig; and Ernst von Bergmann Klinik, Potsdam, Germany. Submitted March 1, 2004; accepted June 24, 2004. Supported by grant No. 01GI9981 from the Bundesministerium für Bildung und Forschung (Kompetenznetz Akute und chronische Leukämien ), Germany. Authors disclosures of potential conflicts of interest are found at the end of this article. Address reprint requests to Hartmut Döhner, MD, Department of Internal Medicine III, University of Ulm, Robert- Koch-Strasse 8, 89081 Ulm, Germany; e-mail: hartmut.doehner@ medizin.uni-ulm.de. A B S T R A C T Purpose To evaluate prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in adult patients with core binding factor (CBF) acute myeloid leukemias (AML). Patients and Methods Individual patient data based meta-analysis was performed on 392 adults (median age, 42 years; range, 16 to 60 years) with CBF AML (t(8;21), n 191; inv(16), n 201) treated between 1993 and 2002 in prospective German AML treatment trials. Results RFS was 60% and 58% and OS was 65% and 74% in the t(8;21) and inv(16) groups after 3 years, respectively. For postremission therapy, intention-to-treat analysis revealed no difference between intensive chemotherapy and autologous transplantation in the t(8;21) group and between chemotherapy, autologous, and allogeneic transplantation in the inv(16) group. In the t(8;21) group, significant prognostic variables for longer RFS and OS were lower WBC and higher platelet counts; loss of the Y chromosome in male patients was prognostic for shorter OS. In the inv(16) group, trisomy 22 was a significant prognostic variable for longer RFS. For patients who experienced relapse, second complete remission rate was significantly lower in patients with t(8;21), resulting in a significantly inferior survival duration after relapse compared with patients with inv(16). Conclusion We provide novel prognostic factors for CBF AML and show that patients with t(8;21) who experience relapse have an inferior survival duration. J Clin Oncol 22:3741-3750. 2004 by American Society of Clinical Oncology 2004 by American Society of Clinical Oncology 0732-183X/04/2218-3741/$20.00 DOI: 10.1200/JCO.2004.03.012 INTRODUCTION Cytogenetically, the group of core binding factor (CBF) acute myeloid leukemias (AML) is defined by the presence of the t(8;21)(q22;q22) or the inv(16)(p13q22)/ t(16;16)(p13;q22). CBFs are a family of heterodimeric transcriptional regulators containing a common beta subunit (CBF ) associated with one of three alpha subunits (CBF ). The t(8;21)(q22;q22) fuses the RUNX1 gene located on chromosome 21 to the CBFA2T1 gene located on chromosome 8. 1 AMLs carrying the t(8;21) are frequently associated with specific characteristics, such as morphologic presentation with the French-American-British subtype M2 with myeloid precursors containing Auer rods, 2 immunophenotypic aberrant expression of the CD19 antigen, 3 and, in some patients, by extramedullary disease (granulocytic 3741

Schlenk et al sarcomas). 2,4 Cytogenetically, the specific translocation may be associated with loss of a sex chromosome (LOS) or deletions of the long arm of chromosome 9 [del(9q)]. 5 Clinically, high complete remission (CR) rates after standard induction therapy and favorable outcome, especially after dose-intensified cytarabine-based postremission therapy, have been reported. 6-9 Inferior outcome has been reported in patients with high WBC or absolute granulocyte count, 9-11 phenotypical expression of the CD56 antigen, 12 additional del(9q), 13 or extramedullary disease. 4 However, these prognostic factors were identified in small retrospective studies, and only high WBC has been confirmed as a prognostically relevant variable in the French AML Intergroup study. 10 In the inv(16) group, the CBF gene located in 16q22 fuses to the MYH11 gene located in 16p13. AMLs carrying the inv(16) are frequently associated with specific characteristics, such as morphologic presentation with the French-American-British subtype M4eo with an abnormal eosinophilic differentiation 14 and, in some patients, extramedullary involvement. 2,15 Cytogenetically, the specific aberration may be associated with trisomies of the chromosomes 8, 21, and 22. 16,17 Clinically, high CR rates after standard induction therapy and favorable outcome have been reported. 6,9,17,18 However, there are conflicting data concerning the dose of cytarabine in postremission therapy. 6,7,18,19 Inferior outcome has been reported in patients presenting with high WBC counts 9,20 and older age. 18 Primary objectives of the present survey were to identify novel prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in a large series of young adults with CBF AML. PATIENTS AND METHODS Patient Selection and Review of the Data Between July 1993 and August 2002, patients were prospectively enrolled in one of the eight following German multicenter treatment trials: Sueddeutsche Haemoblastose Gruppe (SHG)- Hannover AML 2/95, 21 SHG-Hannover AML 1/99, SHG-Dresden AML 96, 22 Acute Myeloid Leukemia Study Group (AMLSG) ULM AMLHD93, 9 AMLSG ULM Acute Myeloid Leukemia Heidelberg 98A, 23 Acute Myeloid Leukemia Cooperative Group (AMLCG) 92, 24 AMLCG99, 25 and Ostdeutsche Studiengruppe Haematologie/Oukologie (033) AML-96. The inclusion criteria of the different trials were concordant, and the inclusion criteria for this survey were as follows: (1) presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16; 16)(p13;q22) on standard karyotypic analysis or presence of the RUNX1-CBFA2T1 or the CBF -MYH11 fusion gene by molecular screening, (2) age 16 to 60 years, and (3) availability of clinical data. Demographic, diagnostic, clinical, and laboratory data, cytogenetics, type of induction, postremission and salvage therapy, and outcome information were collected for each patient, sent to a central coordination center, and reviewed for consistency and completeness before analysis. Immunophenotyping of leukemic cells was not included in the analysis because of lack of consistent data. Cytogenetics In four of the five study groups, cytogenetic and molecular genetic studies were performed in central reference laboratories. Molecular genetic studies included screening for the gene fusion by fluorescence in situ hybridization or polymerase chain reaction in all samples (SHG-Hannover, n 83 26,27 ; SHG-Dresden, n 85 28-30 ; AMLCG, n 85 31,32 ; AMLSG ULM, n 109 33,34 ). In the remaining group (OSHO), cytogenetic analysis was conducted on an institutional level (n 30). The description of the karyotype followed the recommendations of the International System for Human Cytogenetic Nomenclature. 35 Protocols The protocols are summarized in Figure 1 and patient numbers per protocol are given in Table 1. More detailed description of the protocols are provided as online supplemental information. Statistical Analysis The median follow-up for survival was calculated according to the method of Korn. 36 The definition of CR followed the recommended criteria. 37 Because most of the trials followed double induction strategies, evaluation of response was performed after two induction cycles for all trials. OS end points, measured from entry onto one of the prospective studies, were death (failure) and alive at last follow-up (censored). 37 RFS end points, measured from the date of documented CR, were relapse (failure), death in CR (failure), and alive in CR at last follow-up (censored). 37 Consolidation therapy was classified into cytarabine-based chemotherapy (chemotherapy), autologous stem-cell transplantation (SCT), and allogeneic SCT for intention-to-treat analysis. To assess the impact of dosage of cytarabine on RFS and OS in patients receiving chemotherapy, the intended cumulative dosage over all cycles of chemotherapy was calculated. This was done because considerable amounts of cytarabine were given in induction and consolidation therapy. The total dose varied among the different trials between 20.8 g/m 2 and 56.8 g/m 2 and was entered as a continuous variable into univariate and multivariate analysis. Testing and estimation of possible cutoff values for continuous variables were done by maximally selected log-rank statistics. 38 Pairwise comparisons of patient characteristics were performed by the Mann-Whitney U test for continuous variables and by Fisher s exact test for categoric variables. The Kaplan-Meier method was used to estimate the distribution of RFS and OS. CI estimation for the survival curves was based on the cumulative hazard function using Greenwood s formula for the SE estimation. 39 Survival distributions were compared using the logrank test stratified for the variable study. A Cox model was used to identify prognostic variables. 40 Missing data were estimated using a multiple-imputation technique using predictive mean matching with n 100 imputations. 41 A limited backward-selection procedure with was used to exclude redundant or unnecessary variables. 41 To provide quantitative information on the relevance of results, 95% CIs of odds ratios and hazard ratios (HR) were computed. The statistical analyses were performed with the statistical software package R, version 1.7.1 42 together with the Design software library. 41 RESULTS Accrual of Patients and Their Initial Characteristics Between July 1993 and August 2002, 410 patients aged 16 to 60 years with CBF AML were registered. In 18 patients, 3742 JOURNAL OF CLINICAL ONCOLOGY

Core Binding Factor Acute Myeloid Leukemia Fig 1. Summary of the eight prospective treatment trials. IVA, idarubicin, etoposide, cytarabine; HAD, high-dose cytarabine; Auto-SCT, autologous stem-cell transplantation; MAV, mitoxantrone, cytarabine, etoposide; MAMAC, cytarabine, m-amsa; MAC, mitoxantrone and cytarabine; Allo- SCT, allogeneic stem-cell transplantation; ICE, idarubicin, cytarabine, etoposide; HAM, high-dose cytarabine and mitoxantrone; TAD, thioguanine, cytarabine, daunorubicin; S-HAM, sequential HAM; AI, cytarabine and idarubicin; AM, cytarabine and mitoxantrone. no clinical data were available, leading to 392 eligible patients for this survey. Table 2 shows the distribution of clinical variables by type of CBF AML. Patients exhibiting inv(16) showed a statistically significant higher WBC count at diagnosis. Some patients had evidence of extramedullary involvement at diagnosis, including lymphadenopathy, granulocytic sarcoma, and CNS involvement. There was a statistically significant higher rate of lymphadenopathy and skin as well as mucosa involvement in the inv(16) group, whereas chloromas were more frequent in the t(8;21) group. However, diagnostic procedures were symptomorientated, and computed tomography scans and CSF examination were not performed systematically. Cytogenetics In six of 191 patients, t(8;21) was only identified by molecular techniques; in all six patients, there were no assessable metaphases on conventional cytogenetics. The most common additional aberration was LOS in 49%: 31 of 75 female patients had lost of one X chromosome, 58 of 110 male patients had loss the Y chromosome, and only one male patient had loss of the X chromosome. Del(9q) was Table 1. Total Number of Patients and Number of Patients per Postremission Therapy in First Complete Remission in the Various Treatment Trials t(8;21) Treatment Trial Total Chemo Auto Allo Total Chemo Auto Allo SHG-Hannover AML 2/95 30 22 3 1 18 12 2 0 AML 1/99 20 7 7 1 15 7 4 1 SHG-Dresden AML 96 35 29 2 2 50 14 17 9 AMLCG AMLCG92 20 16 0 1 19 17 0 1 AMLCG99 21 13 2 0 25 13 4 2 OSHO (033) AML96 16 10 2 2 14 5 3 3 AMLSG ULM AMLHD93 17 13 2 1 25 19 0 3 AMLHD98A 32 26 0 0 35 11 17 6 Abbreviations: Chemo, chemotherapy; Auto, autologous transplantation; Allo, allogeneic transplantation; SHG, Sueddeutsche Haemoblastose Gruppe; AML, acute myeloid leukemia; CG, Cooperative Group; OSHO, Ostdeutsche Studiengruppe Heamatologie/Oukologie; HD, Heidelberg. inv(16) www.jco.org 3743

Schlenk et al Table 2. Distribution of Factors by Type of Core Binding Factor Acute Myeloid Leukemia at Diagnosis t(8;21) (n 191) No. of Patients % inv(16) (n 201) No. of Patients % Sex Male 114 60 110 55 NS Female 77 40 91 45 Age, years Median 43 42 NS Range 16-60 17-60 FAB type RAEBt 1 0 M0 1 0 M1 11 8 M2 160 7 M3 3 0 M4 11 181 M5 1 3 Missing 3 2 Extramedullary manifestation 17/187 9 42/193 22.005 Lymphadenopathy 0 26 13.001 CNS 1 0.5 2 1 NS Chloroma 10 5 1 0.5.005 Skin/mucosa 4 2 13 7.04 Liver/lung 2 1 0 0 NS WBC count, 10 9 /L No. of patients 178 193 Median 10.8 35.8.001 Range 1.2-152 1.2-395 Platelet count, 10 9 /L No. of patients 185 193 Median 30 34 NS Range 3-470 7-240 Hemoglobin level, g/dl No. of patients 184 192 Median 8.5 8.4 NS Range 1.7-15.1 2.2-14.2 BM blast, % No. of patients 181 186 Median 60 70 NS Range 6.5-100 7-100 Abbreviations: NS, not significant; FAB, French American British; RAEBt, refractory anemia with excess blasts in transformation; BM, bone marrow. P present in 17% (Table 3). Variant t(8;21) was identified by conventional cytogenetics and confirmed by molecular techniques in 11 patients. In 14 of 201 patients, inv(16) was only identified by molecular techniques: no metaphases were available in four patients, a normal karyotype was found in seven patients, and a del(16)(q22) was detected on conventional cytogenetics in three patients. In all these patients, the CBF - MYH11 fusion was confirmed by molecular techniques. The evaluation of additional chromosomal aberrations was restricted to cases with karyotypically confirmed inv(16) (Table 2). The most common additional aberration was trisomy 22 in 22%, followed by trisomy 8 in 12%. There was no association between age, sex, and frequency of additional chromosomal aberrations. Induction Therapy Results of induction therapy were as follows: CR in 87% (166 of 191 patients) and 89% (179 of 201 patients), early or hypoplastic death (ED/HD) in 10% (19 of 191 patients) and 8.5% (17 of 201 patients), and resistant disease in 3% (six of 191 patients) and 2.5% (five of 201 patients) for the t(8;21) and inv(16) groups, respectively. The following variables were analyzed for their potential influence on response to induction therapy: WBC count, platelet count, hemoglobin level, bone marrow and 3744 JOURNAL OF CLINICAL ONCOLOGY

Core Binding Factor Acute Myeloid Leukemia t(8;21) (n 185) Table 3. Additional Chromosomal Aberrations inv(16) (n 179) t(16;16) (n 8) Type No. of Patients % Type No. of Patients % No. of Patients % As sole 58 31 As sole 103 58 6 75 Y 58/110 53 Trisomy 22 39 22 2 25 X 31/75 41 Trisomy 8 23 12 del(9q) 31 17 Trisomy 21 9 5 Trisomy 8 10 5 Other 37 19 Other 33 18 peripheral-blood blast cell count, age, sex, and cytogenetics (t(8;21): LOS, del(9q); inv(16): trisomy 22, trisomy 8). In the inv(16) group, higher WBC count (P.01) and older age (P.04) were associated with an increased ED/HD rate. In the t(8;21) group, no variable had a prognostic impact. Survival Analysis The median follow-up time for survival was 36 months. Sixty-two of 191 and 50 of 201 patients died, resulting in an estimated survival after 36 months of 65% (95% CI, 58% to 73%) and 74% (95% CI, 68% to 81%) for the t(8;21) and the inv(16) groups, respectively (Fig 2). Of the 166 and 179 patients achieving CR after induction therapy, 44 and 58 patients experienced relapse, whereas 11 and 10 patients died in first CR from treatment-related complications in the t(8;21) group and the inv(16) group, respectively. The estimated RFS after 36 months was 60% (95% CI, 53% to 69%) and 58% (95% CI, 51% to 67%) in the t(8;21) and the inv(16) groups, respectively (Fig 2). Of 345 patients achieving CR after induction therapy, four and nine patients were ineligible for postremission therapy, leaving 162 and 170 assessable patients in the t(8;21) and the inv(16) groups, respectively. RFS and OS were analyzed on an intention-to-treat basis for both groups. In the t(8;21) group, only two patients were assigned to an allogeneic SCT, and they were therefore not included in the intention-to-treat analysis. There was no difference in RFS between chemotherapy and autologous SCT in the t(8;21) group (P.81) and between chemotherapy, autologous SCT, and allogeneic SCT in the inv(16) group (P.22; Fig 3). The as-treated analysis for patients receiving chemotherapy as postremission therapy revealed an estimated RFS and OS after 36 months of 62% (95% CI, 53% to 72%) and 75% (95% CI, 68% to 84%) in the t(8;21) group (n 136) and 52% (95% CI, 43% to 64%) and 85% (95% CI, 76% to 92%) in the inv(16) group (n 98), respectively. Forty-one of 44 and 56 of 58 patients who experienced relapse received intensive reinduction therapy, including four and five patients allocated to allogeneic SCT without reinduction therapy in the t(8;21) and the inv(16) groups, respectively. The second CR rate was significantly lower at 33% (15 of 45 patients) in the t(8;21) group, compared with 78% (45 of 58) in the inv(16) group (P.001; odds ratio, Fig 2. Overall treatment results. (A) Relapse-free survival; (B) overall survival. www.jco.org 3745

Schlenk et al Fig 3. Relapse-free survival by postremission therapy. (A) t(8;21); (B) inv(16). SCT, stem-cell transplantation; CHEMO, chemotherapy; AUTO, autologous; ALLO, allogenic. 7.4; 95% CI, 2.8 to 20.7), leading to a significantly (P.05) lower percentage of patients receiving an intensive second consolidation therapy (t(8;21): matched related donor [MRD] SCT, n 17; matched unrelated donor [MUD] SCT, n 2; autologous SCT, n 0; inv(16): MRD-SCT, n 15; MUD-SCT, n 13; autologous SCT, n 3; intensive chemotherapy, n 6). The survival after relapse was significantly better (P.001) in patients exhibiting inv(16) compared with those exhibiting t(8;21) (Fig 4). Evaluation of Prognostic Variables in t(8;21) Analyses of prognostic factors for RFS were performed in the subset of patients receiving chemotherapy for postremission therapy (n 136), as well as in the whole group of patients achieving CR after induction therapy (n 166). The following variables were evaluated: age, sex, WBC count, WBC index (product of WBC count and percentage of bone marrow blasts), 10 bone marrow and peripheralblood blast cell count, platelet count, hemoglobin level, Fig 4. Survival after relapse. cumulative dose of cytarabine over all cycles, additional chromosomal aberrations (LOS, del(9q), trisomy 8), and study. The variable treatment (SCT v no SCT) was included on an intention-to-treat basis in the second analysis. All continuous variables were included in the model either continuously or dichotomized, if a cut point in maximally selected log-rank statistics was evident. For WBC count, a cut point was found at 25.4 10 9 /L (P.05), whereas the cut point selection was done between 10.0 and 40.0 10 9 /L. For platelet count, a cut point was found at 28 10 9 /L (P.05) with a cut point selection between 10.0 and 40.0 10 9 /L. For the other continuous variables, no cut points were evident. The multivariate model for RFS with limited backward selection revealed dichotomized WBC count (high v low) and platelet count (high v low) as prognostic variables in patients receiving chemotherapy as postremission therapy (HR, 2.68; 95% CI, 1.19 to 6.04; and HR, 0.45; 95% CI, 0.23 to 0.90, respectively) as well as in the whole group (HR, 2.39; 95% CI, 1.25 to 4.54; and HR, 0.54; 95% CI, 0.30 to 0.95, respectively). By combining dichotomized WBC and platelet count in a hierarchical model for RFS, three risk groups could be established: a high-risk group with WBC count greater than 25.4 10 9 /L, an intermediate-risk group with WBC count 25.4 10 9 /L and platelet count 28 10 9 /L, and a low-risk group with WBC count 25.4 10 9 /L and platelet count greater than 28 10 9 /L (Fig 5). There was no impact of the total dose of cytarabine given during induction and consolidation therapy on RFS analyzed on an intention-to-treat basis (P.85). In patients who experienced relapse, only univariate analyses were performed because of the low patient number. The analyses revealed that LOS (P.04) and WBC count at diagnosis of greater than 25.4 10 9 /L (P.01) but not duration of first CR with a cut point of 1 year (P.08) were variables associated with an inferior survival after 3746 JOURNAL OF CLINICAL ONCOLOGY

Core Binding Factor Acute Myeloid Leukemia Fig 5. (A) Relapse-free survival; (B) overall survival. Prognostic model for t(8;21) leukemias. Low risk: WBC 25.4 10 9 /L and platelet count 28 10 9 /L; intermediate risk: WBC 25.4 10 9 /L and platelet count 28 10 9 /L; high risk: WBC 25.4 10 9 /L. relapse. There was an interaction between LOS and duration of first CR, with a significantly shorter duration of first CR in patients with loss of the Y chromosome (P.03). Finally, we applied the prognostic factors to OS of all assessable t(8;21) patients. The Cox regression model revealed platelet count 28 10 9 /L (HR, 2.88; 95% CI, 1.62 to 5.14), WBC count greater than 25.4 10 9 /L (HR, 2.15; 95% CI, 1.16 to 3.97), and LOS (HR, 2.42; 95% CI, 1.38 to 4.24) as prognostic factors. The negative impact of LOS on survival was due to a significantly inferior survival of male patients with loss of the Y chromosome (log-rank test P.005), whereas in female patients, LOS had no impact on OS (log-rank test P.59; Fig 6). Evaluation of Prognostic Variables in inv(16) Analyses of prognostic factors for RFS were performed in the subset of patients receiving chemotherapy for postremission therapy (n 98), as well as in the whole group of patients achieving CR after induction therapy (n 178). The following variables were evaluated: age, sex, WBC count, bone marrow and peripheral-blood blast cell count, platelet count, hemoglobin level, cumulative dose of cytarabine over all cycles, additional chromosome aberrations (trisomy 22, trisomy 8), and study. The variable treatment was included on an intention-to-treat basis in the second analysis. For continuous variables no cut points were evident; therefore, they were included in the models as continuous variables. There was an interaction between WBC count and trisomy 22, with significantly lower WBC count at diagnosis in patients exhibiting the additional aberration (P.0002). The multivariate model for RFS with limited backward selection revealed trisomy 22 as the only prognostic variable in patients receiving chemotherapy as Fig 6. Overall survival of patients with t(8;21) grouped by sex and loss of a sex chromosome. (A) Female patients; (B) male patients. www.jco.org 3747

Schlenk et al Fig 7. Relapse-free survival of patients with inv(16) by presence or absence of trisomy 22. postremission therapy (HR, 0.37; 95% CI, 0.13 to 1.04), as well as in the whole group (HR, 0.35; 95% CI, 0.15 to 0.80; Fig 7). There was no prognostic impact of the cumulative dose of cytarabine on RFS analyzed on an intention-to-treat basis (P.19). In patients who experienced relapse, no prognostic variable was evident in univariate analyses. Finally, we applied the prognostic factors for RFS to OS of all inv(16) patients. The Cox regression model did not reveal a prognostic impact of trisomy 22 on OS. DISCUSSION This meta-analysis of several German AML trials was conducted because patient numbers in prospective AML studies are too low to produce conclusive results in cytogenetic subgroups. Individual patient data based meta-analyses are not able to substitute prospective trials, and inhomogeneities such as different randomization strategies among trials have to be taken into account. Entering individual patients characteristics, however, provides a tool to increase statistical power. This survey is based on 392 young adult patients with CBF AML and is, to our knowledge, the largest cohort so far presented. Induction therapies resulted in high CR rates, consistent with data from others. 5,6,17,18 In accordance with the French AML Intergroup, 18 we identified an association between high WBC count and ED/HD in the inv(16) group. In contrast, we were not able to define a cut point for WBC count or to confirm the prognostic value of low platelet count and trisomy 22 for induction failure. The intentionto-treat analysis for postremission therapy revealed no difference in RFS and OS between chemotherapy and autologous SCT in t(8;21) leukemias and similarly no difference in RFS and OS between chemotherapy, autologous transplantation, and allogeneic transplantation in inv(16) leukemias, which is consistent with the data from the MRC AML-10 trial and the French AML Intergroup. 6,18 However, the median follow-up time is still short, especially for patients after autologous and allogeneic transplantation. For patients receiving cytarabine-based consolidation therapy, we were not able to show an interaction between RFS and intended total dose of cytarabine, which ranged from 20.8g/m 2 to 56.8g/m 2. This is at variance with the data reported by Cancer and Leukemia Group B that showed a significantly better RFS for patients assigned to repetitive cycles of high-dose cytarabine in both types of CBF AML. 7,8,19 Our data are in accordance with those of the French AML Intergroup, 10,18 which showed no difference between intermediate- and high-dose cytarabine in postremission therapy with respect to RFS. There were major differences between the two types of CBF AML with respect to outcome after relapse. The CR rate after reinduction therapy was significantly lower in patients with t(8;21) compared with patients with inv(16) (33% and 79%, respectively). As a consequence, there was a significantly lower proportion of patients who experienced relapse with t(8;21) receiving intensive consolidation therapy, resulting in a significantly inferior survival after relapse (Fig 4). The reason why leukemias with t(8;21) are less sensitive to salvage therapy remains unclear. In accordance with the French AML Intergroup, 10 WBC count was identified as a prognostic marker in the t(8;21) group. In our survey, the optimal cut point defined by maximally selected log-rank statistics was 25.4 10 9 /L and nearly reproduced the cut point of 30.0 10 9 /L reported by the French AML Intergroup. 18 The WBC index, defined as product of WBCs and percentage of bone marrow blasts, 10 did not add prognostic information to WBC count in our survey. Thus the definition of high-, intermediate-, and low-risk patients proposed by the French AML Intergroup could not be reproduced in our series. In contrast, we identified platelet count with a cut point at 28 10 9 /L as second prognostic variable for RFS, and bycombining dichotomized WBC and platelet count, we were able to build a prognostic model for RFS that identified high-, intermediate-, and low-risk patients (Fig 5). Two large studies by the British Medical Research Council 6 and Cancer and Leukemia Group B 17 analyzed the impact of additional chromosomal aberrations in CBF AML. In both studies, there was no influence of additional chromosomal aberrations on either RFS or OS. In contrast, in our survey, which is based on a significantly larger patient number, we were able to identify two additional chromosomal aberrations that appeared as independent prognostic variables. LOS in t(8;21) leukemias was a weak but significant prognostic factor for OS. When analyzing by sex, we 3748 JOURNAL OF CLINICAL ONCOLOGY

Core Binding Factor Acute Myeloid Leukemia found that this effect was exclusively due to the negative impact of loss of Y chromosome in male patients (Fig 6). This effect was independent of age (data not shown). Furthermore, we identified trisomy 22 in inv(16) leukemias as an independent predictor for superior RFS (Fig 7). Interestingly, trisomy 22 was the sole independent prognostic factor, whereas clinical variables as previously 9,18,19 reported did not enter the model. Age was not a significant prognostic factor, possibly because our study was restricted to young adults (16 to 60 years) and did not include children. 6,18 In contrast to the French study, trisomy 22 in our series was associated with significantly lower WBC counts. The mechanism by which this aberration improves outcome remains elusive. This meta-analysis comprises a large series of young adult patients with CBF AML receiving state-of-the-art therapy. In this survey, we were able to identify novel biologic risk factors that could help to stratify such patients in the future. Acknowledgment The acknowledgment is included in the full-text version of this article, available online at www.jco.org. It is not included in the PDF (via Adobe Acrobat Reader ) version. Appendix The appendix is included in the full-text version of this article, available online at www.jco.org. It is not included in the PDF (via Adobe Acrobat Reader ) version. Authors Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. REFERENCES 1. Nucifora G, Rowley JD: The AML1 and ETO genes in acute myeloid leukemia with a t(8;21). Leuk Lymphoma 14:353-362, 1994 2. Bloomfield CD, de la Chapelle A: Chromosome abnormalities in acute nonlymphocytic leukemia: Clinical and biologic significance. Semin Oncol 14:372-383, 1987 3. Kita K, Nakase K, Miwa H, et al: Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: Frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34. Blood 80:470-477, 1992 4. Tallman MS, Hakimian D, Shaw JM, et al: Granulocytic sarcoma is associated with the 8;21 translocation in acute myeloid leukemia. J Clin Oncol 11:690-697, 1993 5. Fourth International Workshop on Chromosomes in Leukemia 1982: translocation (8; 21)(q22;q22) in acute nonlymphocytic leukemia. Cancer Genet Cytogenet 11:284-287, 1984 6. 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. Blood 92:2322-2333, 1998 7. Bloomfield CD, Lawrence D, Byrd JC, et al: Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype. Cancer Res 58:4173-4179, 1998 8. Byrd JC, Dodge RK, Carroll A, et al: Patients with t(8;21)(q22;q22) and acute myeloid leukemia have superior failure-free and overall survival when repetitive cycles of high-dose cytarabine are administered. J Clin Oncol 17:3767-3775, 1999 9. Schlenk RF, Benner A, Hartmann F, et al: Risk-adapted postremission therapy in acute myeloid leukemia: Results of the German multicenter AML HD93 treatment trial. Leukemia 17:1521-1528, 2003 10. Nguyen S, Leblanc T, Fenaux P, et al: A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): A survey of 161 cases from the French AML Intergroup. Blood 99:3517-3523, 2002 11. Billstrom R, Johansson B, Fioretos T, et al: Poor survival in t(8;21)(q22;q22)-associated acute myeloid leukaemia with leukocytosis. Eur J Haematol 59:47-52, 1997 12. Baer MR, Stewart CC, Lawrence D, et al: Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). Blood 90:1643-1648, 1997 13. Schoch C, Haase D, Haferlach T, et al: Fifty-one patients with acute myeloid leukemia and translocation t(8;21)(q22;q22): An additional deletion in 9q is an adverse prognostic factor. Leukemia 10:1288-1295, 1996 14. Le Beau MM, Larson RA, Bitter MA, et al: Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia: A unique cytogeneticclinicopathological association. N Engl J Med 309:630-636, 1983 15. Billstrom R, Ahlgren T, Bekassy AN, et al: Acute myeloid leukemia with inv(16)(p13q22): Involvement of cervical lymph nodes and tonsils is common and may be a negative prognostic sign. Am J Hematol 71:15-19, 2002 16. Mrozek K, Heinonen K, de la Chapelle A, et al: Clinical significance of cytogenetics in acute myeloid leukemia. Semin Oncol 24:17-31, 1997 17. Byrd JC, Mrozek K, Dodge RK, et al: Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: Results from Cancer and Leukemia Group B (CALGB 8461). Blood 100:4325-4336, 2002 18. Delaunay J, Vey N, Leblanc T, et al: Prognosis of inv(16)/t(16;16) acute myeloid leukemia (AML): A survey of 110 cases from the French AML Intergroup. Blood 102:462-469, 2003 19. Byrd JC, Ruppert AS, Mrozek K, et al: Repetitive cycles of high-dose cytarabine benefit patients with acute myeloid leukemia and inv(16)(p13q22) or t(16;16)(p13;q22): Results from CALGB 8461. J Clin Oncol 2004 22:1087-1094 20. Martin G, Barragan E, Bolufer P, et al: Relevance of presenting white blood cell count and kinetics of molecular remission in the prognosis of acute myeloid leukemia with CBFbeta/ MYH11 rearrangement. Haematologica 85:699-703, 2000 21. Heil G, Krauter J, Raghavachar A: Riskadapted induction and consolidation therapy in adults with de novo AML aged / 60 years: Results of a prospective multicenter trial. Ann Hematol 83:336-344, 2004 22. Schaich M, Ritter M, Illmer T, et al: Mutations in ras proto-oncogenes are associated with lower mdr1 gene expression in adult acute myeloid leukaemia. Br J Haematol 112:300-307, 2001 23. Schlenk RF, Döhner H: Risk adapted therapy of AML: The AMLSG ULM experience. Ann Hematol 83:S49, 2004 (suppl 1) 24. Büchner T, Hiddemann W, Berdel WE, et al: 6-Thioguanine, cytarabine, and daunorubicin (TAD) and high-dose cytarabine and mitoxantrone (HAM) for induction, TAD for consolidation, and either prolonged maintenance by reduced monthly TAD or TAD-HAM-TAD and one course of intensive consolidation by sequential HAM in adult patients at all ages with de novo acute myeloid leukemia (AML): A randomized trial of the German AML Cooperative Group. J Clin Oncol 21:4496-4504, 2003 25. Büchner T, Hiddemann W, Berdel WE, et al: Upfront randomized TAD-HAM vs HAM-HAM induction, G-CSF priming vs no G-CSF, and prolonged maintenance vs autologous transplantation in de novo AML, secondary AML and highrisk MDS and their subgroups according to cytogenetics and LDH: Interim analysis. Blood 100:345a, 2002 (abstr) 26. Krauter J, Ganser A, Bergmann L, et al: Association between structural and numerical chromosomal aberrations in acute myeloblastic leukemia: A study by RT-PCR and FISH in 447 patients with de-novo AML. Ann Hematol 78: 265-269, 1999 27. Krauter J, Peter W, Pascheberg U, et al: Detection of karyotypic aberrations in acute myeloblastic leukaemia: A prospective comparison www.jco.org 3749

Schlenk et al between PCR/FISH and standard cytogenetics in 140 patients with de novo AML. Br J Haematol 103:72-78, 1998 28. Thiede C, Steudel C, Mohr B, 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 29. Mohr B, Bornhauser M, Thiede C, et al: Comparison of spectral karyotyping and conventional cytogenetics in 39 patients with acute myeloid leukemia and myelodysplastic syndrome. Leukemia 14:1031-1038, 2000 30. Ritter M, Thiede C, Schakel U, et al: Underestimation of inversion (16) in acute myeloid leukaemia using standard cytogenetics as compared with polymerase chain reaction: Results of a prospective investigation. Br J Haematol 98: 969-972, 1997 31. Schoch C, Kohlmann A, Schnittger S, et al: Acute myeloid leukemias with reciprocal rearrangements can be distinguished by specific gene expression profiles. Proc Natl Acad Sci U S A 99:10008-10013, 2002 32. Schoch C, Schnittger S, Klaus M, et al: AML with 11q23/MLL abnormalities as defined by the WHO classification: Incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood 102:2395-2402, 2003 33. Fischer K, Scholl C, Salat J, et al: Design and validation of DNA probe sets for a comprehensive interphase cytogenetic analysis of acute myeloid leukemia. Blood 88:3962-3971, 1996 34. Frohling S, Skelin S, Liebisch C, et al: Comparison of cytogenetic and molecular cytogenetic detection of chromosome abnormalities in 240 consecutive adult patients with acute myeloid leukemia: A study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol 20:2480-2485, 2002 35. Mitelman F: ISCN: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland, Karger, 1995 36. Korn EL: Censoring distributions as a measure of follow-up in survival analysis. Stat Med 5:255-260, 1986 37. Cheson BD, Bennett JM, Kopecky KJ, et al: Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 21:4642-4649, 2003 38. Lausen B, Schumacher M: Maximally selected rank statistics. Biometrics 48:73-85, 1992 39. Therneau TM, Grambsch PM: Modeling Survival Data: Extending the Cox Model. New York, NY, Springer Verlag, 2000 40. Cox DR: Regression models and life tables. J R Stat Soc B 34:187-220, 1972 41. Harrell FE: Regression modeling strategies: With applications to linear models, logistic regression, and survival analysis. New York, NY, Springer Verlag, 2001 42. Ihaka R, Gentleman R: A Language for data analysis and graphics. J Computational Graphical Stat 5:299-314, 1996 3750 JOURNAL OF CLINICAL ONCOLOGY