J Clin Oncol 26: by American Society of Clinical Oncology INTRODUCTION

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1 V O L U M E 2 6 N U M B E R 1 8 J U N E JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T Minimal Residual Disease Values Discriminate Between Low and High Relapse Risk in Children With B-Cell Precursor Acute Lymphoblastic Leukemia and an Intrachromosomal Amplification of Chromosome 21: The Austrian and German Acute Lymphoblastic Leukemia Berlin-Frankfurt- Münster (ALL-BFM) Trials Andishe Attarbaschi, Georg Mann, Renate Panzer-Grümayer, Silja Röttgers, Manuel Steiner, Margit König, Eva Csinady, Michael N. Dworzak, Markus Seidel, Dasa Janousek, Anja Möricke, Carsten Reichelt, Jochen Harbott, Martin Schrappe, Helmut Gadner, and Oskar A. Haas From the Department of Pediatric Hematology and Oncology, St Anna Children s Hospital; the Children s Cancer Research Institute, Vienna, Austria; the Department of Pediatric Hematology and Oncology, Justus- Liebig-University, Giessen; and the Department of Pediatric Hematology and Oncology, Children s University Hospital, University Hospital Schleswig- Holstein, Campus Kiel, Germany. Submitted January 5, 2008; accepted March 17, Supported by the Deutsche Krebshilfe; the Österreichische Kinderkrebshilfe; the Research Program, Genome Research for Health of the Austrian Ministry of Education, Science, and Culture (Grants No. GZ /3-VI/2a/ 2002, and GZ /1-VI/1/2005); and the Fonds zur Förderung der wissenschaftlichen Forschung (FWF Grants No. P15150 and P17551B14). Authors disclosures of potential conflicts of interest and author contributions are found at the end of this article. Corresponding author: Oskar A. Haas, MD, St Anna Children s Hospital, Kinderpitalgasse 6,Vienna, Austria, 1090; oskar.haas@stanna.at by American Society of Clinical Oncology A B S T R A C T Purpose We aimed to identify relapse predictors in children with a B-cell precursor acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iamp21), a novel genetic entity associated with poor outcome. Patients and Methods We screened 1,625 patients who were enrolled onto the Austrian and German ALL Berlin- Frankfurt-Münster (ALL-BFM) trials 86, 90, 95, and 2000 with ETV6/RUNX1-specific fluorescent in situ hybridization probes, and we identified 29 patient cases (2%) who had an iamp21. Minimal residual disease (MRD) was quantified with clone-specific immunoglobulin and T-cell receptor gene rearrangements. Results Twenty-five patients were good responders to prednisone, and all achieved remission after induction therapy. Eleven patients experienced relapse, which included eight who experienced relapse after cessation of front-line therapy. Six-year event-free and overall survival rates were 37% 14% and 66% 11%, respectively. Results of MRD analysis were available in 24 (83%) of 29 patients: nine (37.5%) belonged to the low-risk, 14 (58.5%) to the intermediate-risk, and one (4%) to the high-risk group. MRD results were available in 8 of 11 patients who experienced a relapse. Seven occurred among the 14 intermediate-risk patients, and one occurred in the high-risk patient. Conclusion The overall and early relapse rates in the BFM study were lower than that in a previous United Kingdom Medical Research Council/Childhood Leukemia Working Party study (38% v 61% and 27% v 47%, respectively), which might result from more intensive induction and early reintensification therapy in the ALL-BFM protocols. MRD values were the only reliable parameter to discriminate between a low and high risk of relapse (P.02). J Clin Oncol 26: by American Society of Clinical Oncology X/08/ /$20.00 DOI: /JCO INTRODUCTION Recently, the United Kingdom National Cancer Research Institute (NCRI) Childhood Leukemia Working Party (CLWP) reported a novel genetic entity in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that is characterized by an intrachromosomal amplification of chromosome 21 (iamp21). 1,2 Its common defining feature is the presence of multiple copies of the RUNX1 gene that are usually contained within a morphologically abnormal chromosome 21. This gene amplification was identified by chance during fluorescent in situ hybridization (FISH) screening for ETV6/RUNX1- positive ALL; so far, FISH analysis also remains the only reliable method to identify such occurrences of ALL with an iamp21. The United Kingdom CLWP also provided the first demographic, clinical, and survival data for this specific entity. 2 The results indicated a significantly inferior survival of patients by American Society of Clinical Oncology

2 MRD-Dependent Relapse Risk of Childhood ALL With an iamp21 with an iamp21 compared with other patients with BCP-ALL. According to the highly increased relapse risk, patients with an iamp21 are now assigned into the high-risk arm of the current United Kingdom Medical Research Council (MRC)/CLWP ALL 2003 trial and, in case of a slow early response, are considered for allogeneic stem cell transplantation (SCT) in first complete remission (CR). Because this currently remains the only report of its kind, the extent that the high relapse rate of patients with an iamp21 is influenced by the composition and intensity of the applied treatment, and whether particular parameters may help to predict patients with a low and high risk of relapse, is unknown. We, therefore, report herein our experience with patients with an iamp21 who were enrolled onto the Austrian and German ALL Berlin-Frankfurt-Münster (ALL- BFM) trials. PATIENTS AND METHODS Between September 1986 and January 2007, 1,048 patients with ALL were enrolled onto four Austrian multicenter trials: ALL-BFM 86 (n 142), ALL- BFM 90 (n 256), ALL-BFM 95 (n 230), and ALL-BFM 2000 (n 420). Systematic FISH screening of 885 patient cases (84%) with ETV6/RUNX1- specific probes identified 14 patients (1.6%) with an iamp21. 3,4 An additional 15 patients with an iamp21 were identified in the German ALL-BFM studies. Eleven patient cases were detected by March 2006 (four through FISH confirmation of suggestive karyotypes and seven through retrospective FISH screening of 413 patients), and another four were detected between March 2006 and January 2007, after prospective FISH screening commenced (Table 1). All patient cases were reviewed centrally and were treated after informed consent was obtained from the patient, patient s parents, or the patient s legal guardians. 5 Follow-upwasupdatedinNovember2007.Studieswereconductedwith the approval of the responsible ethic committees. Since trial ALL-BFM 90, minimal residual disease (MRD) analysis with polymerase chain reaction based technologies for the detection of clone-specific immunoglobulin and T-cell receptor gene rearrangements was performed after completion of induction therapy (day 33) and early reintensification treatment (day 78). 6-8 However, MRD results were implemented only for risk-adapted stratification in trial ALL-BFM ETV6/RUNX1 analysis was performed with the dual color-labeled LSI ETV6/RUNX1 ES probe set (Vysis, Downers Grove, IL). An iamp21 was defined as the presence of at least five RUNX1 signals in interphase nuclei and/or the colocalization of at least three copies of the RUNX1 gene on a single chromosome 21. Hyperdiploidy was excluded with additional FISH probes in all patient cases with an iamp21. Event-free survival (EFS) and overall survival (OS) rates were analyzed according to the Kaplan-Meier method, and survival curves were compared with the log-rank test. EFS was defined as the time from diagnosis to the first adverse event or to the date of last follow-up. Inability to achieve remission (ie, early death, refractory disease), relapse at any site, death during CR, or the development of a second malignancy were considered adverse events. Patients who failed to achieve CR were assigned a failure time of zero. OS was Patient BFM Study Table 1. Clinical and Laboratory Characteristics and Outcome of the 29 Patients With ALL and an iamp21 on ALL-BFM Trials Age (years) WBC ( 10 9 /L) Phenotype Prednisone Response BM d 33 MRD Risk Relapse Time to Relapse (years) Outcome 1 A 86 F call Poor CR NA 1st CR G 86 M call Good CR NA BM 0.94 Died as a result of disease after SCT 3 A 90 F pre-b ALL Poor CR IR BM nd CR (SCT) A 90 M call Good CR IR BM nd CR (SCT) G 90 F call Good CR NA 1st CR A 95 M call Good CR IR BM 2.67 Died as a result of disease 7 A 95 F call Good CR HR BM CNS nd CR (SCT) G 95 F BCP ALL Good CR NA BM nd CR (SCT) G 95 M call Good CR NA BM 2.59 Died as a result of disease after SCT 10 A 2000 F call Good CR LR 1st CR A 2000 F call Good CR IR 1st CR A 2000 F pre-b ALL Good CR LR Died as a result of infection 1st CR 13 A 2000 M call Poor CR LR 1st CR A 2000 M call Good CR IR BM nd CR (receiving therapy) A 2000 F pre-b ALL Good CR LR 1st CR A 2000 F pre-b ALL Good CR IR 1st CR A 2000 M pre-b ALL Good CR IR 1st CR A 2000 F call Good CR IR BM 1.04 Died after SCT in 3rd CR 19 G 2000 F pre-b ALL Good CR IR 1st CR G 2000 M pre-b ALL Good CR IR 1st CR G 2000 M pre-b ALL Good CR IR BM CNS 2.28 Died as a result of disease (after SCT) 22 G 2000 M call Good CR LR 1st CR G 2000 M pre-b ALL Good CR LR 1st CR G 2000 M call Good CR LR 1st CR G 2000 M call Poor CR LR 1st CR G 2000 M call Good CR IR BM 1.62 Died as a result of disease (after SCT) 27 G 2000 F call Good CR LR 1st CR G 2000 M call Good CR IR 1st CR G 2000 F call Good CR IR 1st CR 4.47 Abbreviations: ALL, acute lymphoblastic leukemia; iamp21, intrachromosomal amplification of chromosome 21; BFM, Berlin-Frankfurt-Münster; BM, bone marrow; MRD, minimal residual disease; FU, follow-up; A, Austria; F, female; call, common acute lymphoblastic leukemia; CR, complete remission; NA, not available; M, male; G, Germany; SCT, stem-cell transplantation; IR, intermediate risk; HR, high risk; LR, low risk. FU (years) by American Society of Clinical Oncology 3047

3 Attarbaschi et al defined as the time from diagnosis to death from any cause; for surviving patients, OS was censored at the date of last follow-up. The distribution of categoric variables was analyzed by the 2 test. P values.05 were considered statistically significant. RESULTS Of the 29 patients with an iamp21, two patients were identified in trial ALL-BFM 86, three patients in trial ALL-BFM 90, four in trial ALL- BFM 95, and 20 in trial ALL-BFM 2000 (Table 1). The male-to-female ratio was 15:14, and the median age and leukocyte count at diagnosis were 9.00 years (range, 4.13 to years) and /L (range, 0.7 to /L), respectively. Twenty-five patients (86%) were good responders to prednisone and had less than 1,000/ L blasts after a 7-day prephase with prednisone and one intrathecal dose of methotrexate on day 1. All were in CR after induction therapy. Results on MRD analysis were available in 24 patients (83%). Nine patients (37.5%)wereMRD-negativeandhadtwomarkersatthelevelof10 4 after induction therapy (low risk), and 14 patients (58.5%) had levels that were less than after early reintensification therapy (intermediate risk). One patient (4%) fulfilled high-risk MRD criteria ( after early reintensification therapy). Table 2 lists clinical and laboratory characteristics and outcome data of the 29 patients with an iamp21and the 1,596 patient cases without an iamp21. Table 3 lists the data for the 29 patients on ALL-BFM trials compared with the 28 patient cases from the United Kingdom MRC/CLWP. Eleven (38%) of 29 patients experienced relapse after a median time of 2.59 years (range, 0.94 to 5.99 years): one of two patients from trial ALL-BFM 86, two of three from trial ALL-BFM 90, all four from trial ALL-BFM 95, and four of 20 patients from trial ALL-BFM MRD results were available in eight patients who experienced relapse; seven occurred among the 14 intermediate-risk patients, and one occurred in the high-risk patient. Five patients who experienced relapse have survived event-free in second CR after a median follow-up of 4.95 years (range, 0.69 to 7.18 years), which includes four who underwent SCT. Six patients who experienced a relapse died, one as a result of toxicity after SCT in third CR, four as a result of relapse after SCT in second CR, and one as a result of a second relapse after chemotherapy only. Another patient died as a result of an infection in first CR. Median follow-up of the 29 patients was 4.41 years (range, 0.24 to years), but it was less than 1 year in four patients (two each with low and intermediate MRD levels). In the 25 patients with a follow-up of more than 1 year the probabilities of 6-year EFS, relapsefree survival, and OS estimates were 38% 14%, 39% 14% and 66% 11%, respectively (Fig 1). Evaluation of potential prognostic factors (study, sex, age, leukocyte count, prednisone response, MRD levels) among the group of patients with an iamp21 revealed that MRD values were the only parameter able to predict relapsefree survival (MRD low risk [100%] v MRD intermediate risk [37% 16%]; P.02). MRD-based treatment stratification was used for the first time in ALL-BFM 2000, whereas risk assignment in ALL-BFM 86, 90, and 95 was based on conventional parameters only. 4 Conventional and MRD risk group assignment concurred in 19 (79%) of 24 patients. None of the seven patient cases in the MRD low-risk group, but five of 12 in the MRD intermediate-risk group, experienced a relapse. Of the remaining five patient cases, two patients with low MRD risk had a poor response to prednisone and were, thus, assigned to the ALL-BFM 2000 Table 2. Characteristics and Outcomes of Patients With and Without an iamp21 Characteristic Patients With an iamp21 Patients Without an iamp21 No. % No. % Total No. of patients 29 1,596 Male Female Age, years Median Range , WBC, 10 9 /L Median Range , , NA CNS disease Immunophenotype Pro-B ALL Common ALL Pre-B ALL Other T-ALL NA MRD risk group Low risk Intermediate risk High risk NA Prednisone response Good , Poor NA CR after induction therapy , Relapse Death rate year pefs year pos Abbreviations: iamp21, intrachromosomal amplification of chromosome 21; CNS, central nervous system; ALL, acute lymphoblastic leukemia; MRD, minimal residual disease; CR, complete remission; pefs, probability of event-free survival; pos, probability of overall survival. Analysis was restricted to the 877 patients with minimal residual disease data available. Including mainly patients from trials ALL-BFM 86, 90, and 95, who had not been systematically analyzed for minimal residual disease levels. Data was not disclosed because of the ongoing ALL-BFM 2000 trial; for historical comparison, the treatment results of trial ALL-BFM 95 (N 2,169) were as follows: No. of relapses: 356 (16%); 6-year probability of event-free survival: %; 6-year probability of overall survival: %. 9 Including only patients with a follow-up of more than 1 year. high-risk arm. Both are currently in first CR. The only MRD high-risk patient was treated in the ALL-BFM 95 intermediate-risk group and experienced relapse. Two other patients with MRD intermediate risk were treated according to the ALL-BFM 90 standard-risk and highrisk (poor prednisone response) arm, respectively, but both experienced relapse. However, because the ALL-BFM 90 high-risk arm was P by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

4 MRD-Dependent Relapse Risk of Childhood ALL With an iamp21 Table 3. Comparison of the Patients With ALL and an iamp21 Enrolled Onto the BFM and UK MRC/CLWP Studies Characteristic ALL-BFM Trials (86, 90, 95, 2000) UK MRC/CLWP ALL 97 Trial No. % No. % Overall No. of patients with an iamp No. of patients screened 1,625 1,630 % of patients with an iamp Male Female Age, years WBC, 10 9 /L Median CNS disease 0 0 Immunophenotype pro-b ALL 0 0 Common/pre-B ALL T-ALL 0 0 MRD risk grouping Low risk 9 31 NA Intermediate risk NA High risk 1 4 NA NA 5 17 CR after induction therapy Relapse Median time 2.59 NA During front-line therapy After cessation of therapy Death rate Median follow-up, years year pefs, % year pos, % Abbreviations: iamp21, intrachromosomal amplification of chromosome 21; MRC, Medical Research Council; CLWP, childhood leukemia working party; ALL, acute lymphoblastic leukemia; BFM, Berlin-Frankfurt-Münster; CNS, central nervous system; NA, not available; CR, complete remission; pefs, probability of event-free survival; pos, probability of overall survival. Six-year probabilities of event-free survival and of overall survival were not available for the patients from the United Kingdom study. the only one that contained neither an early reintensification nor a late reinduction element, the survival rates were inferior overall to those achieved in trials ALL-BFM 86, 95, and Of the five patients without MRD data, two were treated according to trial ALL-BFM 86 (one in the standard-risk group who experienced relapsed, and one in the high-risk group with a poor response to prednisone who did not experience relapse), one according to trial ALL-BFM 90 (intermediate risk group without relapse), and two were treated according to trial ALL-BFM 95 (one each from the standard- and intermediate-risk groups who both experienced relapse). These observations support the notion that patient cases with an iamp21 certainly benefit from risk-adapted (on the basis of prednisone response and MRD values) treatment intensifications. Event-Free and Overall Survival censored 6-year EFS: 38 ± 14% 6-year OS: 66 ± 11% Time From Diagnosis (years) Fig 1. Six-year event-free survival (EFS) and overall survival (OS) of the 25 patients with iamp21 with a follow-up of at least 1 year. DISCUSSION Compared with the outcome of the patients in the British study, patients with an iamp21 who are treated according to BFM-based regimens seem to experience a lower overall relapse rate (38% v 61%) and a lower early relapse rate (27% v 47%). In the British cohort, eight (47%) of 17 patients who experienced relapse did so during therapy, and nine (53%) experienced relapse between 8 months and 3 years off therapy.incontrast,inthebfmcohortonlythree(27%)of11patients experienced relapse early, whereas the other eight (73%) experienced relapse after cessation of front-line therapy. Moreover, one of the 28 British patients did not achieve CR at all, and three others were not in CR after induction therapy, whereas all 29 patients enrolled onto BFM trials were in CR at this time point. These differences in the overall and early disease recurrences can most likely be explained by a different effectiveness of various treatment components used in the diverse protocols. 4,10-12 For instance, 20 of 28 British patients received a three-drug (prednisone, vincristine, L-asparaginase) induction, no BFM-type early reintensification (cyclophosphamide, cytarabine, mercaptopurine), and no extracompartment therapy that would include high-dose methotrexate, whereas all three elements were consistent components of the ALL-BFM protocols. 4,10,11 Only eight of 20 British patients received a four-drug (with daunorubicine) induction and subsequent early reintensification therapy with both elements similar to the protracted and intense protocols used by the BFM Group. 4,10,11 However, because the United Kingdom CLWP study did not specify the distribution of the 17 patients who experienced relapse in the two treatment-differing trial periods, this issue cannot be additionally assessed. Nevertheless, even a dose-intensified induction treatment with an additional anthracycline in combination with an early postinduction intensification therapy is insufficient to prevent relapses in a certain proportion of patient cases with an iamp21, because the overall relapse rate was still high, even in the ALL-BFM studies. 2 The combined results of the United Kingdom MRC/CLWP and BFM trials also revealed that 16 of 28 patients who experienced relapse by American Society of Clinical Oncology 3049

5 Attarbaschi et al underwent transplantation, eight (50%) of whom are still in CR after a follow-up time between 4.91 and 7.18 years for the BFM and between 4 months and 3.67 years for the British patient cases. The comparatively good outcome of the British patients who did not undergo transplantation is difficult to interpret, because they were apparently not treated in a systematic fashion and because detailed follow-up data is not available. 2 Nevertheless, along with the continuously improved chemotherapy regimens, allogeneic SCT still remains an alternative treatment option for patient cases with an iamp21 who are in second remission. Although the distribution of patient cases with an iamp21 within the three MRD risk groups resembled that of all other BCP-ALL patient cases, it is worth noting that, except for one patient who was MRD high risk and who experienced relapse, all relapses occurred in patients with a MRD intermediate risk. The average relapse risk for all patient cases in this group is 23%, whereas it translates to 50% (7 of 14) for those with an iamp21. 6 This is, so far, one of the highest predictable relapse risks in a specific genetic childhood BCP-ALL subgroup yet identified. The results of our analysis thus demonstrate not only that MRD values reliably discriminate between patients with an iamp21 who have a low and high relapse risk but also that it is possible to extract and define a specific group of patients with clear and distinct risk features that previously remained concealed in the MRD intermediate-risk group. We conclude that, at least in the context of BFM-based treatment regimens, patient cases with an iamp21 who are MRD low risk have an excellent prognostic outlook and that, for this group, the current type of treatment is sufficient. Conversely, early treatment intensification and assignment to a high risk therapy arm is certainly warranted for those with an intermediate MRD risk. AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The authors indicated no potential conflicts of interest. AUTHOR CONTRIBUTIONS Conception and design: Andishe Attarbaschi, Georg Mann, Martin Schrappe, Oskar A. Haas Administrative support: Markus Seidel, Dasa Janousek Provision of study materials or patients: Renate Panzer-Grümayer, Jochen Harbott, Martin Schrappe, Oskar A. Haas Collection and assembly of data: Andishe Attarbaschi, Silja Röttgers, Manuel Steiner, Margit König, Eva Csinady, Dasa Janousek, Anja Möricke, Carsten Reichelt, Jochen Harbott Data analysis and interpretation: Andishe Attarbaschi, Georg Mann, Renate Panzer-Grümayer, Eva Csinday, Michael N. Dworzak, Anja Möricke, Jochen Harbott, Martin Schrappe, Helmut Gadner, Oskar A. Haas Manuscript writing: Andishe Attarbaschi, Michael N. Dworzak, Anja Möricke, Martin Schrappe, Helmut Gadner, Oskar A. Haas Final approval of manuscript: Andishe Attarbaschi, Georg Mann, Martin Schrappe, Helmut Gadner, Oskar A. 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Mitchell CD, Richards SM, Kinsey Se, et al: Benefit of dexamethasone compared with prednisolone for childhood acute lymphoblastic leukemia: Results of the UK Medical Research Council ALL97 randomized trial. Br J Haematol 129: , Vora A, Mitchell CD, Lennard L, et al: Toxicity and efficacy of 6-thioguanine versus mercaptopurine in childhood lymphoblastic leukemia: A randomized trial. Lancet 368: , Eden OB, Harrison C, Richards S, et al: Longterm follow-up of the United Kingdom Medical Research Council protocols for childhood acute lymphoblastic leukaemia, : Medical Research Council Childhood Leukemia Working Party. Leukemia 14: , 2000 Acknowledgment The Acknowledgment is included in the full-text version of this article, available online at It is not included in the PDF version (via Adobe Reader ) by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY