Leukemia (2006) 20, 777–784 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE The MLL recombinome of acute leukemias C Meyer1,23, B Schneider1,23, S Jakob1, S Strehl2, A Attarbaschi2, S Schnittger3, C Schoch3, MWJC Jansen4, JJM van Dongen4, ML den Boer5, R Pieters5, M-G Ennas6, E Angelucci7, U Koehl8, J Greil9, F Griesinger10, U zur Stadt11, C Eckert12, T Szczepan´ski13, FK Niggli14, BW Scha¨fer14, H Kempski15, HJM Brady15, J Zuna16, J Trka16, LL Nigro17, A Biondi18, E Delabesse19, E Macintyre19, M Stanulla20, M Schrappe21, OA Haas2, T Burmeister22, T Dingermann1, T Klingebiel8 and R Marschalek1 1Institute of Pharmaceutical Biology/ZAFES/Diagnostic Center of Acute Leukemia, University of Frankfurt, Frankfurt/Main, Germany; 2CCRI, Children’s Cancer Research Institute, Vienna, Austria; 3Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich, Germany; 4Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; 5Department of Paediatric Oncology/Haematology, Erasmus MC, Sophia Children’s Hospital, Rotterdam, The Netherlands; 6Department of Cytomorphology, University of Cagliari, Cagliari, Italy; 7Haematology and Oncology Hospital ‘A Businco’, Cagliari, Italy; 8Pediatric Hematology and Oncology, University Frankfurt, Frankfurt, Germany; 9Department of Pediatric Hematology and Oncology, University Children’s Hospital, Tuebingen, Germany; 10Department of Hematology and Oncology, Goettingen, Germany; 11Department of Pediatric Hematology and Oncology, Eppendorf, Hamburg, Germany; 12Department of Pediatric Oncology and Hematology, Charite´ – Virchow Campus, Berlin, Germany; 13Department of Pediatric Hematology and Oncology, Silesian Academy of Medicine, Zabrze, Poland; 14University Children’s Hospital, Department of Oncology, Zuerich, Switzerland; 15Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, London, UK; 16Department of Paediatric Haematology/Oncology, Charles University Prague, Prague, Czech Republic; 17Center of Pediatric Hematology Oncology, University of Catania, Catania, Italy; 18M.Tettamanti Research Center, University of Milano-Bicocca, Monza, Italy; 19Biological Hematology, AP-HP and INSERM EMIU210, Paris, France; 20Paediatric Haematology/Oncology, Medical School of Hannover, Hannover, Germany; 21Paediatric Department, University of Schleswig-Holstein, Campus Kiel, Germany and 22Charite´ – Benjamin Franklin Campus, Med. Klinik III, Berlin, Germany Chromosomal rearrangements of the human MLL gene are a leukemias.1,2 For instance, the presence of MLL rearrangements hallmark for aggressive (high-risk) pediatric, adult and therapy- is an independent dismal prognostic factor in infant anti- associated acute leukemias. These patients need to be lymphoblastic factor (ALL), whereas childhood ALL patients identified in order to subject these patients to appropriate . therapy regimen. A recently developed long-distance inverse with MLL AF4 fusions are usually treated according to high-risk PCR method was applied to genomic DNA isolated from protocols. Thus, the identification of MLL gene fusions is individual acute leukemia patients in order to identify chromo- necessary for rapid clinical decisions resulting in specific somal rearrangements of the human MLL gene. We present data therapy regimens. Current procedures to identify MLL rearran- of the molecular characterization of 414 samples obtained from gements include cytogenetic analysis, fluorescence in situ 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and hybridization (FISH) experiments (e.g. MLL split-signal FISH), the involved translocation partner genes (TPGs) was deter- specific reverse transcriptase-PCR (RT-PCR) and genomic PCR mined and several new TPGs were identified. The combined methods. This repertoire of technologies was recently extended data of our study and published data revealed a total of 87 by a long-distance inverse PCR (LDI-PCR) method that uses different MLL rearrangements of which 51 TPGs are now small amounts of genomic DNA to determine any type of MLL characterized at the molecular level. Interestingly, the four gene rearrangement at the molecular level.3 This includes most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in chromosomal translocations, gene internal duplications, chro- histone H3K79 methylation. Thus, translocations of the MLL mosome 11q deletions or inversions, and MLL gene insertions gene, by itself coding for a histone H3K4 methyltransferase, are into other chromosomes, or vice versa, the insertion of presumably not randomly chosen, rather functionally selected. chromatin material into the MLL gene. Leukemia (2006) 20, 777–784. doi:10.1038/sj.leu.2404150; To gain insight into the frequency of MLL rearrangements, published online 2 March 2006 unscreened and prescreened pediatric and adult leukemia Keywords: MLL; chromosomal translocations; partner genes; acute leukemia patients were analyzed. Prescreening tests (cytogenetic analysis, FISH, Southern blot, RT-PCR or NG2-positivity) were performed at different European centers, where acute leukemia patients are enrolled in different study groups (Interfant-99, AMLCG, Introduction GMALL). With the exception of a few patients, all prescreened MLL rearrangements were successfully analyzed and patient- Chromosomal rearrangements involving the human MLL gene specific MLL fusion sequences were obtained. Data obtained are recurrently associated with the disease phenotype of acute from the literature and results obtained in this study are summarized in a color map where all 51 translocation partner Correspondence: Dr R Marschalek, Institute of Pharmaceutical genes (TPGs) and their specific breakpoint regions have been Biology/ZAFES, University of Frankfurt, Biocenter, N230, 303 Marie- assigned. The applied color code will enable all investigators to Curie Str. 9, D-60439 Frankfurt/Main, Germany. identify compatible intron–intron fusions between the MLL gene E-mail: [email protected] 23These authors contributed equally to this work. and all yet characterized TPGs. Moreover, we provide a Received 24 November 2005; revised 3 January 2006; accepted 11 complete list of 87 MLL rearrangements of which 36 still await January 2006; published online 2 March 2006 molecular characterization. The MLL recombinome C Meyer et al 778 Materials and methods decapping enzyme), BCL9L and ARHGEF17. With the exception of BCL9L and ARHGEF17, all of them have been reported in a Patient material technical paper by the authors.3 The BCL9L gene encodes a Genomic DNA was isolated from bone marrrow and/or structural constituent of ribosomes (with similarity to the peripheral blood samples of all patients and sent to our center. Drosophila legless gene) and is located on chromosome Patient samples were obtained from the Interfant-99 study group 11q23.5 The fusion between MLL and BCL9L deletes a (Rotterdam, The Netherlands), the AMLCG study group (Mu- chromosomal area of about 300 kb and fused MLL intron 8 nich, Germany) and the GMALL study group (Berlin, Germany). tail-to-tail with the 30-non-translated region (30-NTR) of BCL9L. Informed consent was obtained from all patients or patients’ Therefore, no functional fusion protein can be produced. The parents/legal guardians and control individuals. ARHGEF17 gene is located on chromosome 11q13 and encodes a protein of 1510 amino acids (164 kDa) that belongs to the family of Rho guanine nucleotide exchange factors involved in Long-distance inverse PCR experiments signaling pathways.6 The in-frame fusion between MLL intron All DNA samples were treated and analyzed as described.3 12 and ARHGEF17 intron 1 occurred on both fusion alleles and Briefly, 1 mg genomic patient DNA was digested with restriction involved two homologous chromosomes 11. enzymes and religated to form DNA circles before LDI-PCR analyses. Restriction polymorphic PCR amplimers were isolated from the gel and subjected to DNA sequence analyses to obtain Different possibilities to cause genetic MLL aberrations the patient-specific fusion sequences. In general, human MLL rearrangements are initiated by DNA cleavage or a DNA damage situation, which induces DNA repair via the non-homologous end joining (NHEJ) DNA repair Results pathway.7,8 Although the MLL gene is predominantly involved in reciprocal chromosomal translocations (Figure 1a), other Identification of MLL rearrangements genetic rearrangements were observed in this study or were Acute leukemia patients carrying MLL rearrangements are not described in the literature. Gene-internal partial tandem exceeding a total of 800 cases per year in Europe (about 300 duplications (PTD) of specific MLL gene portions (duplication pediatric and about 500 adult leukemia patients). To analyze the of introns 2–9, 2–11, 4–9, 4–11 or 3–8; Figure 1b) are frequently recombinome of the human MLL gene, 414 acute leukemia observed in AML patients.9–11 MLL PTD are being discussed to samples from different European centers were analyzed over a mediate dimerization of the MLL N-terminus, a process that period of 24 months (272 pediatric and 142 adult patients). One seems to be sufficient to mediate leukemogenic transforma- Hundred and seventy-six out of 414 leukemia samples were not tion.12 The third possibility is deletions on the long arm