Leukemia (2003) 17, 532–540 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia E Gue´rin1,2, N Entz-Werle´3, D Eyer3, E Pencreac’h1, A Schneider1, A Falkenrodt4, F Uettwiller3, A Babin3, A-C Voegeli1,5, M Lessard4, M-P Gaub1,2, P Lutz3 and P Oudet1,5 1Laboratoire de Biochimie et de Biologie Mole´culaire Hoˆpital de Hautepierre, Strasbourg, France; 2INSERM U381, Strasbourg, France; 3Service d’Onco-He´matologie Pe´diatrique Hoˆpital de Hautepierre, Strasbourg, France; 4Laboratoire Hospitalier d’He´matologie Biologique Hoˆpital de Hautepierre, Strasbourg, France; and 5INSERM U184, Illkirch, France Topoisomerase genes were analyzed at both DNA and RNA segregation.4 These enzymes act by promoting transient DNA levels in 25 cases of newly diagnosed childhood acute breakage in order to allow strand-passage events and DNA lymphoblastic leukemia (ALL). The results of molecular analy- 5 sis were compared to risk group classification of children in relaxation to occur before rejoining the broken DNA ends. order to identify molecular characteristics associated with Two types of DNA topoisomerases have been described. Type response to therapy. At diagnosis, allelic imbalance at topo- I enzymes, encoded in humans by the TOP1, TOP3A and isomerase IIa (TOP2A) gene locus was found in 75% of TOP3B genes,6–8 cleave only one strand of the DNA helix informative cases whereas topoisomerase I and IIb gene loci whereas type II enzymes, encoded by the human TOP2A and are altered in none or only one case, respectively. By semi- TOP2B genes,9,10 cleave both DNA strands. In mammalian quantitative Polymerase chain reaction, we found a 2.5 to 8-fold TOP2A gene amplification in 72% of the children, which was cells, expression of the TOP2A isoform is closely linked to correlated to gene overexpression in every case. These results proliferation state, with maximal protein levels and enzymatic show that TOP2A gene amplification is a frequent event in ALL activity in late S and G2/M phases, whereas TOP2B is at diagnosis. Interestingly, we also identified a small population constitutively expressed at high levels even in quiescent of children that do not present TOP2A gene amplification or cells.11–13 gene overexpression and who are significantly associated with Childhood ALL treatments include essentially type-II- very high risk classified patients showing glucocorticoid resistance. In conclusion, characterization of TOP2A gene specific inhibitors such as anthracyclines (daunorubicin, status in childhood ALL at diagnosis provides useful comple- idarubicin, doxorubicin/adriamycin) or epipodophyllotoxins mentary information for risk assessment. (etoposide, teniposide). During the catalytic reaction of DNA Leukemia (2003) 17, 532–540. doi:10.1038/sj.leu.2402774 topoisomerases, these drugs stabilize enzyme–DNA cleavable Keywords: topoisomerase II alpha; childhood ALL; risk assessment complexes, either by intercalating into the groove between DNA strands or by interfering with the DNA religation reaction of the enzyme.14–16 Consequently, topoisomerase Introduction inhibitors lead to the accumulation of DNA strand breaks and ultimately to cell death.17–19 Acute lymphoblastic leukemia (ALL) represents the most The resistance of several leukemia cell lines to anthracy- common hematological malignancy encountered in children. clines and epipodophyllotoxins has been associated with a As a result of chemotherapy and supportive care improve- decreased protein expression and/or activity of topoisomerase 20–25 ments, the long-term complete remission rate now reaches up II enzymes. In addition, in some particular cell lines, to 80%.1 This increase in long-term event-free survival can be topoisomerase II gene mutations have been identified and attributed to the development of more effective risk-directed suggested to be responsible for the reduced drug sensitivity to 26–29 multidrug protocols, whose goal is to intensify the therapy for DNA topoisomerase inhibitors. Conversely, more recent those patients with a high risk of relapse and to decrease the studies have shown that increasing the intracellular level of the toxicity while maintaining high cure rates for those patients enzyme by transfection of topoisomerase genes can restore the with a lower risk of relapse.2 Current protocols include agents sensitivity of resistant cancer cell lines to DNA topoisomerase 30–34 that have been shown to inhibit DNA topoisomerases and that inhibitors. On the other hand, anthracyclines are well- are combined in the induction regimen with corticosteroids, known chemotherapeutic agents that can cause cardiotoxicity 35 vincristine and l-asparaginase in order to induce a complete in clinical use. In addition, epipodophyllotoxins, as well as remission during the first month of therapy. These agents are other DNA topoisomerase II inhibitors, have been implicated also used during the consolidation therapy, where they are in the occurrence of therapy-related acute myeloid leukemia 36,37 associated with a variety of drugs including methotrexate, 6- and myelodysplastic syndromes. Regarding these data, mercaptopurine, 6-thioguanine, l-asparaginase, aracytine or trying to better adapt the use of DNA topoisomerase inhibitors cyclophosphamide, depending on protocols and risk to leukemia cells biological features could lead to a potential assessment.2,3 reduction of therapy-related cardiotoxic effects as well as DNA topoisomerases are essential nuclear enzymes that secondary leukemias induced by these drugs. In this context, play a crucial role in the control of DNA topology. They are molecular characterization of the cellular target of these involved in the resolution of conformational constraints that inhibitors could represent interesting first information. There- occur during fundamental nuclear metabolic processes such fore, we have analyzed topoisomerase genes at both DNA and as replication, transcription, recombination or chromosome RNA levels, in samples from 25 newly diagnosed children with ALL, using allelotyping and semi-quantitative fluorescent polymerase chain reaction (PCR) approaches. Results were compared to risk group classification of children at diagnosis Correspondence: Pierre Oudet, INSERM U184, ESBS Poˆle API, 67400 Illkirch Cedex, France; Fax: 33 3 88 12 75 39. in order to characterize topoisomerase gene status in different Received 11 April 2002; accepted 12 August 2002 groups of patients. Topoisomerase genes in childhood ALL E Gue´rin et al 533 Materials and methods containing >70% leukemic blast cells (mean: 90%), as determined by May Gru¨nwald Giemsa staining. Oral swab Patients cells were spun at 2500 g for 10 min and washed twice with phosphate buffered saline. Mononucleated cells were Twenty-five children (7 girls and 18 boys) referred for isolated from bone marrow samples by the standard Ficoll diagnosis of primary ALL at the Department of Pediatric technique. Cell aliquots were stored at À801C until nucleic Hematology and Oncology of the Hoˆpital de Hautepierre acid isolation. (Strasbourg, France) were included in the study. Their characteristics are summarized in Table 1. The mean age at presentation was 6.3 years (range: 3 months to 14 years, only Nucleic acid isolation from cytological samples one case being diagnosed in the first 12 months of life). Leukemia (19 B-ALL and 6 T-ALL) was classified according to Genomic DNA was extracted with the classical phenol– the European Group for Immunological Characterization of chloroform procedure and resuspended at 50 ng/ml final Leukemia (EGIL) criteria.38 Children were included in very concentration. Total RNA was isolated from 107 cells with low, average or very high risk treatment groups of the RNeasy kit (Qiagen, Hilden, Germany) following the manu- European Organization for the Research and Treatment of facturer’s instructions. Reverse transcriptase (RT) reaction was Cancer (EORTC) protocols 58881 and 58951, according to performed in a total volume of 33 ml using random hexapri- criteria based on the age at diagnosis, white blood cell count mers and 10 ml total RNA as template (First-Strand cDNA at day 8 after glucocorticoid therapy, immunophenotype and Synthesis Kit, Amersham Pharmacia Biotech, Uppsala, cytogenetic analyses.39 Sweden). Cytogenetic analysis Microsatellite analysis Chromosome analysis was performed according to the R- Two microsatellites surrounding each topoisomerase gene in banding method after short (24 or 48 h) culture of bone close vicinity were analyzed in paired normal and tumoral marrow cells without addition of mitogens. DNA: D20S107 and D20S170 for TOP1 gene, D17S800 and D17S1814 for TOP2A gene, D3S1283 and D3S700 for TOP2B gene (see http://www.gdb.org and http://www.ncbi.nlm.nih. Sample collection for molecular analysis gov/genemap99 for microsatellite primers description). In total, 100 ng of genomic DNA was amplified by PCR in a All samples were taken before induction therapy. Normal total volume of 25 ml using 0.6 units of Taq polymerase and tissue was obtained from buccal swabs or fibroblasts cultures 4 pmol of both forward and Cy5-labelled reverse primers, as whereas ALL cells were recovered from bone marrow aspirates previously described.40,41 PCR was carried out in an Omnigen Hybaid Thermocycler (Hybaid Ltd, Ashford, UK) using the following protocol: initial denaturation of 7 min at 951C, 35 amplification cycles of 1 min at 951C, 1 min at 551C, and Table 1 Patients