Leukemia (2003) 17, 532–540 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Modification of 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 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 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) 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 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 –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, , recombination or 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. (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 analysis

Chromosome analysis was performed according to the R- Two 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 at diagnosis 1 min at 721C, followed by a final elongation of 5 min at 721C. No. of patients (%)

Total 25 (100) Semi-quantitative PCR analysis Age years 0–1 1 (4) TOP2A and TOP2B genes status were analyzed separately 2–9 18 (72) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) 10–15 6 (24) gene co-amplification as an internal standard for quantifica- White blood cell count tion of gene amplification. A total of 100 ng of genomic DNA o10 000/mm3 11 (44) was used in a multiplex PCR amplification performed in a total 10 000 – 99 000/mm3 8 (32) volume of 25 ml using the following optimized primer Z100 000/mm3 6 (24) amounts: 10 pmol of TOP2A and 1.2 pmol of GAPDH primers Immunophenotype for TOP2A gene analysis, 1 pmol of TOP2B and 1 pmol of B II 18 (72) GAPDH primers for TOP2B gene analysis. PCR amplification B III 1 (4) cycles were performed as described above, except that three T II 1 (4) independent PCR reactions of 28, 31 and 35 cycles were done T III 4 (16) in parallel. This procedure allowed us to determine the T IV 1 (4) condition for which, in each sample, topoisomerase and Karyotype GAPDH genes amplifications are simultaneously in the Normal 7 (28) exponential phase, which is the condition required for an Hyperdiploidy 4 (16) accurate quantification of both genes amplification levels. (>50 ) Other 12 (48) Sequences of the primers used for genomic amplification are Not available 2 (8) as follows: TOP2A Cy5-labelled forward primer (exon 11) 50-GCCATTGGCTGTGGTATTG-30 and TOP2A reverse Risk group primer (exon 12) 50-GAGAAGCTTCTCGAACATTGAG-30 Very low and average risk 18 (72) 42 Very high risk 7 (28) were previously described and lead to an amplified fragment of 656 bp on DNA, TOP2B Cy5-labelled forward primer (exon

Leukemia Topoisomerase genes in childhood ALL E Gue´rin et al 534 16) 50-GATTGGGTACTAGTACAGCT-30 and TOP2B reverse laser fluorescent) sequencer (Amersham Pharmacia Biotech, primer (intron 16) 50-GAATAGAAGGTAGGGGGATG-30 (am- Uppsala, Sweden). Fluorescent signals were quantified using plified product of 165 bp), GAPDH forward primer (intron 2) the ALFwin Fragment Analyser software package. Allelic 50-CATCCCTTCTCCCCACACAC-30 and GAPDH Cy5-labelled imbalance at microsatellite markers, which is defined by the reverse primer (intron 2) 50-AGTCCCAGGGCTTTGATTTG-30 partial or complete loss of one of the two alleles or, (amplified product of 104 bp). alternatively, the amplification of one allele compared to the other, was quantified as previously described.40,41 Amplifica- tion levels of topoisomerase genes at both DNA and RNA Semi-quantitative RT-PCR analysis levels are expressed as the ratio of bone marrow cells and paired normal tissue normalized values. TOP2A was analyzed using porphobilinogen deaminase (PBGD) gene as an internal standard for quantifica- tion. A 2.5 ml amount of the total volume of RT reaction Results performed as described above was used in three multiplex PCR reactions of 28, 31 and 35 amplification cycles. Microsatellite topoisomerase genes analysis Experimental conditions were as mentioned for semi- quantitative PCR analysis, except that 2.5 pmol of primers As a first step in evaluating topoisomerase genes status in was used for both TOP2A and PBGD genes and that primers childhood ALL, TOP1, TOP2A and TOP2B genes allelotyping hybridization was performed at 601C. Primers used for TOP2A analysis was performed using two microsatellite markers gene amplification are as described above, leading to an surrounding each topoisomerase gene in close vicinity. The amplified product of 289 bp on cDNA.42 Sequences of the results are detailed in Table 2 for TOP2A gene and primers used for PBGD gene co-amplification are as follows: summarized in Table 3 for the three topoisomerase genes. Cy5-labelled forward primer (exon 1) 50-CTGGTAACGG- A total of 17 children were analyzed for TOP2A genomic CAATGCGGCT-30 and reverse primer (exon 7) 50-GCA- rearrangements using D17S1814 and D17S800 microsatellite GATGGCTCCGATGGTGA-30 (amplified product of 328 bp markers. Interestingly, regions associated with these loci on cDNA). appeared to be highly rearranged in ALL cells. When we compared the microsatellite allele ratio in paired bone marrow and buccal swab cells, allelic imbalance was indeed observed PCR product analysis in 7 out of 14 informative heterozygous cases for D17S1814 TOP2A gene marker, and in 5 out of 12 informative cases Fluorescent PCR products were analyzed by denaturating for D17S800 (Table 2). In order to translate these data polyacrylamide gel electrophoresis on an ALF (automated at the TOP2A gene locus level, we considered patients as

Table 2 Topoisomerase IIa : microsatellite analysis, semi-quantitative PCR and RT-PCR results

Patient ALL Riska Microsatelliteb PCR RT-PCR n1 phenotype (ratio) (ratio) D17S1814 D17S800

1 B II VHR H N 3.0 nd 2 B II AR1 N H 8.0 nd 3 B II AR1 N N 1.0 1.0 4 T III AR2 AI 100% AI 30% 2.5 nd 5 B II AR1 AI 60% H 4.4 7.7 6 B II VLR AI 42% H 5.8 nd 7 T III VHR H N 2.5 nd 8 B II AR1 AI 51% AI 75% 8.0 17.0 9 T IV VHR N N 1.0 1.0 10 B II AR1 H AI 63% 3.5 6.8 11 B II VHR N H 1.0 nd 12 B II VLR nd nd 2.0 nd 13 B II AR1 N H 4.7 10.0 14 T III AR2 nd nd 4.0 4.0 15 T II VHR N AI 38% 1.0 nd 16 B II AR1 AI 54% AI 64% 3.6 3.0 17 B II VHR nd nd 1.0 nd 18 B II VHR nd nd 2.0 2.0 19 B II VLR nd nd 4.5 nd 20 B III AR1 nd nd 3.9 nd 21 B II AR2 nd nd 5.4 3.6 22 B II AR1 nd nd 3.1 12.0 23 B II AR1 AI 25% N 2.5 3.8 24 T III AR2 AI 20% N 5.4 32.0 25 B II VLR N N 2.5 3.5

aVLR: very low risk ; AR1/AR2: average risk 1/2 ; VHR: very high risk; bH: homozygous ; N: no allelic imbalance ; AI: allelic imbalance (%) ; nd: not determined.

Leukemia Topoisomerase genes in childhood ALL E Gue´rin et al 535 Table 3 Topoisomerase genes allelotyping

Gene TOP1 (n=15) TOP2A (n=17) TOP2B (n=15)

Microsatellite D20S107 D20S170 D17S1814 D17S800 D3S700 D3S1283 Informative patients with AI 0 0 7 5 0 1 Informative patients with AI at gene locusa 091 aPatients for which at least one of both microsatellites presents an AI (ie AI/AI, AI/N or AI/H). informative if they were heterozygous for both microsatellite ples. In order to quantify both TOP2A and GAPDH genes in markers or if at least one of these markers was rearranged. In the exponential phase of amplification, several PCR reactions this case, 9 out of 12 informative children (75%) showed were performed in parallel with an increasing number of TOP2A gene allelic imbalance, some children having both cycles. Figure 1 shows two representative examples of TOP2A markers rearranged simultaneously (Table 3). This result gene quantification in ALL children with and without gene contrasts with TOP1 and TOP2B genes allelotyping data. No amplification. alteration at the TOP1 gene locus and only one at the TOP2B TOP2A gene analysis was performed in the 25 children gene locus were indeed observed in the 15 children that were included in the study as well as in 10 control patients without also analyzed for these loci (Table 3). ALL. In this control population, the highest ratio value (mean + From these data, we can conclude that among the three 3 standard deviation) of TOP2A gene levels observed in test topoisomerase genes tested, TOP2A is clearly the most and control tissues was 1.8-fold (data not shown). Therefore, in frequently altered in ALL cells. Therefore, we next focused our experimental conditions, a cut-off of more than 2.0-fold on this gene in order to find out whether modifications was considered as representative of TOP2A gene amplification. observed at microsatellite sequence levels could be associated Using this threshold, results obtained in the 25 ALL children with an amplification or a deletion of TOP2A gene in leukemia showed that 18 of them presented TOP2A gene amplification, cells. whose value ranged between 2.5- and 8.0- fold (Table 2). Among these TOP2A gene amplified patients, five presented a moderate gene amplification that was less than 3-fold, Semi-quantitative PCR analysis of topoisomerase genes whereas TOP2A gene amplification ratio was over 3-fold in the 13 remaining cases. By contrast but in agreement with TOP2A gene amplification levels were evaluated by semi- allelotyping data, TOP2B gene was never found amplified in quantitative PCR performed on paired bone marrow and the 14 children that we analyzed so far (Table 4). buccal samples. The housekeeping gene GAPDH was used as From these data, we can conclude that TOP2A gene an internal co-amplified control, which allowed us to amplification in ALL cells is a frequent event that is observed normalize TOP2A amplification levels between paired sam- in 72% of the children at diagnosis, independently of leukemia

Figure 1 Semi-quantitative PCR analysis of TOP2A gene. Genomic DNA was extracted from paired bone marrow and buccal swab samples. TOP2A and GAPDH genes were co-amplified by multiplex PCR using fluorescent labelled primers. Several reactions were performed in parallel with an increasing number of cycles. Amplified fragments were analyzed by electrophoresis on a sequence analyzer. Fluorescence peak height of TOP2A amplified fragment (656 base pairs) is expressed relative to that of GAPDH fragment (104 bp). Panel a shows a representative example of TOP2A gene amplification in bone marrow cells compared to control cells. Panel b shows a representative sample without TOP2A gene amplification.

Leukemia Topoisomerase genes in childhood ALL E Gue´rin et al 536 Table 4 TOP2A and TOP2B genes status evaluated by semi- quantitative PCR

No. of patients

( n = 25) TOP2A No amplification 7 Amplification ratio 2–3 5 Amplification ratio >3 13

TOP2B ( n = 14) No amplification 14 Amplification ratio 2–3 0 Amplification ratio >3 0

cellular type (14 out of 19 B-ALL and 4 out of 6 T-ALL) (Table 2). Having demonstrated that the TOP2A gene is amplified in ALL cells of most children, the next step was to find out whether this amplification was associated with gene over- expression at the RNA level. Figure 2 Semi-quantitative RT-PCR analysis of TOP2A gene. Total RNA was extracted from paired bone marrow cells and control tissues. After reverse transcription, TOP2A and PBGD gene transcripts were co-amplified by multiplex PCR using an increasing number of cycles. Semi-quantitative RT-PCR analysis of TOP2A gene Fluorescence peak height of TOP2A cDNA amplified fragment (289 bp) is expressed relative to that of PBGD cDNA fragment Total RNA was extracted from bone marrow cells and control (328 bp). This figure shows a representative analysis of a sample with tissues. After reverse transcription, TOP2A and PBGD gene TOP2A gene overexpression in bone marrow cells. transcripts were co-amplified by multiplex PCR. As mentioned for semi-quantitative PCR analysis, several conditions of PCR amplification were used in parallel, in order to compare Comparison of TOP2A gene status and risk group normalized TOP2A gene expression levels in leukemia cells classification and in control tissue. Figure 2 shows a typical profile of a patient with TOP2A gene overexpression in bone marrow In our cohort, seven children were included in the very high cells. RT-PCR ratio values of TOP2A gene overexpression are risk (VHR) group of treatment, two of them (patients 1 and 11) indicated in Table 2 for the 14 children in which we could because of the presence at diagnosis of a translocation t(9;22), obtain RNA from biological samples. four of them (patients 7,9,15 and 18) because of the Among the 14 analyzed children, 11 of them presented persistence of blast cells in peripheral blood after 8 days of TOP2A gene amplification in leukemia cells at the DNA level. glucocorticoid therapy alone (see Table 5), and the last one Interestingly, in every case, this gene amplification was (patient 17) because of the persistence of residual blast cells associated with an overexpression of the gene at the RNA detectable in bone marrow by molecular analysis after 35 days level. However, as shown in Table 2, gene overexpression of induction therapy. In our cohort, we first looked for a levels (3.0 – 32.0- fold) appeared much more variable than correlation between TOP2A gene status and glucocorticoid DNA amplification levels. Finally, the three children in which response, which can be evaluated at day 8 since at this time the TOP2A gene was not amplified did not show any TOP2A point the only systemic treatment that children have received gene overexpression. is intravenous injection of glucocorticoids. If we exclude the two t(9;22) positive VHR cases since the presence of t(9;22) is an independent factor of high risk, we observed that three Cytogenetic analysis (patients 9, 15 and 18) out of the four children that do not respond to 8 days of glucocorticoid therapy do not present any When TOP2A gene amplification results were compared to TOP2A gene amplification, whereas the absence of TOP2A cytogenetic data (Table 5), except in patient 8, no correlation gene amplification is detected in only three (patients 3, 12 and was found between amplification of TOP2A gene located 17) out of the 19 other patients that had a positive response at in 17q21–q22 and duplication. As quantifica- day 8 (P = 0.04 using Fisher exact test). Therefore, the absence tion of TOP2A gene amplification was performed after of TOP2A gene amplification appeared to be significantly normalization with GAPDH gene located in 12p13, it was associated with patients resistant to glucocorticoids. important to check that chromosome 12 was not deleted in We next looked for a correlation between TOP2A gene ALL cells. Cytogenetic analyses showed us that it was the case status and VHR classification at the end of induction therapy for all children, except for patient 16, in which a del(12) (p11) (day 35), independently of the presence of t(9;22). Our results is observed. However, in this patient, TOP2A gene over- showed that four (patients 9, 15, 17 and 18) out of the five expression at the RNA level (3.0- fold) was formally t(9;22) negative children that were VHR classified at day 35 do demonstrated using another reference gene, PBGD, located not present any TOP2A gene amplification whereas the on band q23.3 of chromosome 11, for which no alteration in absence of TOP2A gene amplification is detected in only leukemia cells karyotype was found. Therefore, these cyto- two (patients 3 and 12) out of the 18 other average risk (AR) genetic data allowed us to validate the semi-quantitative and very low risk (VLR) patients (P=0.008 using Fisher exact evaluation of TOP2A gene amplification in all samples. test).

Leukemia Topoisomerase genes in childhood ALL E Gue´rin et al 537 Table 5 Cytogenetic data and white blood cell count at days 0 and 8

Patient Karyotype White blood cell count / ml (blast %)

Day 0 Day 8

1 45, XY, der (4), del (4) (p15), À20, t(9;22) (q34;q11) [10] 23 690 (69%) 1770 (3%) 2 46, XY [8] 1330 (2%) 900 (0%) 3 46, XY, dup (4q) [7] 10 610 (71%) 700 (0%) 4 46, XY [7] 113 600 (86%) 1600 (0%) 5 46, XY [5] 19 100 (74%) 1650 (2%) 6 47, XY, + constitutional surnumary metacentric chromosome [11] 4230 (49%) 2640 (0%) 7 46, XX, der (3) [8] 117 000 (87%) 3270 (63%) 8 56-59, XX, dup (1) (q21;q32), +4, +6, +8, +9, +X, +14, +15, +17, +18, +21, +22 [10] 32 200 (81%) 3590 (18%) 9 46, XY [27] 208 400 (89%) 5850 (41%) 10 63-64, XXY, +3,+4,+5,+6,+8,+9,+10,+12,+14,+15,+18,+21,+21,+22,+22, [10] / 46, XY [10] 6400 (42%) 2580 (0%) 11 46, XX, t(9;22) (q34 ; q11) [10] 21 280 (65%) 1890 (0%) 12 58, XX, +4,+5,+6,+8,+9,+10,+13,+14,+21,+21,+M [4] / 46,XX [21] 1260 (0%) — 13 Not determined ( no cell growth ) 90 000 (88%) 830 (10%) 14 Not determined ( no cell growth ) 100 600 (63%) 1970 (5%) 15 43, ? [6] / 46, XY [6] 271 200 (90%) 31320 (57%) 16 46, XY, del (12) (p11) [10] 5400 (74%) 1500 (21%) 17 57, XXY, [17] / 46, XY [3] 1700 (3%) 2260 (0%) 18 46, XX, der(1) add(4p) der(5) der(12) der(8) [5] / 46, XX [1] 20 910 (78%) 4500 (25%) 19 46, XX 1720 (12%) 710 (6%) 20 46, XY 2640 (45%) 1030 (8%) 21 46, XY, t(12;21) 156 000 (95%) 1520 (12%) 22 46, XY, t(12;21) 7130 (40%) 1900 (0%) 23 46, XY, t(12;21) 18 780 (73%) 3940 (17%) 24 46, XY 4230 (15%) 5400 (12%) 25 47, XX, +21 6100 (45%) 1200 (16%)

Altogether, our results show that TOP2A gene is amplified In order to investigate topoisomerase genes status at the and overexpressed in most of the ALL children at diagnosis DNA level, we first performed an allelotype analysis using (72% of the cases), and that cases without TOP2A gene microsatellite markers surrounding TOP1, TOP2A and TOP2B amplification are significantly associated with most of VHR genes in close vicinity. Allelotyping is a powerful method that patients showing resistance after 8 or 35 days of therapy. discriminates both maternal and paternal alleles and which is therefore very informative to study precisely genomic rearrangements at a defined locus. In blast cells of ALL Discussion children, the TOP2A gene locus is more specifically altered than the two other topoisomerase genes. Allelic imbalance at In recent years, only few studies have addressed the issue of the TOP2A gene locus is indeed found in 75% of informative topoisomerase genes molecular characterization in childhood cases, whereas no alteration at the TOP1 gene locus and only ALL. Most studies have indeed been focused on the one at the TOP2B gene locus are observed. In order to further comparison of topoisomerase genes expression levels between characterize TOP2A genomic rearrangements, we next specimens of newly diagnosed and relapsed ALL which were evaluated gene amplification levels in leukemia cells by often unpaired. Moreover, expression levels were often semi-quantitative PCR. Of the children 72% presented TOP2A indicated as percentages of the value found in a particular gene amplification, which was well correlated with gene locus cell line chosen as a standard, hampering the exact allelic imbalance in informative patients. Only patient 15 quantification of gene expression level in blast cells.43–46 In presented allelic imbalance restricted to one of the two studied two other studies performed on clinical samples of untreated TOP2A microsatellite markers but without concomitant gene childhood ALL, topoisomerase II expression level was amplification. For this patient, a deletion of TOP2A gene determined with rather low-sensitive techniques such as cannot be excluded. Conversely, patient 25 presented TOP2A immunocytochemistry or dot-blot hybridization, although the gene amplification which was not correlated with D17S1814 results were substantiated in some cases by semi-quantitative and/or D17S800 markers allelic imbalance. For this patient, RT-PCR assays.47,48 Finally, none of these studies evaluated the the extent of TOP2A gene amplified region could be more precise status of topoisomerase genes at the DNA level. restricted without affecting those two markers. With the Therefore, the aim of our study was to analyze the status of evolution of the project, closer loci could be topoisomerase genes at both the DNA and RNA levels, using identified in order to precise the status of TOP2A gene in this highly sensitive semi-quantitative fluorescent PCR, in a patient. Altogether, these results show that molecular analysis population of 25 cases of newly diagnosed childhood ALL. of TOP2A gene at the DNA level represents a reliable and We compared the results of molecular analysis to risk group sensitive molecular test that can be easily and rapidly classification of children in order to identify molecular performed at the first day of diagnosis and which is positive characteristics associated with response to therapy, especially in more than 70% of ALL children. in the group of VHR children showing resistance to In ALL cells, except for patient 8, amplification of the glucocorticoids. TOP2A gene, located in 17q21–q22, is observed indepen-

Leukemia Topoisomerase genes in childhood ALL E Gue´rin et al 538 dently of any chromosome 17 duplication. This result suggests evaluate a possible generalized inability of these cells to the presence of a defined and limited localized at undergo apoptosis. the level of the TOP2A gene, which might not be evidenced by In conclusion, our results, in a first series of 25 cases, show conventional cytogenetic analysis. Amplification of the that the absence of TOP2A gene amplification is a comple- TOP2A genomic region was already reported in other mentary and independent factor that can be easily and rapidly malignancies, such as in roughly 25% of breast cancers, in determined at the first day of diagnosis using a reliable which the TOP2A gene was always simultaneously molecular test and which is associated with a high probability co-amplified with the ERBB2 gene, although fiber fluorescent for the children to be resistant mainly to glucocorticoids and to in situ hybridization demonstrated that both genes are be classified in the VHR group. These results now need to be localized on two independent .49,50 In ALL cells, in extended and confirmed in a larger population using more the 16 TOP2A amplified cases that were also analyzed for sensitive techniques like real-time PCR quantification. In ERBB2 gene status, co-amplification of both genes was addition, longer follow-up of children might indicate if TOP2A similarly observed in 13 cases, but isolated TOP2A gene gene status could be considered as a prognostic factor of the amplification could also be observed in three cases (data not patient’s response to the global chemotherapy and/or of the shown). Further studies with additional genomic markers are patient’s relapse. now required to delineate more precisely the extent of TOP2A gene amplicon in ALL cells. In the 14 cases in which we could perform TOP2A gene Acknowledgements analysis at the RNA level, we found a total correlation between gene overexpression and gene amplification. The 11 We thank Eliane Mengus and Evelyne Neuville for expert children in which TOP2A gene was amplified presented technical assistance as well as Guy Hamel and Nicolas Meyer simultaneous gene overexpression and the three remaining for helpful discussions and statistical analysis. Research grants: cases in which TOP2A gene was not amplified were similarly Supported by the Ligue Re´gionale contre le Cancer (Comite´ du not associated with any gene overexpression. These results Bas-Rhin et du Haut-Rhin), the ARAME (Association re´gionale show that analysis of TOP2A gene at the RNA level is as d’action me´dicale et sociale en faveur d’enfants atteints sensitive as DNA analysis. However, as reported in previous d’affection maligne) and the Hoˆpitaux Universitaires de studies,43,45 we also found large interpatient variations in Strasbourg TOP2A gene expression levels, suggesting that in addition to a simple increase in TOP2A gene copy number, transcriptional and/or post-transcriptional regulation mechanisms might also References be implicated in the increased expression of TOP2A gene in ALL cells. 1 Pui CH. Acute lymphoblastic leukemia in children. Curr Opin We also evaluated TOP2A gene status at the DNA level in Oncol 2000; 12: 3–12. 16 samples of acute myelogenous leukemias (AML). In 2 Silverman LB, Weinstein HJ. Treatment of childhood leukemia. contrast to ALL samples, none of these AML presented TOP2A Curr Opin Oncol 1997; 9: 26–33. gene amplification (data not shown). This result confirms 3 Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J Med previous reports tending to show lower TOP2A gene 1998; 339: 605–615. expression levels in primary AML compared to human T- 4 Wang JC. DNA topoisomerases. Annu Rev Biochem 1996; 65: 43,46 635–692. lymphoblastoid cell lines as controls. 5 Roca J. The mechanisms of DNA topoisomerases. 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From a Schmeits JL, Wang J, Shimizu N. One-megabase sequence analysis mechanistic point of view, this link observed between the of the human immunoglobin lambda gene locus. Genome Res absence of TOP2A gene amplification and the in vivo 1997; 7: 250–261. 9 Tsai-Pflugfelder M, Liu LF, Liu AA, Tewey KM, Whang-Peng J, resistance to glucocorticoids in VHR patients could be Knutsen T, Huebner K, Croce CM, Wang JC. Cloning and explained, at least in part, by the known propensity of TOP2A sequencing of cDNA encoding human DNA topoisomerase II to trigger cell death apoptosis when overexpressed in and localization of the gene to chromosome region 17q21–22. mammalian cells.51 Therefore, we speculate that potential Proc Natl Acad Sci USA 1988; 85: 7177–7181. low levels of TOP2A could reduce the pro-apoptotic potential 10 Austin CA, Sng JH, Patel S, Fisher LM. 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