'Upregulation of asparagine synthetase and cell cycle ...

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vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia. Blood 2000; 96: 1094–1099. 2 Stams WA, den Boer ML, Beverloo HB, ...
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AS expression in t(12;21) positive ALL

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cycle-associated) genes of AML1. This matches perfectly with the higher AS expression in these cells at diagnosis compared to t(12;21)-negative ALL cells, since AS transcription is increased in cells that are hampered to proceed through the cell cycle. Nutrition stress caused by L-Asp even enhances this increase in AS expression.2,3 We could not confirm the findings of Krejci in relation to low AS expression in t(12;21)-positive ALL and an unfavorable prognosis.

WAG Stams1 ML den Boer1 HB Beverloo2 ER van Wering3 R Pieters1,3

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1 Erasmus MC, Sophia Children’s Hospital, University Medical Center Rotterdam, Division of Pediatric Oncology/Hematology, Rotterdam, The Netherlands; 2 Erasmus MC, University Medical Center Rotterdam, Department of Clinical Genetics, Rotterdam, The Netherlands; and 3 Dutch Childhood Oncology Group, The Hague, The Netherlands

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Figure 2 Probability of disease-free survival analysis (pDFS) in t(12;21)-positive ALL with high (n ¼ 21, 5-years pDFS 67722%) and low (n ¼ 20, 5-years pDFS 56721%) expression of AS. The median value (1.2) was used as a cutoff for the separation into high and low expression of AS (a). AS expression in t(12;21)-positive ALL patients at diagnosis with (n ¼ 10) subsequent relapse compared to t(12;21)positive ALL patients in continuous complete remission (CCR) (n ¼ 31) (MWU: P40.05) (b). AS mRNA was determined by real-time quantitative PCR using the same conditions as described previously.2

cohort, however, we could not confirm this: high AS expression did not correlate with a higher disease-free survival (Figure 2a) and AS expression did not differ between t(12;21)-positive ALL patients with and without subsequent relapse (Figure 2b). We hypothesize that t(12;21)-positive ALL cells already at diagnosis have impaired potential to proceed from G1 into S phase due to the fact that TEL/AML1 represses the target (ie cell

1 Ramakers-van Woerden NL, Pieters R, Loonen AH, Hubeek I, van Drunen E, Beverloo HB et al. TEL/AML1 gene fusion is related to in vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia. Blood 2000; 96: 1094–1099. 2 Stams WA, den Boer ML, Beverloo HB, Meijerink JP, Stigter RL, van Wering ER et al. Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21)+ pediatric ALL. Blood 2003; 101: 2743–2747. 3 Krejci O, Starkova J, Otova B, Madzo J, Kalinova M, Hrusak O et al. Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells. Leukemia 2004; 18: 434–441. 4 Aslanian AMFB, Kilberg MS. Asparagine synthetase expression alone is sufficient to induce L-asparaginase resistance in MOLT-4 human leukaemia cells. Biochem J 2001; 357: 321–328. 5 Strom DK, Nip J, Westendorf JJ, Linggi B, Lutterbach B, Downing JR et al. Expression of the AML-1 oncogene shortens the G(1) phase of the cell cycle. J Biol Chem 2000; 275: 3438–3445. 6 Bernardin-Fried F, Kummalue T, Leijen S, Collector MI, Ravid K, Friedman AD. AML1/RUNX1 increases during G1 to S cell cycle progression independent of cytokine-dependent phosphorylation and induces cyclin D3 gene expression. J Biol Chem 2004; 279: 15678–15687. 7 Hiebert SW, Sun W, Davis JN, Golub T, Shurtleff S, Buijs A et al. The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription. Mol Cell Biol 1996; 16: 1349–1355. 8 Colletta G, Cirafici AM. TSH is able to induce cell cycle-related gene expression in rat thyroid cell. Biochem Biophys Res Commun 1992; 183: 265–272.

Reply to ‘Upregulation of asparagine synthetase and cell cycle arrest in t(12;21) positive ALL’ by Stams et al

Leukemia (2005) 19, 319–320. doi:10.1038/sj.leu.2403574 Published online 4 November 2004 6

Current address: Experimental Hematology, Cincinnati Children’s Hospital Medical Center, OH, USA Correspondence: Dr O Krejci, Cincinnati Children’s Hospital Medical Center, Division of Experimental Hematology, Stem Cell Biology Program, 3333 Burnet Avenue, Mail Location 7013, TCHRF 7552, Cincinnati, OH 45229, USA; Fax: þ 1 513 636 3768; E-mail: [email protected] Received 9 August 2004; accepted 9 September 2004; Published online 4 November 2004

TO THE EDITOR

In the March issue of Leukemia, we reported a difference in disease-free survival in t(12;21) patients based on asparagine synthetase expression level at initial diagnosis.1 Our group of patients was treated according BFM protocols (ALL BFM 90 and ALL BFM 95). In this issue of Leukemia Stams et al2 present no such difference in a similar group of patients treated according to different protocols. The difference in t(12;21) group behaviour under different therapeutic protocol was observed previously.3 Moreover Stams et al used GAPDH as a control gene in their Leukemia

Correspondence

320 study. This gene is located on chromosome 12p13, a region frequently deleted in leukaemic cells with t(12;21).4,5 Deletions on genomic level could interfere with GAPDH expression and thus have effect on asparagine synthetase to GAPDH ratio presented by Stams et al. In addition, Stams et al present a small but significant difference between the t(12;21) positive and t(12;21) negative untreated ALL samples regarding the percentage of cells in the Sphase. In our original study we reported a decrease of cell percentage in S þ G2/M phases after in vitro L-asparaginase treatment selectively in t(12;21)-positive cells. The observed difference was at the order of one magnitude. The small difference observed by Stams et al in the untreated patient specimens might be of interest, especially when confirmed in an independent cohort. However, there are unanswered issues like why the proportion of G2M phase is not correspondingly depressed and whether this difference might be modulated by the same mechanisms as the L-asparaginase-induced cell cycle arrest.

O Krejci1,6 J Starkova1,2 B Otova3 J Madzo1,2 M Kalinova1,4 O Hrusak1,5 J Trka1,2

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CLIP – Childhood Leukaemia Investigation Prague, Charles University Prague, Czech Republic; 2 Department of Paediatric Haematology and Oncology, 2nd Medical School, Charles University Prague, Czech Republic; 3 Department of Biology and Medical Genetics,

1st Medical School, Charles University Prague, Czech Republic; 4 Department of Pathology and Molecular Medicine, Charles University Prague, Czech Republic; and 5 Department of Immunology, 2nd Medical School, Charles University Prague, Czech Republic

References 1 Krejci O, Starkova J, Otova B, Madzo J, Kalinova M, Hrusak O et al. Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells. Leukemia 2004; 18: 434–441. 2 Stams WA, den Boer ML, Beverloo HB, van Wering ER, Pieters R. Upregulation of asparagine synthetase and cell cycle arrest in t(12;21) positive ALL. Leukemia 2005; 19: 318–319. 3 Loh ML, Silverman LB, Young ML, Neuberg D, Golub TR, Sallan SE et al. Incidence of TEL/AML1 fusion in children with relapsed acute lymphoblastic leukemia. Blood 1998; 92: 4792–4797. 4 Raynaud SD, Dastugue N, Zoccola D, Shurtleff SA, Mathew S, Raimondi SC. Cytogenetic abnormalities associated with the t(12;21): a collaborative study of 169 children with t(12;21)-positive acute lymphoblastic leukemia. Leukemia 1999; 13: 1325–1330. 5 Zuna J, Ford A, Peham M, Patel N, Saha V, Eckert C et al. TEL deletion analysis supports a novel view of relapse in childhood acute lymphoblastic leukemia. Clin Cancer Res 2004; 10: 5355–5360.

Reply to Krejci et al

Leukemia (2005) 19, 320–321. doi:10.1038/sj.leu.2403576 Published online 4 November 2004 TO THE EDITOR

As an explanation for the difference in prognostic relevance of AS expression in our study1 and Krejci’s study,2 Krejci et al commented that GAPDH as a reference gene might be inappropriate, since this gene is located at 12p13. However, GAPDH is located telomeric from TEL with a distance of approximately 5 Mb. The interstitial deletion of the nontranslocated TEL gene in t(12;21) positive ALL usually encompasses an area centromeric from TEL.3 Furthermore, GAPDH was equally expressed in t(12;21) positive and t(12;21) negative ALL in a former study.4 Moreover, no difference in GAPDH expression was found between t(12;21) positive patients with and without a deletion of TEL.5 Therefore, we believe that GAPDH can be used as a reference gene in RTQ-PCR. Even if we correlate the AS expression of t(12;21) positive ALL patients with clinical outcome, without correcting for GAPDH, opposite results from Krejci et al occur: 5-years pDFS of 27% with high AS expression (expression above the median AS expression (P450)) compared to 5-years pDFS of 94% with low AS expression (Po50); P ¼ 0.002. Correspondence: Dr M den Boer, Erasmus MC – Sophia Children’s Hospital, University Medical Center Rotterdam, Division of Pediatric Oncology/Hematology, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands; Fax: þ 31 10 4089433; E-mail: [email protected] Received 18 August 2004; accepted 9 September 2004; Published online 4 November 2004 Leukemia

The main message of our letter, however, was to extend the findings of Krejci et al concerning the relationship between TELAML1 and cell cycle distribution. Our data show that t(12;21) positive ALL already at diagnosis show cell cycle retardation prior to L-Asp treatment. A possible explanation for this is the repression of AML1 target genes by TEL-AML1.1 Further support of our findings are the fact that TEL-AML1 affects somatic recombination of antigen receptor genes, which is closely related to the cell cycle6 and the fact that t(12;21) positive ALL have higher AS expression at initial diagnosis compared to t(12;21) negative ALL.2,4

WAG Stams1 ML den Boer1 HB Beverloo2 ER van Wering3 R Pieters1,3

1 Erasmus MC – Sophia Children’s Hospital, University Medical Center Rotterdam, Division of Pediatric Oncology/Hematology, Rotterdam, The Netherlands; 2 Erasmus MC – University Medical Center Rotterdam, Department of Clinical Genetics, Rotterdam, The Netherlands; and 3 Dutch Childhood Oncology Group, The Hague, The Netherlands

References 1 Stams WA, den Boer ML, Beverloo HB, Van Wering ER, Pieters R. Upregulation of AS and cell cycle arrest in t(12;21) positive ALL. Leukemia 2005; 19: 318–319. 2 Krejci O, Starkova J, Otova B, Madzo J, Kalinova M, Hrusak O et al. Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells. Leukemia 2004; 18: 434–441. 3 Kobayashi H, Montgomery KT, Bohlander SK, Adra CN, Lim BL, Kucherlapati RS et al. Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human