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
1
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
Correspondence
321 chromosome 12 in malignant hematologic diseases. Blood 1994; 84: 3473–3482. 4 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. 5 Stams WA, den Boer ML, Beverloo HB, Meijerink JP, Van Wering ER, Janka-Schaub GE et al. Expression levels of TEL, AML1
and the fusion products TEL-AML1 and AML1-TEL versus drug sensitivity and clinical outcome in t(12;21) positive pediatric ALL. submitted. 6 Hubner S, Cazzaniga G, Flohr T, van der Velden VH, Konrad M, Potschger U et al. High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1-positive leukemias. Leukemia 2004; 18: 84–91.
Reply to Stams et al
Leukemia (2005) 19, 321. doi:10.1038/sj.leu.2403577 Published online 4 November 2004
TO THE EDITOR
In their original letter1 commenting to our paper,2 Stams et al showed they failed to confirm statistical difference in asparagine synthetase (AS) expression levels in TEL/AML1pos paediatric ALL patients with or without subsequent relapses. This difference might be considered clinically relevant since data on patients from different protocols are compared. Variant dosage of specific chemotherapeutics and their combinations used in the different protocols may lead to dissimilar treatment results (as it was clearly shown with E2A/ PBX1 aberration).3 Therefore, this discrepancy might be exposing a potentially very significant biological feature of TEL/ AML1pos patients, hitherto unknown. However, data on treatment protocols are unavailable in the study of Stams et al. Such data are needed to substantiate their doubts about the prognostic significance of AS in TEL/AML1pos ALL. Another explanation of a contrast of these studies is the difference in reference genes. In their justification of GAPDH use as a reference gene, Stams et al neglect that the deletions were reported to include regions centromeric as well as telomeric from the TEL gene.4,5 Even if the coding sequence of the GAPDH gene would be intact, the deletion could affect regulating sequences often localised at a larger distance. The reported differences of AS are subtle, and normalised expression is sensitive to minor changes in reference gene expression. Showing the raw AS expression data or any other non-normalised RQ-PCR data might only blur the real picture. It would help the discussion and justify the use of GAPDH if the expression of various reference genes in a set of samples with t(12;21) are compared. Then, we can proceed to look whether AS has a different prognostic significance in different chemotherapeutic protocols.
O Krejci1,6 J Starkova1,2 B Otova3 J Madzo1,2 M Kalinova1,4 O Hrusak1,5 J Trka1,2
1
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 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. 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 Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004; 350: 1535–1548. 4 Andreasson P, Johansson B, Billstrom R, Garwicz S, Mitelman F, Hoglund M. Fluorescence in situ hybridization analyses of hematologic malignancies reveal frequent cytogenetically unrecognized 12p rearrangements. Leukemia 1998; 12: 390–400. 5 Kanerva J, Niini T, Vettenranta K, Riikonen P, Makipernaa A, Karhu R et al. Loss at 12p detected by comparative genomic hybridization (CGH): association with TEL-AML1 fusion and favorable prognostic features in childhood acute lymphoblastic leukemia (ALL). A multi-institutional study. Med Pediatr Oncol 2001; 37: 419–425.
6 Current address: Dr O. Krejci, Experimental Hematology, Cincinnati Children’s Hospital Medical Center, OH, USA Received 3 September 2004; accepted 9 September 2004; Published online 4 November 2004
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