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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0144. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0144v1 100/1/347    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Panzer-Grümayer, E. R. Articles by Haas, O. A. Search for Related Content PubMed PubMed Citation Articles by Panzer-Grümayer, E. R. Articles by Haas, O. A. Related Collections Neoplasia Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 July 2002, Vol. 100, No. 1, pp. 347-349 BRIEF REPORT Nondisjunction of chromosomes leading to hyperdiploid childhood B-cell precursor acute lymphoblastic leukemia is an early event during leukemogenesis E. Renate Panzer-Grümayer, Karin Fasching, Simon Panzer, Klaudia Hettinger, Klaus Schmitt, Sylvia Stöckler-Ipsiroglu, and Oskar A. Haas From the Children's Cancer Research Institute; St Anna Kinderspital; Clinic for Blood Group Serology, University of Vienna; Landeskinderkrankenhaus Linz; and Austrian Newborn Screening Laboratory, Department of Pediatrics, University of Vienna; all of Austria.     Abstract Top Abstract Introduction Patient, materials, and methods Results and discussion References A hyperdiploid karyotype is found in 30% of B-cell precursor acute lymphoblastic leukemias in childhood. The time of nondisjunction of chromosomes leading to hyperdiploidy during leukemogenesis is unknown. We used the 3 clonotypic immunoglobulin heavy chain (IgH) gene rearrangements as molecular markers for each of the 3 chromosomes 14 in a case with hyperdiploid acute lymphoblastic leukemia to define the order of eventsnamely, somatic recombination and nondisjunction of chromosomesduring leukemia development. A partial sequence homology of the incomplete DJH rearrangement with 1 of the 2 nonfunctional VDJH rearrangements suggests that the doubling of chromosomes had occurred after this DJH rearrangement and thus during early B-cell differentiation. The occurrence of the nondisjunction of chromosomes as well as ongoing rearrangement processes in utero were confirmed by the presence of all 3 IgH rearrangements in neonatal blood spots, providing the first evidence that hyperdiploidy formation is an early event in leukemogenesis in these leukemias. (Blood. 2002;100:347-349) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Patient, materials, and methods Results and discussion References A hyperdiploid karyotype is detected in about 30% of childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and is usually associated with low-risk features and good prognosis.1 Among these leukemias, the group with more than 50 chromosomes has a nonrandom pattern of chromosomal gain. There is evidence that the hyperdiploid karyotype arises by a simultaneous gain of multiple chromosomes from a diploid karyotype during a single abnormal cell division.2 One of the frequently doubled chromosomes is chromosome 14, which is commonly present in 3 but sometimes also in 4 copies in hyperdiploid BCP ALL.3 On this chromosome the immunoglobulin heavy chain (IgH) genes are located. Following their somatic recombination they can be used as clonal markers in B-lineage leukemias.4,5 During normal B-cell development, IgH is the first antigen receptor gene that is rearranged, joining first a DH to a JH segment on both alleles, followed by a VH to DJH rearrangement on one of these alleles. If this latter recombination is not productive, the other incomplete rearrangement undergoes further recombination.6,7 The somatic recombination of IgH genes in childhood BCP ALL follows the normal B-cell development, but most of the clonotypic IgH rearrangements are not productive, which suggests that a first transforming event has occurred in a very immature cell.4,5 These IgH rearrangements can be used as clonal markers to investigate the time of nondisjunction of chromosomes 14 in hyperdiploid BCP ALL, if the rearrangements of each of the 3 IgH genes are unique. This may occur if one chromosome 14 duplicates before its respective IgH rearrangement or a rearrangement continues to rearrange after formation of hyperdiploidy. In line with these considerations are the results from a recent report showing that a subgroup of hyperdiploid ALL has 3 IgH rearrangements, while another subgroup has 2 rearrangements.5 These data support the assumption that nondisjunction of chromosomes can occur before or after the initiation of IgH recombination, respectively. We therefore selected a hyperdiploid BCP ALL with 3 chromosomes 14 and 3 IgH gene rearrangements to investigate whether the nondisjunction had occurred before or after the IgH rearrangements. Moreover, neonatal blood spots were investigated for the presence of the leukemia clone-specific IgH rearrangements. We thereby provide the first evidence that nondisjunction leading to hyperdiploid BCP ALL in young children is an event early in B-cell differentiation during leukemogenesis in utero.     Patient, materials, and methods Top Abstract Introduction Patient, materials, and methods Results and discussion References Patient We selected one hyperdiploid BCP ALL (CD10+, CD19+, cµ) case with a trisomy 14 and 3 IgH rearrangements, a 2.6-year-old boy. Hyperdiploidy was identified by routine cytogenetics in bone marrow cells at diagnosis. The presence of trisomy 14 was confirmed by fluorescence in situ hybridization in 76% of cultured interphase cells. The study was approved by the ethics committee of the Children's Cancer Research Institute, St Anna Kinderspital, and informed consent was obtained from the parents. DNA preparation from fresh cells and Guthrie cards High molecular weight DNA was extracted by proteinase K digestion followed by phenol chloroform extraction. Guthrie spots were routinely cut into 3 pieces, and DNA was extracted from each piece separately at different occasions. DNA from Guthrie cards was extracted using the DNAzol kit (Vienna Labs, Vienna, Austria) as described previously.8 Integrity of the DNA was confirmed by amplification of the exon 16 of the RET proto-oncogene.8 Single-cell sorts Single leukemic cells were sorted on a FACStar Plus (BD Bioscience, San Jose, CA) using the light scatter profile of the mononuclear cells from bone marrow at diagnosis, which contained more than 95% blasts. Cells were sorted in a 200-µL Eppendorf tube containing 20 µL 1 × buffer and stored at 20°C until further use. Determination of leukemia clone-specific IgH gene rearrangements Polymerase chain reaction (PCR) amplification of incomplete and complete IgH rearrangements was performed using family-specific DH and VH primers, respectively, and one JH consensus primer, as described previously.9 Amplified products were directly sequenced in both directions, and involved gene segments were identified by BLAST sequence similarity searches and by comparison with published sequences of all known human Ig genes (http://www.ebi.ac.uk). Clone-specific PCR A first-round PCR was performed using DH3 and VH3 family-specific primers as described above. One microliter of a 1:20 dilution of the first-round 50 µL PCR reaction was used for a second-round nested PCR. For the DH3-22/J5 rearrangement, the primers were positioned into the intron region 5' of the DH3-22 segment overlapping the 3' end of the DH3 primer to avoid coamplification of the VH3/DH3-22/J5 rearrangement. For the 2 different complete rearrangements, 3' primers were designed homologous to the individual VND regions and amplified with internal VH3 specific primers, respectively. PCR products were size-separated on agarose and polyacrylamide gels and visualized by ethidium bromide staining. Precautions to avoid contamination with amplified material were followed as previously described.8 Amplifications were performed on a PTC 300 Thermocycler (Techne, Cambridge, United Kingdom). All PCR products of the expected size were directly sequenced after gel purification using QIAEX II Gel Extraction kit (Qiagen, Valencia, CA).     Results and discussion Top Abstract Introduction Patient, materials, and methods Results and discussion References The hyperdiploid leukemia with a trisomy 14 selected for our study has 3 different nonfunctional IgH rearrangements (Table 1). It can be assumed that each of these IgH rearrangements is located on a different chromosome 14. To confirm that all 3 alleles are contained within a single leukemia clone, we sorted individual leukemic cells and performed the clone-specific nested PCR for each IgH rearrangement in 10 sorted cells each. We obtained PCR products of the expected size for rearrangements A, B, and C in 9, 10, and 7 cells, respectively (Figure 1). Sequencing of these obtained amplicons revealed the respective rearrangements, thus confirming monoclonality of the leukemia. Two of these rearrangementsthe incomplete DJH and one of the complete VDJH rearrangementswere found to have homologous DJH joinings (Table 1). Thus, the chromosome harboring the original DJH rearrangement must have duplicated before the subsequent joining of a VH segment to one of these incomplete rearrangements.10 These findings suggest that an incomplete DJH rearrangement on one allele and presumably a nonfunctional VDJH rearrangement on the second allele were present in the diploid cell before mitosis, leading to the assumption that the nondisjunction of chromosome 14 is an early event in B-cell differentiation. Whereas in mice clonal expansion at an early stage of B-cell development does not require pre-B-cell receptor formation,11 IgH expression is a prerequisite for initiating early cell division in normal B lymphocytes in humans.7 Thus, an earlier transforming eventpreceding the abnormal mitosiswas necessary for this diploid cell to enter the cell cycle.                                View this table: [in this window] [in a new window]   Table 1. Nucleotide sequences of the clonotypic IgH rearrangements in a hyperdiploid ALL View larger version (27K): [in this window] [in a new window]   Figure 1. Analysis of leukemia-specific IgH rearrangements in single leukemic cells and neonatal blood spots of a patient with hyperdiploid ALL. A 2-round nested clone-specific PCR was performed, and products were size fractionated on 2.5% agarose (A) or 4% to 12% polyacrylamide gels (B). (Ai) DH3-22/J5 rearrangement of 224 base pairs; (Aii) VH3/DH3-22/J5 rearrangement of 169 base pairs; and (Aiii) VH3/DH2-22/J4 rearrangement of 125 base pairs. (A) SM size marker VIII (Roche, Mannheim, Germany), lane 0, no DNA; lanes 1 to 5, dilutions of leukemic cell DNA into peripheral blood DNA 102 to 106; lane 6, DNA from control Guthrie card; lane 7, DNA from the patient's Guthrie card; lane 8, PCR product from a representative single cell. (B) SM size marker, lane 1, peripheral blood DNA from healthy donors; lane 2, DNA from the patient's Guthrie card; lane 3, DNA from control Guthrie card; lane 4, single leukemia cell; lane 5, dilution of DNA from leukemic cells into peripheral blood DNA 105. Arrows indicate the size of specific PCR products. Sequencing of all PCR products of the expected size confirmed identity with the original rearrangements. These data were reproduced in a second experiment (data not shown). We and others have shown the prenatal origin of leukemia-specific chromosomal translocations in children with ALL by retrospective evaluation of neonatal blood spots.8,12,13 Using this source of the earliest available hematopoietic cells in this patient for the retrospective analysis of clonotypic IgH rearrangements, we addressed the question of whether nondisjunction leading to hyperdiploidy had occurred prenatally. Indeed, all 3 rearrangements were present already at birth (Figure 1), indicating that doubling of chromosome 14 as well as the ongoing rearrangement processes occur early during leukemogenesis, while the leukemia became clinically apparent only 2.6 years later. Because it was shown that hyperdiploidy results from one single abnormal mitosis, nondisjunction of chromosome 14 is likely to concur with that of the other multiplied chromosomes present in a hyperdiploid leukemia.2 However, the causes for nondisjunction as well as the impact of hyperdiploidy on leukemia development still remain elusive.14     Footnotes Submitted January 16, 2002; accepted February 23, 2002. Prepublished online as Blood First Edition Paper, [April 17, 2002]; DOI 10.1182/blood-2002- 01-0144. Supported by a grant from the Österreichische Nationalbank Jubiläumsfonds no. 8438 to E.R.P.-G. and from the "Österreichische Kinderkrebshilfe." The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: E. Renate Panzer-Grümayer, CCRI, St Anna Kinderspital, Kinderspitalg.6, A-1090 Vienna, Austria; e-mail: panzer{at}ccri.univie.ac.at.     References Top Abstract Introduction Patient, materials, and methods Results and discussion References 1. Raimondi SC, Pui C-H, Hancock ML, Behm FG, Filatov L, Rivera GK. Heterogeneity of hyperdiploid (51-67) childhood acute lymphoblastic leukaemia. Leukemia. 1996;10:213-224[Medline] [Order article via Infotrieve]. 2. Onodera N, McCabe NR, Rubin CM. Formation of hyperdiploid karyotype in childhood acute lymphoblastic leukemia. Blood. 1992;80:203-208[Abstract/Free Full Text]. 3. Pui C-H, Crist WM, Look AT. Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood. 1990;76:1449-1463[Abstract/Free Full Text]. 4. Height SE, Swansbury GJ, Matutes E, Treleaven JG, Catovsky D, Dyer MJS. Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia. Blood. 1996;87:5242-5250[Abstract/Free Full Text]. 5. Szczepanski T, Willemse MJ, van Wering ER, van Weerden JF, Kamps WA, van Dongen JJM. Precursor-B-ALL with DH-JH gene rearrangements have an immature immunogenotype with a high frequency of oligoclonality and hyperdiploidy of chromosome 14. Leukemia. 2001;15:1415-1423[CrossRef][Medline] [Order article via Infotrieve]. 6. Alt FW, Oltz EM, Young F, Gorman J, Taccioli G, Chen J. VDJ recombination. Immunol Today. 1992;13:306-314[CrossRef][Medline] [Order article via Infotrieve]. 7. LeBien TW. Fates of human B-cell precursors. Blood. 2000;96:9-23[Abstract/Free Full Text]. 8. Fasching K, Panzer S, Haas OA, Marschalek R, Gadner H, Panzer-Grümayer ER. Presence of clone-specific antigen receptor gene rearrangements at birth indicates an in utero origin of diverse types of early childhood acute lymphoblastic leukemia. Blood. 2000;95:2722-2724[Abstract/Free Full Text]. 9. Szczepanski T, Pongers-Willemse MJ, Langerak AW, et al. Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor   lineage. Blood. 1999;93:4079-4085[Abstract/Free Full Text]. 10. Steenbergen EJ, Verhagen OJHM, van den Berg H, et al. Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement of V to DJ joining) in childhood B precursor acute lymphoblastic leukemia. Leukemia. 1997;11:1258-1265[CrossRef][Medline] [Order article via Infotrieve]. 11. Kline GH, Hayden TA, Riegert P. The initiation of B cell clonal expansion occurs independently of pre-B cell receptor formation. J Immunol. 2001;167:5136-5142[Abstract/Free Full Text]. 12. Gale KB, Ford AM, Repp R, et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A. 1997;94:13950-13954[Abstract/Free Full Text]. 13. Wiemels JL, Cazzaniga G, Daniotti M, et al. Prenatal origin of acute lymphoblastic leukemias in children. Lancet. 1999;354:1499-1503[CrossRef][Medline] [Order article via Infotrieve]. 14. Greaves M. Molecular genetics, natural history and the demise of childhood leukemia. Eur J Cancer. 1999;35:173-185[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: M. Eguchi-Ishimae, M. Eguchi, H. Kempski, and M. Greaves NOTCH1 mutation can be an early, prenatal genetic event in T-ALL Blood, January 1, 2008; 111(1): 376 - 378. [Abstract] [Full Text] [PDF] S. Fischer, G. Mann, M. Konrad, M. Metzler, G. Ebetsberger, N. Jones, B. Nadel, O. Bodamer, O. A. Haas, K. Schmitt, et al. Screening for leukemia- and clone-specific markers at birth in children with T-cell precursor ALL suggests a predominantly postnatal origin Blood, October 15, 2007; 110(8): 3036 - 3038. [Abstract] [Full Text] [PDF] K. Paulsson, I. Panagopoulos, S. Knuutila, K. J. Jee, S. Garwicz, T. Fioretos, F. Mitelman, and B. Johansson Formation of trisomies and their parental origin in hyperdiploid childhood acute lymphoblastic leukemia Blood, October 15, 2003; 102(8): 3010 - 3015. [Abstract] [Full Text] [PDF] M. F. Greaves, A. T. Maia, J. L. Wiemels, and A. M. Ford Leukemia in twins: lessons in natural history Blood, October 1, 2003; 102(7): 2321 - 2333. [Abstract] [Full Text] [PDF] M. Konrad, M. Metzler, S. Panzer, I. Ostreicher, M. Peham, R. Repp, O. A. Haas, H. Gadner, and E. R. Panzer-Grumayer Late relapses evolve from slow-responding subclones in t(12;21)-positive acute lymphoblastic leukemia: evidence for the persistence of a preleukemic clone Blood, May 1, 2003; 101(9): 3635 - 3640. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0144v1 100/1/347    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Panzer-Grümayer, E. R. Articles by Haas, O. A. Search for Related Content PubMed PubMed Citation Articles by Panzer-Grümayer, E. R. Articles by Haas, O. A. 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