GENES, CHROMOSOMES & CANCER 48:725–736 (2009) V(D)J Targeting Mistakes Occur at Low Frequency in Acute Lymphoblastic Leukemia Katrina Vanura, 1 * Maruska Marusic Vrsalovic, 2 Trang Le, 1 Rodrig Marculescu, 3 Rajko Kusec, 2 Ulrich Ja ¨ger, 1 and Bertrand Nadel 4 1 Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna,Vienna, Austria 2 Department for Molecular Diagnostics and Genetics, Dubrava University Hospital, Zagreb,Croatia 3 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna,Vienna, Austria 4 Centre d’Immunologie de Marseille-Luminy,CNRS-INSERM-Universite¤ de la Me¤ diterrane¤ e, Marseille, France Translocations of proto-oncogenes to the B-cell or T-cell antigen receptor loci in acute T- or B-cell leukemia and lym- phoma have been, in most cases, accredited to V(D)J or switch recombination depending on the location of the breakpoint at the receptor locus. Only in rare instances, the reports take into account mechanistic characteristics of the translocation mechanism. To assess the functional ability of several sites implicated in supposedly V(D)J-mediated translocations, we tested five sites at four proto-oncogene loci in an ex vivo recombination substrate assay for their potential to act as direct target for V(D)J recombination. Our results show that the LMO2/RBTN2/TTG2 site and one LCK/P56 site readily engage in recombination with a genuine TCR element with the majority of breakpoint junctions showing the characteristics of V(D)J recombination, which strongly supports the involvement of this mechanism in the pathogenesis of the corresponding trans- locations in vivo. The site at the TLX1/HOX11 locus yielded 0.8% V(D)J-specific junctions. Sites at the LCK/P56 and TCF3/ E2A proto-oncogenes resulted in exclusively unspecific breakpoints scattered over part of or the entire proto-oncogene region tested, marking them as unlikely V(D)J recombination targets. Our data suggest that, while being a potentially dan- gerous mechanism due to the introduction of DNA breaks, V(D)J recombination is a tightly controlled mechanism allowing for only few direct mistakes. V V C 2009 Wiley-Liss, Inc. INTRODUCTION Lymphoid neoplasia is characterized by a vari- ety of genetic changes in chromosome structure ranging from simple reciprocal translocations to complex karyotypes (Rabbitts, 1994, 2001). Usu- ally, these changes are accompanied by the over- expression of one to many genes, in most cases, due to their erroneous placement under specific genetic regulative elements, or by expression of fusion proteins with particular oncogenic features (Nambiar et al., 2008). Translocations of proto-oncogenes to the B-cell or T-cell antigen receptor loci (BCR, TCR) very often are accompanied by overexpression of the proto-oncogene involved. For these transloca- tions, either V(D)J or class switch recombination are held responsible depending on whether the breakpoints lie within the coding regions for the antigen receptors or within the IGH switch regions (Marculescu et al., 2008). In addition, so- matic hypermutation has been suggested as caus- ative mechanism for a number of B-cell malignancies (Ku ¨ ppers and Dalla-Favera, 2001). In early reports, however, statements regarding the culpable recombination mechanisms are solely based on the morphological characteristics of the translocation junctions, and identification and designation of translocation mechanisms sometimes occurred indiscriminately, partly due to the incomplete mechanistic knowledge avail- able at the time. The accumulated, more detailed knowledge on V(D)J, switch recombination, and somatic hypermutation with their inherent rules helped to create a more refined classification. Still, functional tests are necessary to estimate Additional Supporting Information may be found in the online version of this article. Supported by: Austrian Ministry of Education, Science and Cul- ture [through the projects Gen-AU-Child (GZ 200.136/1–VI/1/ 2005), CLEXO (GZ 200.062/2-VI/1/2002)]; Center of Molecular Medicine of the Austrian Academy of Sciences (Grant number: 20010); ‘‘Cancer Research Initiative’’ of the Medical University of Vienna, Austria; Austrian Exchange Service. *Correspondence to: Katrina Vanura, Division of Hematology and Hemostaseology, Department of Medicine I, Medical Univer- sity of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: katrina.vanura@meduniwien.ac.at Received 28 November 2008; Accepted 15 April 2009 DOI 10.1002/gcc.20677 Published online 19 May 2009 in Wiley InterScience (www.interscience.wiley.com). V V C 2009 Wiley-Liss, Inc.