Raffini, L. J. et al. Panhandle and reverse-panhandle PCR enable cloning of der(11) and der(other) genomic breakpoint junctions of MLL translocations and identify complex translocation of MLL, AF-4, and CDK6. Proc. Natl Acad. Sci. USA 99, 4568-4573

Division of Oncology, Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2002; 99(7):4568-73. DOI: 10.1073/pnas.062066799
Source: PubMed


We used panhandle PCR to clone the der(11) genomic breakpoint junction in three leukemias with t(4;11) and devised reverse-panhandle PCR to clone the breakpoint junction of the other derivative chromosome. This work contributes two elements to knowledge on MLL translocations. First is reverse-panhandle PCR for cloning breakpoint junctions of the other derivative chromosomes, sequences of which are germane to understanding the MLL translocation process. The technique revealed duplicated sequences in one case of infant acute lymphoblastic leukemia (ALL) and small deletions in a case of treatment-related ALL. The second element is discovery of a three-way rearrangement of MLL, AF-4, and CDK6 in another case of infant ALL. Cytogenetic analysis was unsuccessful at diagnosis, but suggested t(4;11) and del(7)(q21q31) at relapse. Panhandle PCR analysis of the diagnostic marrow identified a breakpoint junction of MLL intron 8 and AF-4 intron 3. Reverse-panhandle PCR identified a breakpoint junction of CDK6 from band 7q21-q22 and MLL intron 9. CDK6 encodes a critical cell cycle regulator and is the first gene of this type disrupted by MLL translocation. Cdk6 is overexpressed or disrupted by translocation in many cancers. The in-frame CDK6-MLL transcript is provocative with respect to a potential contribution of the predicted Cdk6-MLL fusion protein in the genesis of the ALL, which also contains an in-frame MLL-AF4 transcript. The sequences in these three cases show additional MLL genomic breakpoint heterogeneity. Each breakpoint junction suggests nonhomologous end joining and is consistent with DNA damage and repair. CDK6-MLL is a new fusion of both genes.

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    • "In addition, we find that etoposide increases the proportion of cells in which RUNX1 and RUNX1T1 loci are closely juxtaposed and that this effect is TOP2B dependent. Unlike MLL, where multiple t-AML patient breakpoints have been published (Aplan et al., 1996; Atlas et al., 1998; Strissel et al., 2000; Raffini et al., 2002; Langer et al., 2003; Whitmarsh et al., 2003; Libura et al., 2005; Felix et al., 2006; Cowell et al., 2012), only one RUNX1-RUNX1T1 translocation breakpoint has been mapped at base pair level from a t- AML patient to date (Zhang et al., 2002). Without this positional information it is difficult to hypothesize on specific mechanisms involved in therapy-related versus de novo RUNX1-RUNX1T1 leukemia cases (Shimizu et al., 1992; Xiao et al., 2001; Zhang et al., 2002). "
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    • "The present patient had a translocation at 7q22 in addition to t(4;11)(q21;q23). The same chromosome abnormalities detected in the present case, including threeway translocation of MLL-AF4, have been reported by Raffini et al. [9]. Three-way or more frequent chromosomal translocations have been reported in 1.8% of patients with the 11q23 translocation [10]. "
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    • "This leads to a DNA damage-repair model in which various naturally occurring DNA topoisomerase II poisons induce DNA topoisomerase II-mediated damage in leukemia in utero (Gilliland et al., 2004). The large deleted regions observed in other infant cases are consistent with multiple sites of breakage or, alternatively, more extensive processing (Raffini et al., 2002). "

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