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

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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Available from: Luca Lo Nigro, Sep 29, 2015
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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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    ABSTRACT: Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment. © 2013 Wiley Periodicals, Inc.
    Genes Chromosomes and Cancer 02/2014; 53(2):117-28. DOI:10.1002/gcc.22124 · 4.04 Impact Factor
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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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    ABSTRACT: A 68-year-old man was admitted to our hospital in September 2008 because of a left-sided chest pain. Bone marrow examination showed that 85.5% of leukemic cells were positive for myeloperoxidase (MPO) and were negative for esterase stain. Flow cytometric analysis (FCM) revealed the expression of CD19, CD79a, CD13, CD33, CD34, and HLA-DR on the blasts. Cytogenetic analysis of bone marrow cells using the G-banding technique demonstrated 47, XY, +X, t(4;11;7)(q21;q23;q22) in five of the 20 analyzed cells. The patient was diagnosed as having mixed biphenotypic acute leukemia according to the European Group for Immunologic Classification of Leukemia criteria. Mixed-phenotype acute leukemia is a rare, difficult to diagnose entity. Whether patients with mixed-phenotype acute leukemia should be treated with regimens designed for acute myeloid leukemia, acute lymphoblastic leukemia, or both remains unclear.
    11/2011; 2011(8):148482. DOI:10.1155/2011/148482
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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). "
    DNA Repair and Human Health, 10/2011; , ISBN: 978-953-307-612-6
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