The MLL gene (11q23) is frequently involved in genetic aberrations in de novo and therapy-related acute myeloid leukemia (t-AML). In t-AML, MLL translocations are detectable in up to 10% of all cases.1 Typically, these t-AML are characterized by a short latency period of up to 2 years and are associated with previous topoisomerase inhibitor treatment.2 The initiating event is thought to be a breakage at the topoisomerase binding site within the breakpoint cluster region (bcr; exons 5–11) of the MLL gene. In more than 70% of MLL-positive t-AML, the MLL breakpoint is localized in the telomeric region of the bcr, whereas in de novo AML, it is in the centromeric region.3 MLL aberrations (deletions, insertions, translocations) can be induced in normal hematopoietic progenitors in vitro by exposure to topoisomerase II inhibitors,4 and by retrospective analysis, t-AML-associated MLL aberrations have been backtracked up to a few months after topoisomerase II inhibitor therapy in clinical samples.5
As the incidence of t-AML may be up to 10% in high-dose, myeloablative treatment schedules, it is of certain interest to identify patients who are at an increased risk to attract this complication. Recently, Ng et al.6 investigated clinical samples of children who were treated with topoisomerase inhibitors/anthracyclines by Southern blot and panhandle PCR and observed MLL aberrations (MLL breakage and illegitimate recombination) in 7% (5/71) of the patients.
We sought to investigate prospectively whether MLL aberrations are detectable in adult patients with aggressive non-Hodgkin's lymphoma treated with a myeloablative topoisomerase II inhibitor-containing regimen (MegaChoep trial of the German high-grade lymphoma study group, DSNHL).
Samples, collected after informed consent and as approved by the Ethics Committee of the University of Goettingen, were subjected to the isolation of mononuclear cells, DNA isolation by DNAeasy System (Quiagen, Hilden, Germany), Xba1 digest and inverse genomic MLL-PCR (igPCR) for the therapy-associated MLL breakpoint cluster/topoisomerase binding site region as described.4 Processing of samples was carried out according to GLP guidelines for PCR. The mononuclear cells count was 5 × 105–5 × 106 and the DNA yield ranged between 4.5 and 300 μg. Four microgram was used for igPCR. We first studied a control group of adult (peripheral blood, n=8) and newborn (cord blood, n=10) individuals not exposed to chemotherapy and 18 peripheral blood samples of patients who underwent therapy. Surprisingly, aberrant amplification products (deletions, insertions), indicating MLL breakage and illegitimate recombination, were observed in mononuclear cells of the adult (5/8) and newborn cohort (8/10). The incidence of MLL aberrations in this healthy control cohort (13/18) was comparable to that of the patients (12/18) (see Figure 1). As the samples were processed up to 30 h after being taken, we supposed that MLL breakage and religation might occur during spontaneous apoptosis.
We then performed a sequential investigation of 10 peripheral blood samples of healthy adults. After being taken, aliquots were either submitted directly to the isolation of mononuclear cells and further processing or were stored as whole blood at room temperature. The incidence of aberrant amplification products increased depending on the time interval between sample taking and sample processing. At 0 h, 0/10 samples, at 24 h, 5/10 samples and at 48 h, 7/10 samples exhibited aberrant amplification products (see Figure 2) parallel to the increase of apoptotic cells as determined by the annexin binding assay (data not shown). Sequencing of the aberrant amplification products in the control and patients cohort showed internal rearrangements in the MLL bcr (deletions and insertions), which were not MLL partial tandem duplications. No translocations have been observed so far.
We conclude that MLL breakage and religation in the topoisomerase-sensitive bcr, leading to MLL rearrangements (deletions, insertions) in clinical samples, may occur not only after exposure to topoisomerase II inhibitors but also during spontaneous apoptosis of clinical samples. The MLL gene is sensitive to site-specific cleavage in the t-AML-associated bcr during the initial stages of apoptosis owing to a colocalized scaffold attachment region.7 Religation of cleaved MLL, implicating an at least partially intact DNA repair, has however never been observed during spontaneous apoptosis in clinical samples. Our observation suggests that the rapid processing of samples (less than 24 h) is a prerequisite to discern therapy-induced MLL rearrangements from those that are associated with spontaneous apoptosis.
Recent findings of Betti et al.8 provided experimental evidence that the initial MLL cleavage upon treatment with topoisomerase II inhibitors is not directly mediated by the inhibitors. In concordance with Betti et al., our observations indicate that further research concerning the proapoptotic/leukemogenic role of topoisomerase inhibitors within the complex network of post-breakage DNA processing is necessary.
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Basecke, J., Karim, K., Podleschny, M. et al. MLL rearrangements emerge during spontaneous apoptosis of clinical blood samples. Leukemia 20, 1193–1194 (2006) doi:10.1038/sj.leu.2404211
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