Abstract
Patients with t(11;19) leukemias have one of two different translocations resulting in gene rearrangement with the MLL gene on 11q23. The ELL (eleven-nineteen lysine-rich leukemia) gene at 19p13.1, is rearranged in individuals with t(11;19) and acute myeloid leukemia, while the ENL (eleven-nineteen leukemia) gene at 19p13.3, is rearranged most frequently in those patients with t(11;19) and B-cell acute lymphoid leukemia. However, AML and rare T-cell ALL patients have been reported with the ENL gene rearrangement. At the cytogenetic level of resolution, it is not always possible to distinguish which of the two genes is involved in these translocations and precise molecular classification may be necessary for appropriate chemotherapy regimens to be devised and administered. We therefore developed a dual-color FISH (fluorescent in situ hybridization) assay using BACs (bacterial artificial chromosomes) for ELL and ENL. ELL is directly labeled in spectrum orange and ENL is directly labeled in spectrum green and the probes are co-hybridized. The strategy for the assay is to assess the interphase nuclei of patients for gain of an orange signal, indicating an ELL gene rearrangement (3O2G signal pattern), or gain of a green signal, indicating an ENL gene rearrangement (3G2O signal pattern). The gain of signal is due to splitting of the respective locus during the translocation of distal 19p to 11q23. As constructed, the probes showed a 100% specificity for the respective translocations, based on metaphase and interphase analysis of ELL and ENL t(11;19) positive patients; all of the patients were diagnosed with myeloid leukemia. In addition, further comparative validation was performed using the commercially available dual-color breakapart MLL probe (Vysis). Analysis of the ELL positive AML patients with the MLL probe showed the expected abnormal interphase signal pattern (1O1G1Fusion), confirming MLL gene rearrangement. However, the MLL pattern for the single patient with an ENL gene rearrangement and AML showed an alternate interphase pattern, indicating an alternate MLL breakpoint. Analysis of additional patients is required to determine if those with AML and ENL gene rearrangements routinely have a different MLL breakpoint; this difference could potentially contribute to a myeloid disease presentation. In order to establish clinically valid cut-offs for residual disease analysis, interphase validation was performed. Probe specificity was 100%, with a sensitivity of greater than 97% for both loci. This results in a normal cut-off (plus 2 S.D.) of 0.9/200 abnormal cells for ENL gene rearrangement and 2.18/200 abnormal cells for ELL gene rearrangement. In this laboratory's use, the probe set is slightly more sensitive for disease detection than the commercially available MLL gene probe (Vysis), which has a sensitivity of 96% and a normal cut-off of 1.84/200 cells. This probe set allows quick distinction between ELL and ENL gene rearrangements in patients with acute leukemia.
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