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Mol Cancer Res. 2018 Feb;16(2):279-285. doi: 10.1158/1541-7786.MCR-17-0569. Epub 2017 Nov 13.

Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia.

Author information

The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
Institute of Pharm. Biology/DCAL, Goethe-University, Frankfurt/Main, Germany.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Children's Cancer Institute, University of New South Wales, Sydney, Australia.
Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia.
The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
Oncology Services Group, Children's Health Queensland Hospital and Health Service, Brisbane, Australia.
UQ Child Health Research Centre, The University of Queensland, Brisbane, Australia.
Contributed equally


Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the "gold standard" technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10-7 and transcripts down to 0.005 copies/ng.Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279-85. ©2017 AACR.

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