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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

1
The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia.
2
Institute of Pharm. Biology/DCAL, Goethe-University, Frankfurt/Main, Germany.
3
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
4
Children's Cancer Institute, University of New South Wales, Sydney, Australia.
5
Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia.
6
The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia. andrew.moore@uq.edu.au.
7
Oncology Services Group, Children's Health Queensland Hospital and Health Service, Brisbane, Australia.
8
UQ Child Health Research Centre, The University of Queensland, Brisbane, Australia.
#
Contributed equally

Abstract

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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