Targeted Next-Generation Sequencing for Detecting <i>MLL</i> Gene Fusions in Leukemia

Abstract

Mixed lineage leukemia (<i>MLL</i>) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence <i>in situ</i> hybridization (FISH) fail to detect <i>MLL</i> translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the "gold standard" technique that is used to characterize <i>MLL</i> 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 <i>MLL</i> fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (<i>n</i> = 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 <i>MLL</i> fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a <i>MLL-MLLT1</i> fusion where LDI-PCR had failed to determine the breakpoint, whereas a <i>MLL-MLLT3</i> fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect <i>MLL-AFF1</i> in MV4-11 cell dilutions of 10^-7 and transcripts down to 0.005 copies/ng.<b>Implications:</b> This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for <i>MLL</i>-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies <i>MLL</i> partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. <i>Mol Cancer Res; 16(2); 279-85. ©2017 AACR</i>.