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

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