American Association for Cancer Research
15417786mcr170569-sup-190431_2_supp_4345686_zygc20.pdf (162.82 kB)

Figure S1 from Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia

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journal contribution
posted on 2023-04-03, 17:23 authored by Sadia Afrin, Christine R.C. Zhang, Claus Meyer, Caedyn L. Stinson, Thy Pham, Timothy J.C. Bruxner, Nicola C. Venn, Toby N. Trahair, Rosemary Sutton, Rolf Marschalek, J. Lynn Fink, Andrew S. Moore

Expression of the exon 10 fusion transcript in patient 17 and patient 32. Standard curve was established using patient 32 sample in a range from 25ng/µl to 0.1 ng/µl in 1 in 2 serial dilutions.


The Kids' Cancer Project

Children's Hospital Foundation



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.