TY - JOUR
T1 - Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia
AU - Afrin, Sadia
AU - Zhang, Christine R.C.
AU - Meyer, Claus
AU - Stinson, Caedyn L.
AU - Pham, Thy
AU - Bruxner, Timothy J.C.
AU - Venn, Nicola C.
AU - Trahair, Toby N.
AU - Sutton, Rosemary
AU - Marschalek, Rolf
AU - Lynn Fink, J.
AU - Moore, Andrew S.
N1 - Publisher Copyright:
2017 American Association for Cancer Research.
PY - 2018/2/1
Y1 - 2018/2/1
N2 - 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 ¼ 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 107 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.
AB - 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 ¼ 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 107 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.
UR - http://www.scopus.com/inward/record.url?scp=85041473555&partnerID=8YFLogxK
U2 - 10.1158/1541-7786.MCR-17-0569
DO - 10.1158/1541-7786.MCR-17-0569
M3 - Article
C2 - 29133595
AN - SCOPUS:85041473555
SN - 1541-7786
VL - 16
SP - 279
EP - 285
JO - Molecular Cancer Research
JF - Molecular Cancer Research
IS - 2
ER -