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

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• Mixed lineage leukemia (MLL) gene rearrangements are critical in leukemia pathogenesis, occurring in ~70% of infant and 10% of adult/therapy-related cases.
• Conventional diagnostics like cytogenetics and FISH often fail to detect MLL translocation partner genes (TPGs).
• Long-distance inverse PCR (LDI-PCR), the gold standard, is laborious and requires substantial genomic DNA input.

Purpose of the Study:

• To develop and evaluate a targeted next-generation sequencing (NGS) approach for rapid and sensitive detection of MLL fusions.
• To overcome the limitations of current diagnostic methods for MLL gene rearrangements.

Main Methods:

• Anchored multiplex PCR-based enrichment (AMP-E) was employed for targeted MLL fusion identification using low RNA input.
• Libraries were generated with Archer FusionPlex Heme and Myeloid panels and sequenced on the Illumina platform.
• AMP-E was validated against LDI-PCR using 39 pediatric leukemia diagnostic specimens.

Main Results:

• The AMP-E method successfully identified TPGs in concordance with LDI-PCR, detecting 10 different MLL fusions across 39 samples.
• AMP-E identified an MLL-MLLT1 fusion missed by LDI-PCR, demonstrating superior breakpoint characterization.
• Sensitivity assays showed AMP-E can detect MLL-AFF1 down to 0.005 copies/ng, indicating high sensitivity.

Conclusions:

• NGS-based AMP-E offers improved sensitivity and efficiency for diagnosing MLL-rearranged leukemia compared to conventional methods.
• This assay rapidly identifies MLL partner genes and patient-specific fusion sequences.
• The identified fusion sequences hold potential for monitoring minimal residual disease in leukemia patients.