Development and validation of sensitive BCR::ABL1 fusion gene quantitation using next-generation sequencing

Area of Science:

• Molecular Biology
• Genomics
• Hematology

Background:

• BCR::ABL1 fusion is crucial for chronic myeloid leukemia (CML) prognosis and monitoring.
• Current real-time quantitative polymerase chain reaction (RQ-PCR) methods have limitations for BCR::ABL1 quantification.
• Next-generation sequencing (NGS) offers a potential alternative for precise molecular testing.

Purpose of the Study:

• To design and validate an NGS-based assay for quantifying BCR::ABL1 and ABL1 transcript copy numbers.
• To overcome the limitations associated with conventional RQ-PCR in CML monitoring.
• To establish a sensitive and reliable method for tracking BCR::ABL1 levels.

Main Methods:

• Developed and validated a Next-generation Quantitative sequencing (NQ-seq) assay using Illumina Nextseq 550Dx.
• Employed an in-house bioinformatics pipeline for deep sequencing analysis.
• Validated analytical performance (precision, linearity, limit of detection) and compared with RQ-PCR using 145 clinical samples.

Main Results:

• NQ-seq demonstrated a limit of detection of MR 5.6 (BCR::ABL1 0.00028% IS).
• The assay showed excellent precision and a linear range from MR 2.0 to 5.0.
• NQ-seq results for International Scale (IS) values highly correlated with conventional RQ-PCR.

Conclusions:

• NQ-seq is a highly sensitive and reliable tool for monitoring BCR::ABL1 transcripts in CML patients.
• The NGS-based assay provides an effective alternative for molecular disease monitoring.
• Further prospective studies on large patient cohorts are needed to confirm the clinical impact of this NGS strategy.