Development and validation of sensitive BCR::ABL1 fusion gene quantitation using next-generation sequencing
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Published:2023-05-29
Issue:1
Volume:23
Page:
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ISSN:1475-2867
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Container-title:Cancer Cell International
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language:en
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Short-container-title:Cancer Cell Int
Author:
Lee Hyeonah,Seo Jieun,Shin Saeam,Lee Seung-Tae,Choi Jong Rak
Abstract
Abstract
Background
BCR::ABL1 fusion has significant prognostic value and is screened for chronic myeloid leukemia (CML) disease monitoring as a part of routine molecular testing. To overcome the limitations of the current standard real-time quantitative polymerase chain reaction (RQ-PCR), we designed and validated a next-generation sequencing (NGS)-based assay to quantify BCR::ABL1 and ABL1 transcript copy numbers.
Methods
After PCR amplification of the target sequence, deep sequencing was performed using an Illumina Nextseq 550Dx sequencer and in-house–designed bioinformatics pipeline. The Next-generation Quantitative sequencing (NQ-seq) assay was validated for its analytical performance, including precision, linearity, and limit of detection, using serially diluted control materials. A comparison with conventional RQ-PCR was performed with 145 clinical samples from 77 patients.
Results
The limit of detection of the NQ-seq was the molecular response (MR) 5.6 [BCR::ABL1 0.00028% international scale (IS)]. The NQ-seq exhibited excellent precision and linear range from MR 2.0 to 5.0. The IS value from the NQ-seq was highly correlated with conventional RQ-PCR.
Conclusions
We conclude that the NQ-seq is an effective tool for monitoring BCR::ABL1 transcripts in CML patients with high sensitivity and reliability. Prospective assessment of the unselected large series is required to validate the clinical impact of this NGS-based monitoring strategy.
Funder
National Research Foundation of Korea
Publisher
Springer Science and Business Media LLC
Subject
Cancer Research,Genetics,Oncology
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