PloS one · 2025
Detection of leukemia gene fusions on DNA-level through targeted Next-Generation Sequencing.
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Chen Bin, Gao Zhihui, Chen Long, Zhang Hong, Lin Yani, Li Jing
Аннотация
BACKGROUND: Targeted Next-Generation Sequencing (tNGS) is commonly used to detect genetic mutations in patients with leukemia on DNA-level, necessitating additional methods to confirm genomic rearrangements and gene fusions, resulting in a substantial sample volume requirement and significant labor expenses.
METHODS: A novel custom leukemia tNGS panel, independently developed by Sino-US Diagnostics Lab, includes all exons from 302 genes closely associated with leukemia and 95 introns from 26 genes, thereby facilitating the detection of gene fusion alterations on DNA-level. Additionally, the common breakpoint regions of IGH and MYC are employed for the detection of IGH or MYC rearrangements. Commercial quantitative reverse transcription polymerase chain reaction (qRT-PCR) reagents were used simultaneously to detect 45 gene fusions in RNA samples. A total of 357 adults diagnosed with leukemia were included in the study.
RESULTS: The qRT-PCR method detected a total of 102 gene fusions, encompassing 23 distinct types. The tNGS method identified the same gene fusions on DNA-level in 98 samples, achieving a Positive Percent Agreement (PPA) of 96.1% (98/102) when compared to the qRT-PCR method, and no false positive findings. Additionally, it revealed the presence of two gene fusions, KMT2A::ELL and KMT2A::MLLT3, which had gone undetected by qRT-PCR. The tNGS can also identify IGH or MYC gene rearrangements in patients with B-ALL, achieving a PPA of 93.8% (15/16) when compared to the FISH. Moreover, tNGS can accurately identify the specific partner genes associated with these rearrangements, facilitating a more precise analysis of the impact of mutations on prognosis.
CONCLUSION: This study confirms the feasibility of employing tNGS methods to concurrently identify gene mutations and fusions (including IGH and MYC rearrangements) at the DNA level in adults with leukemia.
METHODS: A novel custom leukemia tNGS panel, independently developed by Sino-US Diagnostics Lab, includes all exons from 302 genes closely associated with leukemia and 95 introns from 26 genes, thereby facilitating the detection of gene fusion alterations on DNA-level. Additionally, the common breakpoint regions of IGH and MYC are employed for the detection of IGH or MYC rearrangements. Commercial quantitative reverse transcription polymerase chain reaction (qRT-PCR) reagents were used simultaneously to detect 45 gene fusions in RNA samples. A total of 357 adults diagnosed with leukemia were included in the study.
RESULTS: The qRT-PCR method detected a total of 102 gene fusions, encompassing 23 distinct types. The tNGS method identified the same gene fusions on DNA-level in 98 samples, achieving a Positive Percent Agreement (PPA) of 96.1% (98/102) when compared to the qRT-PCR method, and no false positive findings. Additionally, it revealed the presence of two gene fusions, KMT2A::ELL and KMT2A::MLLT3, which had gone undetected by qRT-PCR. The tNGS can also identify IGH or MYC gene rearrangements in patients with B-ALL, achieving a PPA of 93.8% (15/16) when compared to the FISH. Moreover, tNGS can accurately identify the specific partner genes associated with these rearrangements, facilitating a more precise analysis of the impact of mutations on prognosis.
CONCLUSION: This study confirms the feasibility of employing tNGS methods to concurrently identify gene mutations and fusions (including IGH and MYC rearrangements) at the DNA level in adults with leukemia.