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Rapid detection of PML::RARA fusions in acute promyelocytic leukemia using CRISPR/Cas9-mediated nanopore sequencing with adaptive sampling  

Cancer Genetics and Therapeutics
  • Primary Categories:
    • Cancer
  • Secondary Categories:
    • Cancer
Introduction:
Acute promyelocytic leukemia (APL) represents approximately 10% - 15% of newly diagnosed cases of acute myeloid leukemia and is characterized by coagulopathy and bleeding complications. Early diagnosis and prompt treatment are critical to reduce early mortality in APL, as the initiation of all-trans retinoic acid therapy can rapidly reverse coagulopathy and improve outcomes. The PML::RARA fusion gene is a defining feature of APL, and its swift identification is crucial for the timely initiation of targeted therapy to prevent fatal bleeding episodes and optimize patient prognosis. Current diagnostic approaches for detecting gene fusions in APL include conventional karyotyping, FISH, real-time quantitative reverse transcription PCR (RT-PCR), and next-generation sequencing (NGS)-based assays. While these methods are widely used, they each have limitations, including varying sensitivity and turnaround times that range from several days to weeks.

Methods:
We developed a novel nanopore sequencing-based approach for the rapid detection of PML::RARA fusions in APL patients. This approach utilizes CRISPR/Cas9 for targeted enrichment of regions of interest, bypassing PCR amplification before sequencing. During the nanopore sequencing process, we employed adaptive sampling to selectively prioritize sequencing of only the regions associated with the PML and RARA genes (designated as regions of interest). This enables real-time data collection and analysis focused solely on relevant fusion sites. We named this approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). The amplification-free nature of this method provides several key advantages, including lower upfront costs, reduced per-sample expenses, and rapid turnaround times—approximately 4 hours from patient sample to fusion detection. Additionally, CENAS provides long read lengths and real-time sequencing data, all achievable with a portable nanopore MinION system. This makes it a highly accessible and efficient solution for clinical diagnostics.

Results:
Using CENAS, we successfully sequenced the breakpoints of both typical and atypical PML::RARA fusions in APL cell lines and clinical samples of APL patients. When compared to standard genetic diagnostic methods (FISH, RT-PCR, NGS), CENAS demonstrated excellent concordance in detecting PML::RARA fusions, highlighting its accuracy and reliability. In addition to confirming known fusion breakpoints, CENAS provided detailed sequence information for both typical and atypical PML::RARA fusions. Remarkably, CENAS also identified previously unreported genomic regions and genes, including ANKFN1, JOSD1, and a region on 13q14.13, associated with atypical fusions (three-way translocations or insertional fusions). To our knowledge, the involvement of ANKFN1, JOSD1, and the 13q14.13 region—flanking the SIAH3 and ZC3H13 genes—has not been previously documented in the context of atypical PML::RARA fusions. Among patients with atypical fusions, the presence of JOSD1 or the 13q14.13 region did not appear to influence treatment outcomes or prognosis. However, the APL patient with ANKFN1 involvement exhibited an aggressive disease course, characterized by treatment resistance and a poor prognosis.

Conclusion:
CENAS holds significant promise as a point-of-care diagnostic tool for the rapid, low-cost, and bedside detection of PML::RARA fusions in APL patients. With early mortality rates still as high as 15% and approximately 10% of patients either resistant to initial therapy or experiencing relapse, there is a pressing need for more efficient diagnostic and therapeutic strategies. CENAS’s ability to identify novel genomic factors associated with atypical PML::RARA fusions offers critical insights that could significantly enhance prognosis and inform treatment decisions in APL. These findings suggest that CENAS not only serves as an effective diagnostic tool for detecting fusion status, but also has the potential to uncover new genetic markers that may influence disease progression, therapeutic resistance, and treatment response. This could pave the way for more personalized, targeted therapies, ultimately improving outcomes for APL patients. By enabling the identification of previously undetected genomic factors, CENAS has the potential to transform both the diagnosis and management of APL.

 

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