Rapid Detection of PML::RARA Fusions in Acute Promyelocytic Leukemia: CRISPR/Cas9 Nanopore Sequencing with Adaptive Sampling

Acute promyelocytic leukemia (APL) accounts for approximately 10-15% of newly diagnosed acute myeloid leukemia cases and presents with coagulopathy and bleeding. Prompt diagnosis and treatment are required to minimize early mortality in APL as initiation of all-trans retinoic acid therapy rapidly reverses coagulopathy. The <i>PML::RARA</i> fusion is a hallmark of APL and its rapid identification is essential for rapid initiation of specific treatment to prevent early deaths from coagulopathy and bleeding and optimize patient outcomes. Given limitations and long turnaround time of current gene fusion diagnostic strategies, we have developed a novel amplification-free nanopore sequencing-based approach with low cost, easy setup, and fast turnaround time. We termed the approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). Using CENAS, we successfully sequenced breakpoints of typical and atypical <i>PML::RARA</i> fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting <i>PML::RARA</i> fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical <i>PML::RARA</i> fusions and identified additional genes (<i>ANKFN1</i> and <i>JOSD1</i>) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the <i>ANKFN1</i> gene, the <i>JOSD1</i> gene, and the 13q14.13 genomic region flanking with the <i>SIAH3</i> and <i>ZC3H13</i> genes have not been reported in the atypical <i>PML::RARA</i> fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a <i>PML::RARA</i> fusion. Given the early death rate in APL patients still reaches 15%, and ~10% of APL patients are resistant to initial therapy or prone to relapse, further sequencing studies of typical and atypical <i>PML::RARA</i> fusion might shed light on the pathophysiology of the disease and its responsiveness to treatment. Understanding the involvement of additional genes and positional effects related to the <i>PML</i> and <i>RARA</i> genes could shed light on their role in APL and may aid in the development of novel targeted therapies.