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

Abstract

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 PML::RARA 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 PML::RARA fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting PML::RARA fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical PML::RARA fusions and identified additional genes (ANKFN1 and JOSD1) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the ANKFN1 gene, the JOSD1 gene, and the 13q14.13 genomic region flanking with the SIAH3 and ZC3H13 genes have not been reported in the atypical PML::RARA fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a PML::RARA 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 PML::RARA 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 PML and RARA genes could shed light on their role in APL and may aid in the development of novel targeted therapies.

Keywords: CRISPR/Cas9; PML::RARA fusions; acute promyelocytic leukemia; adaptive sampling; nanopore sequencing.

Figure 1

The CRISPR/Cas9-enriched nanopore sequencing with the adaptive sampling (CENAS) approach to reveal PML::RARA fusions in APL patients. The entire procedure includes DNA extraction from blood or marrow, CRISPR/Cas9-guided enrichment of targeted PML and RARA genomic regions, nanopore sequencing with adaptive sampling, and sequencing data analysis to reveal sequences involving PML::RARA fusions. hr: hours; min: minutes.

Figure 2

Atypical PML::RARA fusions in case #13. (a) Partial karyogram shows a t(15;22;17) three-way translocation (white arrows for abnormal derivative chromosomes). Below: Interphase FISH shows an atypical FISH signal pattern (2R2G1F). The red arrows point to a PML::RARA fusion. (b) CENAS shows sequence reads of a PML::RARA fusion involving the PML gene on chromosome 15q and the RARA gene on 17q (red arrows), and a JOSD1::RARA fusion involving the RARA gene on 17q and the JOSD1 gene on chromosome 22 (green arrows). Sequences were aligned to human genome builder GRCh37/hg19. (c) Diagram of the t(15;22;17) three-way translocation and atypical PML::RARA fusion. Black arrows point to derivative chromosomes involved in this translocation.

Figure 3

Cryptic PML::RARA fusion in case #12. (a) Partial karyogram shows a t(13;15) reciprocal translocation (white arrows for abnormal derivative chromosomes) with two normal chromosomes 17. Interphase FISH shows an atypical FISH signal pattern (1R2G1F). The red arrows point to a PML::RARA fusion. Metaphase FISH shows an insertional PML::RARA fusion into the derivative chromosome 15 formed by t(13;15) translocation, ish der(15)t(13;15)(q14;q24) ins(15;17)(q24;q21q21) (PML +, RARA +). (b) CENAS shows sequence reads of a complex atypical PML::RARA fusion involving chromosomes 13, 15, and 17. Red arrows point to a PML::RARA fusion/t(15;17) translocation, green arrows point to a fusion of the PML gene and 13q14.13 (chr13:46,515,010), and black arrows point to fusions of the RARA gene and 13q14.13 (chr13:46,515,891). These data support the presence of complex fusions and rearrangements. Sequences were aligned to human genome builder GRCh37/hg19.

Figure 4

Atypical PML::RARA fusion in case #11. (a) Interphase FISH shows an atypical FISH signal pattern (1R2G1F). The red arrows point to a PML::RARA fusion. (b) CENAS reveals an atypical PML::RARA fusion (a likely insertional fusion). Red arrows point to a PML::RARA fusion, green arrows point to the PML gene fused to the ANKFN1 gene on 17q22, and black arrows point to the RARA gene fused to the ANKFN1 gene on 17q22. These data suggest the presence of a likely insertional PML::RARA fusion into a derivative chromosome 17. Sequences were aligned to human genome builder GRCh37/hg19.

Figure 5

Multiple PML::RARA fusions in case #14. (a) Partial karyogram shows der(15), −16, +17, ider(17)(q10)t(15;17)x2, and +22 (white arrows for abnormalities). The red box shows isoderivative (ider) chromosome 17q involving t(15;17) translocations on both 17q arms. Interphase FISH reveals multiple fusions. (b) CENAS displays a higher number of sequencing fusion reads (indicated by red arrows) compared to non-fusion reads, due to the presence of multiple RARA::PML fusions in this case. Sequences were aligned to human genome builder GRCh37/hg19.