Whole genome sequencing uncovers novel BCR::ABL1 breakpoints and variants in leukemia: Implications for personalized medicine

Abstract

Abstract Background: The BCR::ABL1 fusion gene is a hallmark of chronic myeloid leukemia (CML) and is also present in a subset of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL) cases. Traditional methods for identifying this important biomarker, such as FISH and RT-PCR, are limited in the ability to detect novel or complex variants and also do not assess additional genetic alterations that could impact therapeutic decision making. Whole Genome Sequencing (WGS) is a comprehensive alternative, with the ability to detect a wide range of genetic alterations, including single nucleotide variants, insertions, deletions, and structural rearrangements such as BCR::ABL1 fusions. Methods: We retrospectively analyzed 215 hematological clinical samples sent for testing at Tempus that received both WGS via Tempus xH and RNA-seq via Tempus xR. Patients had historical diagnoses of AML (n=146), MDS (n=41), CML (n=25), and other blood cancers (n=3). Results: We detected BCR::ABL1 fusions in 29 samples (20 CML, 7 AML, 0 MDS, 2 others) using a combination of WGS and RNA-seq. Of these, 90% (n=26) were detected by both methods. For the three discordant samples, one was detected by WGS and not RNA-seq and 2 were detected by RNA-seq with a low total read support not detected by the WGS assay. Of the 26 samples that had support from both WGS and RNA-seq, BCR breakpoints largely aligned with expectation based on the known p210 (major) and p190 (minor) variants, which differ between AML and CML. RNA-seq confirmed all (100%) of the exon 1-3 breakpoints in p190 and exons 13-14 in p210. Most notably, our analysis uncovered 2 abnormal breakpoints with clinical implications, which were confirmed via RNA-seq. One specimen had a breakpoint within exon 2 of ABL1 resulting in an in frame transcript with a shortened exon 2. The other abnormal breakpoint was in intron 2 of ABL1 that results in exon 2 loss and exon 3 retention. It is known that the next generation allosteric tyrosine kinase inhibitor asciminib loses efficacy without exon 3, suggesting the use of an alternative therapeutic inhibitor is more appropriate highlighting the clinical utility of breakpoint resolution of WGS. Lastly, we used the WGS data to assess the presence of any pathogenic or likely pathogenic mutations in ASXL1 and RUNX1, both of which may impact survival and treatment outcomes. In this small cohort (n=26), we found ASXL1 alterations in 27% and RUNX1 in 12%, providing additional insights WGS can play in identifying key molecular markers. Conclusions: Our findings demonstrate that WGS can not only identify known BCR::ABL1 fusion events but also uncover novel breakpoints and variants of clinical significance, particularly when used in conjunction with RNA-seq. These data suggest that WGS is a powerful tool for the comprehensive genomic profiling of leukemia, with potential implications for personalized medicine and targeted therapy.