Development and Validation of a Whole Genome Sequencing-Based Test for Cytogenetic Characterization of Acute Myeloid Leukemia

Molecular assessment is a mainstay in the diagnosis and management of acute myeloid leukemia and currently includes a variety of approaches spanning analysis of small genomic variants via next generation sequencing (NGS) as well as large genomic rearrangements and copy number gains/losses via fluorescent in situ hybridization (FISH) and/or cytogenetics. With current advances in next generation sequencing throughput and decreasing costs, it is possible to characterize all of these variant types in a single assay using whole genome sequencing (WGS). Here we report the development and validation of a novel whole genome sequencing assay for AML characterization. Briefly, we curated a cohort of over 100 patient blood or bone marrow samples that were previously characterized by clinical cytogenetics, FISH and/or small panel NGS testing for sequencing via WGS. NGS libraries were prepared using a PCR-free and tagmentation-based protocol. Sequencing was performed on a Novaseq 6000 sequencer to an average per base coverage of over 100x, and variation was characterized using DRAGEN-SV, DRAGEN-CNV and small variant callers. WGS-based assessment showed strong concordance with prior assays. Key examples include accurate calling of common large deletions such as losses of chromosome arms 5q and 7q, as well as accurate detection of common inversions such as inv(16). Unlike karyotyping, WGS has significantly higher resolution than the cytogenetic band level, and as such we could observe specific breakpoints for interstitial deletions as well as detected additional structural variants that are likely too small for observation by cytogenetics. While additional validation work is ongoing our initial results as well as results from other groups show strong promise for the use of WGS for comprehensive genomic characterization in AML.

Piening:Merck: Research Funding; Illumina: Honoraria, Research Funding; Eli Lilly: Research Funding. Brundu:Illumina: Current Employment. Sayyari:Illumina: Current Employment. Lee:Illumina: Current Employment. Truong:Illumina: Current Employment. Han:Illumina: Current Employment, Current equity holder in publicly-traded company. Sathyan:Illumina: Current Employment. Bifulco:SironaDx: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; PrimeVax: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; BioAI: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.