A Comprehensive Whole Genome Sequencing Assay Provides Robust Characterization of Clinically Relevant Genomic Alterations across Myeloid Malignancies Concordant with Matched Results from Targeted DNA, Whole Transcriptome RNA and Cytogenetic Profiling
Authors: Huether Robert , Hoskinson Derick , Anur Pavana , Torres Raul , Beutner Karl R , Yang Yan , Kaneva Kristiyana , Potts Kelly A , Frazier Andrew , Braunstein Iris , Mahon Brett , Thompson Michael A , Sasser Kate , Nimeiri Halla , Kraft Lewis J , De La Vega Francisco M , Dinner Shira N. , Garcia-Manero Guillermo 
Published: December 2024

Background:
Identifying genetic alterations, as recommended by NCCN and WHO guidelines, is crucial for managing patients with myeloid malignancies including diagnosis, risk assessment, and therapeutic decisions. Traditional methods such as small mutation panels, cytogenetics, and fluorescence in situ hybridization require separate tests and have limitations.
Whole-genome sequencing (WGS) is revolutionizing the assessment of genomic alterations in hematological malignancies (HM) by identifying the full suite of genomic changes with a single test. Studies by Duncavage et al. (2021) and re-analysis by Deshpande et al. (2023) showed that WGS offers a complementary, potentially superior, approach to traditional risk stratification in myeloid neoplasms. However, their study was partially constrained by focusing on risk-defining translocations, reducing the diversity of structural variants (SV) they report.
To test the sensitivity of WGS in capturing a diverse array of mutation types, including SVs, we developed a comprehensive WGS assay optimized for clinically relevant alterations in Acute Myeloid Leukemia (AML), Chronic myelogenous leukemia (CML), Myeloproliferative neoplasm (MPN) and Myelodysplastic Syndromes (MDS). This tool is tailored to characterize recurrent SVs, extensive copy number alterations (CNAs), and single nucleotide variants/insertions and deletions (SNVs/indels).
In this pilot study, we assess the performance of the WGS profiling assay in 135 patients with AML, MDS, CML, and small subset of other HM to evaluate the sensitivity of WGS identification of selected genetic alterations. We compared these results to matched data from targeted DNA panel, whole RNA transcriptome sequencing, and selected samples with available cytogenetic results.
Method:
To compare genomic alteration results and assess concordance on the same sample with the WGS assay, 135 patients were sequenced via Tempus xT-Heme (a 648 gene targeted DNA sequencing panel), 125 patients had additional RNA-seq results via Tempus xR (a whole transcriptome RNA sequencing assay). Ten samples also had cytogenetics data. For the WGS assay, DNA obtained from blood and bone marrow aspirates was used to construct paired-end libraries via tagmentation and whole-genome sequenced to 80X mean coverage with the Illumina NovaSeq-X platform (2x150bp reads). Data were analyzed in a tumor-only modality using the DRAGEN Bio-IT Platform with custom post-processing filters. We filtered SNV/Indel alterations to 40 genes (targeted by Duncavage et al.) with VAF&gt;=10%, 608 recurrent rearrangements, and CNAs &gt;5MB.
Result:
In our cohort (65% AML, 19% CML, 13% MDS and 2% other HM) the WGS assay identified 218 reportable SNVs/indels, 104 SVs, and 14 large CNAs, showing high concordance with xT-Heme, RNA or cytogenetic assays. Specifically, 99.5% of SNVs/indels and 98.9% of SVs were concordant between xT-Heme and WGS. A subset of guideline recommended SVs (e.g. RUNX1-RUNX1T1, ELN adverse risk fusion) were uniquely detected by the WGS assay alone and were confirmed with RNA (12.5% [13/104]). All large CNAs detected by WGS were 100% concordant with available clinically reported cytogenetics. Notably, our cohort included nine FLT3 internal tandem duplications (ITD) ranging from 12 to 97 nucleotides, (8/9) were identified by WGS-the missed ITD had an xT-Heme VAF of 1.9%. WGS missed some variants identified by xT-Heme with low VAFs &lt;5% (targeted sequencing depths of ~600x) that were likely subclonal. WGS showed exceptionally high concordance to traditional techniques in identification of clinical relevant findings in myeloid neoplasms.
Conclusions:
We demonstrate high concordance (&gt;98.9%) in identifying guideline recommended genomic alterations, including large CNAs, in myeloid malignancies by a single WGS test compared to parallel conventional methods. The ability to obtain large CNA results, historically reserved for cytogenetic testing, has the potential to save costs and fill an unmet clinical need globally where cytogenetic resources are limited. Additionally, WGS can identify unique SVs that may be missed by conventional methods and enables clinical benefits such as HLA typing for potential transplant (alloHCT) or diagnostic refinement by retroviral insertion (e.g. HTLV-1). These findings demonstrate the potential for integration of WGS into clinical practice to enhance personalized treatment strategies.