Whole Genome Sequencing Reveals Novel, Clinically Relevant Findings Missed by Standard Of Care for Patients with Myelodysplastic Syndrome

Background
Genomic analysis is critical for accurate diagnosis and risk stratification for patients with myeloid malignancies. Whole genome sequencing (WGS) has the ability to capture a breadth of alterations and has the potential to complement or replace current genomic profiling techniques. We sought to validate the molecular findings from Tempus xH, a WGS based assay, with standard of care [SOC] results from a major academic institution (MDACC).

Methods
We retrospectively sequenced 43 patient samples from MDACC using the Tempus xH WGS assay. SOC data from cytogenetics (karyotype +/- FISH), 81-gene panel sequencing, and/or optical genome mapping OGM (n = 10), was regarded as the source of truth. We performed a two-part study: an initial unblinded analysis (n = 10) comparing WGS to SOC, followed by a blinded unbiased comparative review (n = 33). Clinically relevant alterations were compared between the SOC and xH reportable range workflows. We excluded mutations with < = 10% VAF and filtered copy number alterations (CNAs) < 5Mb, and combined OGM and cytogenetics (10 > = metaphases). Structural variants (SVs) were included if they overlapped orthogonal truth or were recurrent myeloid fusions.

Results
In the unblinded analysis (n = 10), WGS detected 53 clinically reported alterations, yielding 94% sensitivity. Single nucleotide variants (SNVs) had 100% sensitivity, and 18/20 CNA events and 6/7 SVs were detected. The single missed SV event was a derivative chromosome (chr), between chr1 and chr20, identified by cytogenetics with breakpoints over the masked centromere region of the WGS. However, WGS detected CNAs in chr arms of 1 and 20, signalling the event was captured yet not resolved as a derivative chr. Two copy losses on chr 19 (complete loss and p-arm) and an ASXL1 frameshift were detected by WGS but not by SOC. In the blinded study (n = 33), WGS detected 143 events, including 124 true positives and no alterations from SOC went undetected by WGS demonstrating 100% sensitivity. WGS identified 24/24 of the CNAs detected by cytogenetics. Among all specimens, 21 novel, high confidence variants were identified in 11 specimens (39%) by WGS that were not detected by SOC; including 14 CNAs, two SNVs, and five SVs, including diagnostically and/or prognostically critical alterations: 2 MECOM rearrangements, 1 KMT2A-PTD, an 11MB deletion in chr 17p (TP53 locus), and a focal RUNX1 deletion highlighting the potential clinical benefit of WGS.

Conclusions
WGS accurately recapitulates results from targeted NGS panels, OGM and cytogenetics and advances our understanding of MDS biology. xH WGS uncovered a significant number of novel, clinically relevant and potentially targetable findings undetected by SOC. WGS provides a complementary view of the genome, identifying actionable and prognostic markers helping clinicians make more informed treatment decisions.