ALTseqTM, a rapid (< 48 hours) whole genome sequencing clinical test for Acute Myeloid Leukemia (AML)

Abstract

Abstract AML accounts for one-third of adult leukemias. With an incidence of approximately 22, 000 new cases/year, AML is projected to cause over 11,000 deaths in the United States in 2025. Hence, new and more effective diagnostic, prognostic, and therapeutic approaches are highly needed. Genomic profiling is crucial to provide a proper prognosis and predict treatment response to emerging new therapeutics for both younger adult (<60 y/o) or older (>60 y/o) AML patients. This information is key to determine the best course of treatment and to make earlier decisions of whether to proceed to allogeneic hematopoietic cell transplants. However, the turnaround time for traditional “gold-standard” diagnostics — including FISH, cytogenetics, and targeted gene panels — can often exceed two weeks, depending on laboratory capacity and healthcare system logistics. Because many patients with AML require immediate initiation of therapy, clinicians are frequently forced to make treatment decisions without the benefit of genomic insights that could optimize therapy selection or trial eligibility — particularly in patients with high-risk, refractory, or relapsed disease. To address this unmet need, we developed ALTseqTM, a rapid whole genome sequencing (rWGS) assay for AML, at the TGen Clinical Laboratory (CAP: 8713783, CLIA: 03D2215886). ALTseqTM was specifically designed to deliver clinically actionable genomic results in under 48 hours, significantly faster than conventional testing. This accelerated turnaround time was enabled by streamlining laboratory workflows, enhancing bioinformatic pipelines, and expediting variant approval for reporting. The assay captures single nucleotide variants (SNVs), insertions and deletions (indels) — including FLT3-ITDs and KMT2A-PTDs —, 155 distinct structural variants (SVs including translocations or inversions) along with KMT2A rearrangements, and genome-wide copy number alterations, all in a single test. Specimens received must be whole blood or bone marrow in an EDTA tube and contain ≥20% blasts. DNA is extracted using the Qiagen DNA Blood Mini Kit (Qiagen, Hilden, Germany), prepared using the Watchmaker Genomics DNA Library Prep Kit (Watchmaker Genomics, Denver, CO, USA), and sequenced on the NovaSeq X Plus (Illumina, San Diego, CA, USA). The analytical pipeline utilizes both commercially available and proprietary algorithms with resulting VCFs compared to a knowledge base built in-house to create a json file used by the LIMS to produce the final clinical report. The curated report covers the genomic status of 41 genes with validated detection of key AML-related aberrations in both peripheral blood and bone marrow specimens. These include mutations in NPM1, TP53, RUNX1, IDH1/2, FLT3, MEN1, rearrangements involving KMT2A, MECOM, NUP98, and canonical translocations such as PML::RARA, RUNX1::RUNX1T1, and BCR::ABL1, among others. ALTseqTM has a limit of detection of 9%, 8%, 10%, and 7% for SNVs, indels, CNAs, and SVs, respectively, with sensitivities of 96%, 96.4%, 95.7%, and 100%, respectively. The positive predictive value for all variant types is ≥99.5%. Since implementation, ALTseqTM has been used in 35 AML cases, achieving an average turnaround time (TAT) of 35.2 hours, with a range of 29.4 to 46.3 hours from sample receipt to report delivery. Physician satisfaction with the assay's speed and clinical utility has been consistently high at City of Hope where the initial launch has occurred. In summary, we describe the successful deployment of a high-throughput, fast-turnaround rWGS platform for AML, capable of delivering comprehensive genomic profiling of 41 genes in <48 hours from sample receipt to clinical reporting. This approach has enabled earlier, more informed treatment decisions, and we are actively evaluating its impact on hospital length of stay and clinical trial enrollment, particularly where molecular eligibility criteria are required. Building on this success, we are now validating a similar rWGS approach for patients with multiple myeloma and plan to expand ALTseqTM to include acute lymphoblastic leukemia (ALL).