Improved Variantplex workflow reduces assay time and enhances sensitivity for acute myeloid leukemia targets

Introduction: Acute myeloid leukemia (AML) is a hematologic malignancy with high mortality. Detecting AML minimal residual disease (MRD) throughout the patient's care continuum can be challenging. Anchored Multiplex PCR (AMP™) VARIANT Plex assay solutions have been previously used to assess mutations in AML samples; however, the need for faster and more sensitive assay solutions warrants the development of new chemistry workflows to deliver on the need to identify mutations at low variant allele frequencies (≤0.01% VAF) in AML MRD samples. To meet this need, we developed an optimized VARIANT Plex workflow to reduce assay turnaround time and increase sensitivity for AML-relevant targets. Methods: Seraseq Myeloid Mutation Mix was spiked into Genome-in-a-Bottle DNA at frequencies of 1-0.01% and processed using the standard low allele frequency (AF) VARIANT Plex workflow and the new optimized workflow. The Archer panel utilized for prepping all samples was designed to target hotspots within 8 genes relevant to AML. Libraries were pooled and sequenced on multiple sequencing platforms. Data were analyzed with Archer Analysis v7.4 using targeted mutation files and customized filters to enhance variant identification in analytical sensitivity. Results: The new VARIANT Plex chemistry workflow reduced total assay time from ~1.5 days to ~7 hours and decreased the number of required bead cleanups from five to three. Workflow improvements increased DNA-to-library conversion resulting in substantial increases to Unique On-Target reads that yielded >100% increase in unique base coverage across AML panel targets. Utilizing tailored PCR conditions for low variant identification also boosted assay performance. The newly developed VARIANT Plex assay solution identified the expected (7/7) AML-relevant SNVs/indels and FLT3-ITDs down to 0.01%, with full target space powered to at least 0.1% variant identification. Conclusions: This improved VARIANT Plex workflow enables rapid, highly sensitive identification of AML mutations, supporting MRD identification applications.