Abstract 1699: Accurate detection of low AF variants relevant to AML by Anchored Multiplex PCR and next generation sequencing
Authors: Johnson Verity , Moore Kaitlyn E. , Griffin Laura M. , Berlin Aaron , Licon Abel , Walters Ryan 
Published: August 2019

Abstract
Introduction: Acute Myeloid Leukemia (AML) is clinically and biologically heterogeneous, requiring the detection of multiple mutations for characterization. For instance, FLT3-ITDs and CEBPA mutations represent important markers in AML, however they are difficult to detect by NGS due to the highly variable nature of ITDs, the high GC content of CEBPA, and the difficulty in mapping repeated sequences to a wild-type reference. Tracking low frequency mutations is also of growing importance. The ability to accurately detect variants at low allele fractions (AFs) using a single test can be used to assess treatment efficacy and potential relapse.
Methods: We developed Archer® VariantPlex® myeloid assays based on Anchored Multiplex PCR (AMP™) to detect important mutations in myeloid malignancies. AMP is a target enrichment strategy that uses molecular-barcoded adapters and gene-specific primers for amplification, permitting open-ended capture of DNA fragments from a single end. This approach enables flexible and strand-specific primer design to provide better coverage of ITD-containing and GC-rich regions. We also developed a method to assess SNV sensitivity taking into account both unique coverage depth and noise for single base substitutions. This strategy enables utilization of position-specific detection thresholds and maximizes sensitivity and specificity. We tested this approach using the VariantPlex® Core Myeloid panel, by titrating reference inputs into background normal samples to examine detection of low AF variants.
Results: Our assay enables calling of a 30bp FLT3-ITD down to sub-0.05% allele frequencies. Using optimized low AF conditions improves coverage depth, consistency of low AF FLT3-ITD detection, and sensitivity (98.5% of bases are powered to call a true variant at an allele frequency of 3.0% with 1M reads and 200ng of input). We show &gt;1000X unique molecule coverage across the coding region of CEBPA and use this challenging region to visualize the minimum detectable AF (MDAF) at which a variant has a &gt;95% probability of being detected above the noise (95MDAF). Finally, we show consistent single nucleotide variant (SNV), insertion and deletion (indel), and ITD calling at sub-0.5% allele frequencies, and demonstrate the utility of reporting variant-specific MDAFs and normal dataset P-values when analyzing low AF variants.
Conclusion: AMP provides NGS-based detection of complex mutation types that are relevant in AML. We demonstrate robust calling of FLT3-ITDs and other variants at low AFs. We also demonstrate full coverage of CEBPA with high depth and low noise such that the 95MDAF predicts confident variant calling at low AFs. This approach is accurate and scalable, enabling simultaneous detection of multiple mutation types across multiple target genes in a single assay.
Citation Format: Verity Johnson, Kaitlyn E. Moore, Laura M. Griffin, Aaron Berlin, Abel Licon, Ryan Walters. Accurate detection of low AF variants relevant to AML by Anchored Multiplex PCR and next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1699.