Abstract LB254: Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation
Authors: Schmidt Elizabeth , Fitzgerald Devon M. , Zanette Camila , Meredith Gavin D. , McElwain Mark A. , Burciaga Raul , Vavra Kevin C. , Smith Thomas H. , Salk Jesse J. , Higgins Jake 
Published: April 2023

Sensitive and specific detection of low frequency mutations in acute myeloid leukemia (AML) is critical in research of minimal residual disease (MRD). At variant allele frequencies (VAF) below approximately 1%, PCR and sequencing errors result in prohibitive signal-to-noise ratios with next generation sequencing (NGS). Duplex Sequencing (DS) relates the original top and bottom DNA strands to make double stranded consensus sequences to greatly reduce errors. In two experiments we show further improvement of DS of AML-related genes by the use of enzymatic fragmentation (EF) and an updated gene panel that incorporates 2022 European LeukemiaNet (ELN) recommendations. In the first DS study, 29 AML-related genes (59 kb) were targeted in hybrid capture. Mutant cell line DNA was mixed into DNA from a healthy young donor to simulate MRD. Expected VAFs in mixtures were 1.0-0.003%. Samples comprised a mix of 4 insertions and deletions (indel), a mix of 15 single nucleotide variants (SNV), and 4 serial dilutions of a FLT3 ITD plus an NPM1 insertion. Pure diluent DNA was used as a negative control. Each sample was prepared in quadruplicate with mechanical fragmentation (MF) vs EF. DNA input mass was 1,500 ng per replicate, except for 50-250 ng for the FLT3/NPM1 mixes with expected VAF of 1% and 0.1%, respectively. Input masses were set to ensure &gt;95% probability of detection of all mutations in the combined data. All targeted variants down to 0.003% VAF were detected in spike-in mixtures, with expected vs observed VAF highly correlated whether using MF or EF (R2 &gt;0.98 for all mixtures). Duplex molecular depth was 1.2-2.0x higher with EF vs MF across input masses. Panel-wide mean duplex depth per 1,500 ng replicate was 30,376x for MF and 48,036x for EF, for combined mean depths of 121,508x for MF and 192,144x for EF. In the negative control, background mutational calls at spike-in positions were 4/2,993,429 duplex bases using MF and 0/4,780,491 duplex bases with EF, reflecting increased specificity with EF. Next, a revised panel targeting 36 AML-related genes (80 kb) was used for DS with EF. Here differing mutant cell line DNA was mixed into the same diluent from above (the negative control). This mixture harbors 27 variants with expected VAFs of 0.125-0.006% (4 indels, 21 SNVs, a FLT3 ITD and an NPM1 insertion), 17 of which (2 indels, 14 SNVs and the NPM1 insertion) span VAFs of 0.011-0.009% to establish a limit of detection of 0.01%. With 2,000 ng (+/- 10%) input, for 14 samples, panel-wide mean duplex depth was 56,528x. For variants with expected VAF ≥ 0.009%, the assay had &gt;98% sensitivity, &gt;96% specificity, and &gt;98% accuracy at genomic positions of known true positive variants. In summary, DS with EF yields more data per nanogram of DNA than MF, maximizing the use of precious samples. This method exhibits extremely low background mutation signal and a low limit of detection across targets that are valuable in AML MRD research.

Citation Format: Elizabeth Schmidt, Devon M. Fitzgerald, Camila Zanette, Gavin D. Meredith, Mark A. McElwain, Raul Burciaga, Kevin C. Vavra, Thomas H. Smith, Jesse J. Salk, Jake Higgins. Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB254.