Development of an NGS assay to detect MRD in hematologic malignancies.

Abstract

e24177 Background: There are significant prognostic and therapeutic benefits in monitoring minimal/measurable residual disease (MRD) in hematologic malignancies. However, monitoring of MRD requires the detection of minute cell populations with mutations present at very low allele frequencies (AFs), and is currently performed using low throughput flow cytometry or qPCR/ddPCR methodologies which are limited in their ability to monitor multiple mutations. An approach that can assay mutations in multiple genes may provide a broader understanding of MRD and patient prognosis. We have developed a Next Generation Sequencing (NGS) MRD assay using the Illumina NextSeq platform to simultaneously monitor a number of genes associated with hematologic malignancies (e.g. NPM1, RUNX1, FLT3). Here we present studies demonstrating detection of mutations at the low AFs required for the monitoring of MRD in hematologic malignancies. Methods: A custom target capture NGS assay was developed using Unique Molecular Identifier-based error correction enabling mutation detection of AFs down to 0.01%–0.1%. This assay incorporates barcoded duplex adapters and xGen Lockdown Probes (Integrated DNA Technologies) to enrich for relevant genes. Following sequencing on the NextSeq500, a custom bioinformatics pipeline was utilized for the error correction and false-positive reduction necessary to accurately detect mutations at the low AFs required for MRD detection. Results: Initial studies utilized Horizon Discovery’s TruQ Reference standards with mutations characterized at 1.0%–1.3% by ddPCR. These samples were diluted with a wildtype standard to demonstrate detection at even lower AFs. Subsequent studies were performed on peripheral blood samples from patients diagnosed with myelodysplastic syndromes and acute myeloid leukemia. Conclusions: This NGS MRD assay can accurately detect low AF mutations in both control and clinical MDS and AML samples. These results show that the NGS MRD assay described can be effectively utilized to monitor and study the course of disease in hematologic malignancies. Subsequent studies are underway to further increase the sensitivity of the assay and demonstrate its utility in a broad range of high-sensitivity applications.