Abstract 1904: Cell-based reference standards for the development and validation of assays for the assessment of minimal residual disease in acute myeloid leukemia
Authors: Colt W. Nash, Matthew G. Butler, Jayanthi Ramprakash, Ojaswee Dahal, Andrew T. Anfora, David E. Merriam, Russell K. Garlick, Yves Konigshofer
Journal: Cancer Research (2025)

Abstract Here, we describe the generation of novel cell-based reference standards for the development and validation of molecular diagnostic assays that assess measurable residual disease (MRD) in acute myeloid leukemia (AML). Unlike other hematological malignancies that are characterized by a defined genetic abnormality - such as the BCR::ABL1 translocation in chronic myelogenous leukemia - AML is associated with numerous different mutations that vary between patients. While about third of AML patients harbor a mutation in NPM1 and/or an internal tandem duplication (ITD) in FLT3, this is not the case for the majority of patients. This complicates the monitoring of the presence or absence of MRD during and after treatment because assays need to be very sensitive but inexpensive enough to allow for repeated measurements over time. For example, in the absence of an NPM1 or FLT3-ITD mutation, it may be beneficial to design patient-specific assays that focus on only the mutations found in that patient’s AML. As an initial proof of concept, we engineered an AML cell line to add NPM1 and/or FLT3-ITD mutations. The number of mutations per cell was quantified by digital PCR. This allowed for the generation of clones that represented AML cells with only a defined NPM1 or FLT3-ITD mutation, as well as AML clones with both. These were combined with peripheral blood mononuclear cells to dilutions as low as 1:100, 000 and analyzed by flow cytometry (a traditional way of monitoring for MRD in AML), electrophoresis (FLT3-ITD), digital PCR, and NGS. Compatibility was observed with all methods, but digital PCR and NGS appeared to be more sensitive. We then expanded the NPM1 variants beyond type A (c.860_863dup) to include types B, D, and J, and added additional FLT3-ITDs to include duplicated lengths as long as 228 bp. In order to increase the information obtained from a single sample in NGS, which is capable of independently measuring multiple variants, we combined these into a pool that was quantified by digital PCR and analyzed by NGS but also retained compatibility with flow cytometry to enable sorting and single-cell analyses. In conclusion, we have generated reference standards that incorporate multiple variants in NPM1 and FLT3, which are designed for the development and validation of molecular biology-based assays. In order to expand beyond the mutations found in only a third of AML patients, we are applying the same method to add mutations in additional genes such as IDH1, IDH2, and TP53. Citation Format: Colt W. Nash, Matthew G. Butler, Jayanthi Ramprakash, Ojaswee Dahal, Andrew T. Anfora, David E. Merriam, Russell K. Garlick, Yves Konigshofer. Cell-based reference standards for the development and validation of assays for the assessment of minimal residual disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1904.