Abstract 3332: Reference materials for the development of assays for the detection of measurable residual disease in acute myeloid leukemia
Authors: Elango Rajula , Butler Matthew G. , Dahal Ojaswee , Ramprakash Jayanthi , Merriam David , Konigshofer Yves , Garlick Russell K. 
Published: June 2023

Abstract
The detection of Minimal Residual Disease (MRD) in Acute Myeloid Leukemia (AML) can present a challenge because it can require detecting just several malignant cells within thousands - if not millions - of normal cells. AML is a rare form of cancer that affects blood and bone marrow cells, and AML accounts for ~2 % of all cancer deaths in the United States with a 5-year relative survival rate of ~30 %. Early diagnosis of AML is crucial in determining targeted treatment plans that allow for better prognosis. At that point, tracking the response of AML to a therapy and monitoring for a future relapse becomes important. While nearly half of AML cases contain mutations in either NPM1 or FLT3, nearly half of AML cases appear to have driver mutations in other genes. One of the major challenges associated with the development and validation of assays for MRD in AML is the absence of cell-based reference materials that feature diverse mutations that may be encountered clinically.
To begin to address this concern, we developed full process cell-based reference materials that contain a number of mutations, including nucleophosmin (NPM1) c.860_863dup, an FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD), and SNVs in IDH1 and IDH2. We formulated them at concentrations down to 1 in 100,000 in a background of normal leukocytes. Mutations were quantified by digital PCR assays as well as by a targeted Next Generation Sequencing (NGS) panel. We also prepared similar reference materials in a circulating cell-free DNA (ccfDNA) format and analyzed them similarly.
Using digital PCR, we were able to detect the expected mutations in extracted DNA - although reference materials with mutations present in fewer than 1 in 1,000 cells were difficult to analyze using an input of 50 ng of gDNA per sample. Similarly, error corrected NGS data of this gDNA and ccfDNA became challenging to analyze when the mutation-containing cells were present at fewer than 1 in 1,000 cells due to the low frequency of the mutations.
Overall, we have generated cell-based and ccfDNA-based reference materials for the development and validation of assays for the detection of MRD in AML. While our own assays were unable to reliably detect the mutations at the lowest concentrations, these materials should be of use for assay developers who are trying to push and establish the sensitivities of their assays.
Citation Format: Rajula Elango, Matthew G. Butler, Ojaswee Dahal, Jayanthi Ramprakash, David Merriam, Yves Konigshofer, Russell K. Garlick. Reference materials for the development of assays for the detection of measurable residual disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3332.