Single cell genotypic and phenotypic analysis of measurable residual disease in acute myeloid leukemia

Measurable residual disease (MRD), defined as the population of cancer cells which persists following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia (AML) and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones and normal precursors remains a challenge with current MRD tools. Herein, we developed a single cell (sc) MRD assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated scDNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/pre-leukemic and leukemic cells surviving AML therapy. Statement of significance ScMRD assay integrates mutation and immunophenotype at single cell resolution and therefore distinguishes clonal hematopoiesis/preleukemic vs. leukemic clones. This study serves as a framework for identifying high-risk MRD clones and improving our understanding of both the molecular drivers and vulnerabilities of therapy resistant AML clones. ### Competing Interest Statement L.A.M. has received honoraria from Mission Bio and serves on the Mission Bio Speakers Bureau. S.F.C. is a consultant for and holds equity interest in Imago Biosciences, none of which are directly related to the content of this paper. A.S. and A.L. hold employment at Mission Bio. A.D.G. received research funding from Celularity, ADC Therapeutics, Aprea, AROG, Pfizer, Prelude, and Trillium; received research funding from and served as a consultant for Aptose and Daiichi Sankyo; served as a consultant and member of advisory committees for Astellas, Celgene, and Genentech; received research funding from, served as a consultant for, and was a member of advisory committees for AbbVie; and received honoraria from Dava Oncology. RLL is on the supervisory board of Qiagen and is a scientific advisor to Imago, Mission Bio, Syndax. Zentalis, Ajax, Bakx, Auron, Prelude, C4 Therapeutics and Isoplexis for which he receives equity support. RLL receives research support from Ajax and Abbvie and has consulted for Incyte, Janssen, Morphosys and Novartis. He has received honoraria from Astra Zeneca and Kura for invited lectures and from Gilead for grant reviews. W.X. has received research support from Stemline Therapeutics.