A Multiomic, Single-Cell Measurable Residual Disease (scMRD) Assay for Simultaneous Assessment of DNA Mutations and Surface Immunophenotypes in Acute Myeloid Leukemia

The small population of cancerous cells that remain following treatment, known as measurable residual disease (MRD), is the major cause of relapse in acute myeloid leukemia (AML). Usually, these refractory cells have gained additional resistance mutations or changed their surface immunophenotypes in ways that preclude detection and phasing by current gold standard flow cytometry or bulk next-generation sequencing assays. For this reason, a multiomic single-cell MRD (scMRD) assay could offer a more comprehensive indicator of relapse and the potential for faster response. Here, we present a new scMRD assay with a 0.01% limit of detection that provides single-cell clonal architecture and immunophenotyping to not only identify residual leukemia cells, but also identify putative DNA or protein targets for novel biomarker insights. The assay enables rare-cell detection on a standard Mission Bio Tapestri® run by adding (i) an upfront enrichment for blast cells, (ii) a DNA and protein panel specifically designed for characterization of clonal architecture in AML MRD hotspots and biomarkers, (iii) an automated analysis pipeline to evaluate single-cell multiomics output, and (iv) multiplexing for simultaneous analysis of up to three patient samples in a single run via germline variant identification. By utilizing Mission Bio's technology for sequencing single cells, this pipeline can identify and correlate co-occurring de novo variants, thereby reducing false positive rates over bulk assays that do not correlate variants. Furthermore, it can create phylogenetic trees of the detected MRD cells and present their surface protein signature and arm-level copy number. To demonstrate assay features and reproducibility, surrogate MRD samples were constructed using positive control cell lines (expressing CD34 and CD117 markers) or diseased cells spiked into healthy bone marrow cells before processing them with the scMRD assay. We detected the 0.01% cell line spike-ins in 20 of 20 samples tested, with an average blast enrichment of 30.8x. We further applied the scMRD assay to bone marrow aspirate samples from AML patients and achieved 0.01% diseased cell spike-in detection. The scMRD assay resolved the genotype clonal architecture, identifying multiple leukemic clones with co-occurring mutations and readily distinguishing pre-leukemic from leukemic clones. The integration of genotype and immunophenotype further enhanced MRD detection by identifying genotype-specific protein expression patterns. By combining high reproducibility with multiomics, this assay offers a potential scalable solution for comprehensive MRD detection that could possibly guide patient stratification and therapeutic decision-making in the future.

Thompson:Mission Bio: Current Employment. Geller:Mission Bio: Current Employment. Nousheen:Mission Bio, Inc: Current Employment. Krishnan:Mission Bio, Inc: Current Employment. Wang:Mission Bio: Current Employment. Mendoza:Mission Bio, Inc: Current Employment. Druley:Mission Bio, Inc: Current Employment. Sciambi:Mission Bio, Inc: Current Employment.