Clinical Application of Single-Cell MRD By Genotype and Phenotype in AML

Measurable residual disease (MRD) is a powerful prognostic indicator in acute myeloid leukemia (AML). While some genotyping assays with high sensitivity and specificity exist (i.e., mutated NPM1 or FLT3-ITD), current clinical tools to evaluate MRD in most AML patients include multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) panels. MFC-based MRD assays require experienced investigators and are prone to false negative results. NGS-based assays cannot distinguish clonal hematopoiesis from residual disease, and therefore are prone to false positive results. A single-cell MRD (scMRD) assay incorporating multi-omics (both genotype and phenotype) in AML, addresses these limitations and has recently been reported to have a sensitivity of approximately 0.01% in retrospective samples (Robinson et al., 2023). Here, we employed an scMRD multi-omics assay in a prospective cohort of AML patients to assess its clinical applicability.

Between July 2023 and June 2024, we prospectively enrolled sequential patients receiving routine induction therapy for AML at the Sylvester Comprehensive Cancer Center (SCCC) of the University of Miami. A total of 21 patients had samples collected and processed for scMRD assay using Mission Bio's TapestriⓇ platform. Briefly, bone marrow aspirates were enriched for CD34+ and CD117+ cells (Miltenyi). Enriched samples were multiplexed, encapsulated, and barcoded to generate single-cell genomic and protein libraries using Tapestri's v3 chemistry, before being sequenced on an Illumina NovaSeqⓇ X Plus. The median age was 60 (range 42-89). The majority of patients (61.9%) received induction with cytarabine and anthracycline, while 28.6% received hypomethylating agents and venetoclax while 9.5% received other therapies. The median follow-up time was 205 days.

A total of 25 samples were collected from 21 patients, 17 had AML and 4 had other myeloid malignancies (CMML, MDS, or MPN). A total of 61,858 cells were called (mean 2690 cells/sample). The scMRD multi-omics assay was compared to both the clinical MFC- and NGS-based MRD calls. Two cases that highlight the clinical utility of the scMRD multi-omics assay include an AML patient with IDH1 and NPM1 baseline mutations. After induction therapy with cytarabine and anthracycline, 31 mutant cells with an IDH1 R132S mutation were detected using scMRD. Following three cycles of consolidation, the scMRD assay detected additional mutations and an aberrant cell surface phenotype, despite the patient testing MRD negative by MFC and NGS. Additionally, qPCR for mutated NPM1 were negative at both timepoints. The second case illustrating the scMRD assay's sensitivity was a patient harboring TP53, FLT3-TKD, DNMT3A, CDKN2A, U2AF1, IDH2, and NPM1 baseline mutations, collected after induction therapy with cytarabine and anthracycline. Despite testing MRD negative by MFC and NGS clinical assays, as well as by qPCR for the NPM1 mutation, the scMRD assay was able to successfully detect mutant cells with aberrant cell surface phenotypes. Several other informative cases will be presented but were omitted here due to space limitations.

In summary, we have shown that an scMRD multi-omics assay can be utilized in clinical practice, resulting in >90% success rates in a range of myeloid neoplasms. In AML, discordance with clinical MRD assays by MFC and NGS was seen in patient samples. In discordant cases, scMRD was more sensitive in detecting residual disease, particularly in IDH1/2 and NPM1 co-mutant cases. In both of these cases, the NPM1 mutation was cleared as confirmed by qPCR and scMRD; however, the scMRD detected IDH1 and other mutations in addition to an aberrant cell surface phenotype in the CD34+/CD117+ enriched population. Due to the study's short follow-up period and ongoing therapy, the clinical outcomes in these cases are yet to be determined. While scMRD's future impact on AML clinical care remains to be seen, its rapid turnaround time (<1 week) in high-volume centers or a central location, suggests its clinical utility may be vast given its applicability across genotypes. In conclusion, we report that single-cell MRD multi-omics in AML was feasible in clinical practice and offered additional information above that received from MFC- and NGS-based MRD assays, either alone or in combination. Updated results from clinical outcomes will be presented.

Otero:Mission Bio: Current Employment. Gucwa:Mission Bio: Current Employment. Novoa:GSK: Other: Speaker; BMS: Other: Speaker; SERVIER: Other: Speaker. Watts:Immune Systems Key: Research Funding; Celgene/BMS: Consultancy; Reven Pharma: Consultancy; Daiichi Sankyo: Consultancy; Rafael Pharma: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Takeda: Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Other: safety monitoring or advisory boards, Research Funding.