Combined Single-Cell DNA Genotyping and Protein Quantification (DAb-seq) in Acute Myeloid Leukemias Reveals Distinct Immunophenotypic Subsets Among Pathogenic Clones
Authors: Demaree Benjamin , Delley Cyrille , Peretz Cheryl , Vasudevan Harish , Ruff David , Ooi Aik , Smith Catherine C. , Abate Adam 
Published: November 2019

Genetic and immunophenotypic diversity is a hallmark of hematopoietic malignancies, and clonal evolution has been implicated as a driver of tumor initiation, progression, treatment response, and relapse. Consequently, a deeper understanding of the complex relationship between mutational profiles and disease phenotypes is critical to better understand the underlying disease processes. Probing the question of genotype-immunophenotype interplay at scale has historically proven difficult due to technical limitations permitting capture of only a single datatype from an individual cell. In acute myeloid leukemia (AML), flow cytometry for detection of measurable residual disease (MRD) has become standard of care for monitoring of treatment response and risk for relapse. At the same time, DNA-based molecular profiling of AML provides prognostic information and can guide therapeutic decisions at the time of diagnosis and disease progression. However, it remains a challenge to effectively integrate these assessments into a comprehensive model of disease state that can be used for clinical decision-making. Here we present DNA and Antibody sequencing (DAb-seq), a novel technology for simultaneously performing high-throughput, single-cell measurements of both DNA genotype and protein markers from >5,000 single cells. To our knowledge, DAb-seq is the first technology to directly integrate readouts of DNA genotype and cellular phenotype at the single-cell level. The experimental workflow leverages the single-cell microfluidic technology on the Mission Bio Tapestri platform, a system optimized to conduct multiplex single-cell targeted genomic DNA amplification. In our modified protocol, cells are stained with a pool of monoclonal antibodies conjugated with barcoded oligonucleotides, enabling a sequencing-based readout of single-cell protein signatures. The sequencing data is analyzed with a bioinformatic pipeline that separates antibody signal from targeted genotyping data on a cell-by-cell basis. We apply DAb-seq to the analysis of multiple AML patient samples, using a panel of 50 DNA targets comprising recurrently mutated genes in AML and 23 antibodies commonly used in flow cytometry-based MRD detection. In a single test case of pediatric AML, we identify cells harboring known pathogenic KRAS and FLT3 mutations at diagnosis and relapse. Among these genetically defined cell subsets, we observe differential protein expression of CD56, CD11b, and CD15. Specifically, we find a significant positive correlation between KRAS mutational status and combined expression of CD56 and CD11b. Similarly, FLT3 mutant cells, the dominant clone at relapse, are found to express elevated levels of CD15. We further apply DAb-seq technology to the profiling of mixed phenotype leukemia and monitoring the phenotypic dynamics of genetic clones in response to CD33 targeted therapy. Our data suggest cell populations defined by single variants in reality comprise multiple immunophenotypic subpopulations, underscoring the complex interplay between genotype and protein expression. While the precise mechanism underlying this immunophenotypic diversity remains to be elucidated, our work demonstrates the utility of DAb-seq in uncovering broader patterns of genotypic and phenotypic evolution in large AML patient cohorts. Ultimately, such studies could support further stratification of disease subtypes and increase the precision with which therapies are applied.

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Disclosures

Ruff: Mission Bio Inc.: Employment. Ooi:Mission Bio: Employment, Equity Ownership. Smith:Astellas Pharma: Research Funding; Abbvie: Research Funding; fujiFilm: Research Funding; Revolution Medicines: Research Funding. Abate:Mission Bio, Inc.: Equity Ownership.