Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a group of blood cancers with high mortality. Even though complete remission is often achieved, majority of the AML patients still relapse. Measurable residual disease (MRD), defined as the population of cancer cells which persists following chemotherapy, is responsible for AML relapse. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of curative treatments more effectively. Discriminating residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. We developed a single cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single cell DNA sequencing (scDNA-seq) and immunophenotyping (Tapestri, Missionbio). The platform utilizes a custom amplicon panel covering 109 amplicons across 31 genes frequently mutated in AML and a panel of 42 oligo-conjugated antibodies against surface makers. We applied our scMRD assay to sequence 30 MRD samples (multiplexed into 6) from 29 AML patients to study the clonal architecture and protein correlation. In addition, 2 MRD samples were sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. Five patients with AML at diagnosis were also sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. In order to establish the sensitivity of our scMRD assay, we spiked AML cells into normal bone marrow cells. We sequenced 12 such spiked samples after multiplexing.]]>
Adult patients with AML were studied between 2020 and 2021. All samples from MSKCC underwent genetic sequencing with a targeted amplicon panel of 685 genes (HemePACT) or by an NGS platform panel comprised of 49 genes frequently mutated in myeloid disorders (RainDance Technologies ThunderBolts Myeloid Panel). Single point variants and short insertions/deletions not identified in database of known non-somatic variants (dbSNP and 1000 genomes) were included in analysis. Samples were selected based on mutation coverage by the Mission Bio Custom amplicon panel with variant allele frequencies of all covered mutations being > 2% by bulk sequencing. Samples with low cell numbers (< 0.1x106) were excluded. ]]>
In May 2021, we initiated this study by identifying and sequencing 37 samples from AML patients to investigate the clonal framework of myeloid malignancies, using the Tapestri scDNA sequencing platform. All samples had been previously sequenced by bulk molecular profiling.]]>