Memorial Sloan Kettering (MSKCC) Single Cell Mutational Profiling in Myeloid Malignancies

Myeloid malignancies have previously been characterized by large scale bulk molecular profiling studies on patient samples. These studies, while clinically informative, are unable to delineate mutation order as well as clonal architecture and complexity. To investigate the clonal framework of myeloid malignancies, the Single Cell Mutational Profiling in Myeloid Malignancies study was designed. This study performed sequencing of 148 samples from 117 adult patients diagnosed with clonal hematopoiesis (n = 21), myeloproliferative neoplasms (MPN; n = 14), and acute myeloid leukemia (AML; n = 82) between 20 and 87 years of age. Sequencing of all samples was performed using the Tapestri single cell DNA sequencing platform from Mission Bio, Inc. The platform utilizes a custom amplicon panel covering 109 amplicons across 31 genes frequently mutated in myeloid malignancies (CH, MPN, AML). Using the single cell DNA sequencing data, we were capable of elucidating clonal architecture and clonal complexity in each sample. In a subset of samples, we also performed simultaneous single cell DNA sequencing and cell surface protein expression analysis using the Tapestri DNA + Protein platform. This study allowed us to determine the expression of 7 cell surface proteins (CD3, CD11b, CD19, CD34, CD38, CD45RA, and CD90) at single cell resolution. We performed DNA+ Protein sequencing on samples from CH patients (n = 7) as well as AML patients (n = 17).]]>
Adult patients with normal karyotype myeloid neoplasms between 2014 and 2019 were studied. Samples from four patients with complex karyotype/p53 mutant AML were studied for comparison. 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 >5% by bulk sequencing. Samples with low cell number (<5 x 10*6 cells) per frozen aliquot were excluded. Samples were prioritized if they contained 1) more than one protein coding mutation in epigenetic modifier genes DNMT3A, TET2, ASXL1, or IDH1/2, 2) an NPM1 mutation, 3) protein coding mutations in NRAS, KRAS and/or FLT3 (either internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations). ]]>
In May 2019, we initiated this study by identifying and sequencing 115 samples from CH, MPN, and AML to investigate the clonal framework of myeloid malignancies using the Tapestri single cell DNA sequencing platform. All samples had been previously sequenced by bulk molecular profiling. In July 2019, a subset of AML samples (n=17) were sequenced using the Tapestri DNA+Protein single cell sequencing platform, which simultaneously genotypes and immunophenotypes cells at single cell resolution. All samples had been previously sequenced by bulk molecular profiling and all patients selected had been previously sequenced using the Tapestri DNA platform. In February 2020, we sequenced an additional 9 samples from 5 AML patients, with samples being selected pre- and post- therapy. Additionally, 7 samples from 7 CH patients were sequenced using the Tapestri DNA+Protein sequencing platform. All samples were previously sequenced by bulk molecular profiling.]]>