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Morita, K.1; Wang, F.1; Jahn, K.2; Yan, Y.3; Durruthy-Durruthy, R.4; Parikh, A.4; Matthews, J.1; Little, L.1; Gumbs, C.1; Zhang, J.1; Song, X.1; Thompson, E.1; Patel, K.1; Bueso-Ramos, C.1; DiNardo, C.1; Ravandi, F.1; Jabbour, E.1; Andreeff, M.1; Cortes, J.1; Konopleva, M.1; Garcia-Manero, G.1; Kantarjian, H.1; Eastburn, D.4; Futreal, P. A.1; Beerenwinkel, N.2; Takahashi, K.1

doi: 10.1097/01.HS9.0000561612.73874.67
Simultaneous Sessions II: Acute myeloid leukemia - Biology & translational research - Towards molecular therapies

1The University of Texas MD Anderson Cancer Center, Houston, United States

2Swiss Federal Institute of Technology in Zurich, Zürich, Switzerland

3The University of Texas Health Science Center at Houston, Houston

4Mission Bio, Inc., South San Francisco, United States

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Assessment of genetic intratumor heterogeneity using next generation sequencing (NGS) can underestimate the complexity of subclonal architecture, since it is confounded by tumor purity, zygosity, and cell-level co-occurrence and exclusivity among multiple mutations.

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To thoroughly dissect the subclonal architecture, we performed single cell DNA sequencing (scDNA-seq) in 98 bone marrow samples from 80 patients with acute myeloid leukemia (AML).

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We used a novel microfluidics-based scDNA-seq platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). As a reference, all samples were concurrently sequenced by the bulk NGS using 295-gene exome capture sequencing. Allele-specific copy number data was obtained from SNP array data (Illuminia Omni 2.5 array). Droplet digital PCR (Bio-Rad QX200 Droplet Digital PCR system) was used to estimate the sensitivity of our scDNA-seq platform.

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Median 6,786 cells/sample were sequenced with median allele drop-out rate of 7.2% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 26x/cell. The scDNA-seq detected all of the bulk NGS-confirmed mutations. RUNX1 and FLT3 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss-of-heterozygosity of the mutant loci, which likely resulted in homozygous calls. The scDNA-seq also uniquely detected driver mutations that were not detected by the bulk NGS but were confirmed by droplet digital PCR, suggesting that scDNA-seq is more sensitive than the conventional bulk NGS.

scDNA-seq data unambiguously visualized the single-cell level co-occurrence of driver mutations (i.e. DNMT3A/FLT3-ITD/NPM1 and SRSF2/IDH2), confirming the cooperative function of these mutations. scDNA-seq data also revealed the cellular-level mutual exclusivity between functionally redundant mutations such as IDH1/IDH2, FLT3-ITD/TKD and NRAS/KRAS (Figure 1).

Inference of phylogenetic trees using SCITE algorithm (Jahn, et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the cases showed a linear evolution pattern where the founder mutations linearly acquired sub-clonal mutations in a step-wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 15 patients revealed the remodeling of clonal architecture in AML. For example, scDNA-seq data for a previously-untreated therapy-related AML case revealed that a FLT3p.D835Y clone, which was originally presented as a small sub-clone, survived the induction therapy consisting of azacitidine and sorafenib, and significantly expanded at relapse, while all other FLT3 and KRAS mutations were eradicated. This clonal remodeling is consistent with differential sensitivity of various FLT3 mutations to sorafenib (Smith et al. Leukemia 2015), of which FLT3 p.D835Y mutation has been shown to be more resistant to sorafenib compared to p.D835E and ITD mutations, demonstrating the differential behavior of sub-clones and clonal selection under molecularly targeted therapy (Figure 2).



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We performed a high-throughput scDNA-seq and described a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single-cell resolution.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.