Abstract 5348: Single-cell analysis of mutational heterogeneity in acute myeloid leukemia tumors with high-throughput droplet microfluidics

To make possible the routine characterization of genetic diversity within cancer cell populations, we developed a novel two-step microfluidic droplet workflow that enables efficient and massively-parallel single-cell PCR-based genomic barcoding for single-cell DNA sequencing applications. We demonstrate that the two-step microfluidic approach is required for robust DNA amplification on thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic loci. To apply our single-cell sequencingtechnology to human tumor samples, we developed a targeted panel to partially sequence 26 genes frequently mutated in acute myeloid leukemia (AML) including TP53, DNMT3A, FLT3, NPM1, NRAS, IDH1and IDH2. Using this panel, we were able to sensitively identify SNV and indel-defined clones within AML samples and assess their distribution at the time of diagnosis, remission and relapse. We also used single cell SNVs to monitor host and donor cell populations during bone marrow transplantation (BMT), which allowed us to accurately evaluate engraftment and disease relapse. Collectively, our single-cell data indicates that clonal populations inferred from VAFs obtained from bulk sequencing data may not fully resolve the heterogeneity within tumors; moreover, the single-cell nature of our approach enabled the unambiguous identification of multiple co-occurring mutations within subclones that is not possible with bulk measurements. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with traditional sequencing paradigms and demonstrate the value of single-cell analysisfor AML. Citation Format: Dennis J. Eastburn, Maurizio Pellegrino, Adam Sciambi, Sebastian Treusch, Liwen Xu, Robert Durruthy-Durruthy, Kaustubh Gokhale, Jose Jacob, Tina X. Chen, William Oldham, Jairo Matthews, Hagop Kantarjian, Andrew Futreal, Keyur Patel, Keith W. Jones, James L. Zehnder, Koichi Takahashi. Single-cell analysisof mutational heterogeneity in acute myeloid leukemia tumors with high-throughput droplet microfluidics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5348.