Clonal Evolution of Acute Myeloid Leukemia Revealed by High-Throughput Single-Cell Genomics

One of the pervasive features of cancer is the diversity of mutations found in malignant cells within the same tumor, a phenomenon called clonal diversity or intratumor heterogeneity. Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection inside the tumor ecosystem and is believed to contribute to treatment resistance and cancer recurrence. The ability to precisely delineate the clonal substructure of a tumor, including the evolutionary history of its development and the co-occurrence of its mutations, is necessary to understand and overcome treatment resistance. However, DNA sequencing of bulk tumor samples cannot accurately resolve complex clonal architectures. Here, we performed high-throughput single-cell targeted DNA sequencing to quantitatively assess the clonal architecture of 123 acute myeloid leukemia (AML) patient samples. Single-cell genotyping of a large cohort of AML samples provided cell-level evidence of co-occurrence and mutual exclusivity among AML driver mutations. Reconstruction of the tumor phylogeny uncovered history of tumor development that was characterized by linear and branching clonal evolution. Some of the samples showed evidence of convergent evolution. Single-cell DNA sequencing of xenotransplanted samples revealed clonal diversity in leukemia initiating cell populations. We have also performed simultaneous single-cell profiling of mutations and cell surface proteins that revealed cellular genotype-phenotype associations. Analysis of longitudinal samples in 15 patients by single-cell sequencing revealed a precise picture of treatment response on the level of individual clones illustrating the underlying evolutionary process of therapeutic resistance and recurrence in AML. Together, these data portray clonal diversity, architecture, and evolution of AML with unprecedented resolution, and highlight their clinical relevance in the era of precision medicine.