Longitudinal Monitoring of AML Tumors with High-Throughput Single-Cell DNA Sequencing Reveals Rare Clones Prognostic for Disease Progression and Therapy Response

AML (acute myeloid leukemia) is increasingly being treated with precision medicine. To better inform treatment, the mutational content of patient samples must be determined. However, current tumor sequencing paradigms are inadequate to fully characterize many instances of the disease. A major challenge has been the unambiguous identification of potentially rare and genetically heterogeneous neoplastic cell populations, capable of critically impacting tumor evolution and the acquisition of therapeutic resistance. Standard bulk population sequencing is unable to identify rare alleles and definitively determine whether mutations co-occur within the same cell. Single-cell sequencing has the potential to address these key issues and transform our ability to accurately characterize clonal heterogeneity in AML. Previous single-cell studies examining genetic variation in AML have relied upon laborious, expensive and low-throughput technologies that are not readily scalable for routine analysis of the disease.

We applied a newly developed platform technology to perform targeted single-cell DNA sequencing on over 140,000 cells and generated high-resolution maps of clonal architecture from AML tumor samples. Marrow and/or peripheral blood samples were collected prior to, during treatment, and at clinical progression to the FLT3 inhibitor gilteritinib given on a clinical trial for relapsed/refractory AML with FLT3 mutation. Single-cell sequencing of multiple patient samples demonstrated that relapse clones acquired oncogenic RAS mutations. We utilized the high-throughput and sensitivity of our single-cell approach to more definitively assess where in the course of treatment these RAS mutated clones were acquired. Oncogenic RAS harboring clones, comprising between 0.4%, and 0.1% of tumor populations, were identified in patient samples either prior to or shortly after onset of treatment. Significantly, these RAS variant alleles were not detectable with targeted bulk sequencing. Throughout the course of treatment with the FLT3 inhibitor gilteritinib, the RAS mutant clones selectively expanded and were responsible for resistance to therapy and relapse. These findings point to the presence of underlying genetic heterogeneity in AML and demonstrate the utility of sensitively assaying clonal architecture to better inform patient stratification and therapy selection.