Single-Cell Mutational Profiling of Clonal Evolution in De Novo AML during Therapy and Relapse
Authors: Aleshin Alexey , Durruthy-Durruthy Robert , Corces M. Ryan , Stafford Melissa , Liedtke Michaela , Medeiros Bruno C. , Eastburn Dennis J. , Majeti Ravindra 
Published: October 2019

Background: De novo acute myeloid leukemia (AML) is a molecularly heterogeneous disorder with clinically variable outcomes. Recent studies on the mutational landscape of AML have been informative in better stratifying risk of relapse. However, bulk sequencing techniques have been limited in their ability to delineate the true complexity of tumoral molecular heterogeneity and allow for efficient identification of drug resistant subclones. Here, we applied high-throughput single cell sequencing technique to identify patterns of clonal heterogeneity and evolution in longitudinal samples from patients with AML undergoing induction chemotherapy.

Methods: Matched diagnosis, remission, and relapse samples were examined for 20 de novo AML cases including 15 relapsed and 5 non-relapsed controls. Mutational bulk sequencing was performed by NGS panel sequencing and exome sequencing was available in select cases. Single cell processing was performed using the Tapestri (Mission Bio) platform. Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes were then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual AML samples, genotyping the most clinically relevant loci across upwards of 10,000 individual cells.

Results: Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. We observed high concordance between bulk VAFs and sample level VAFs derived from single cell sequencing data. Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. Rare subclones associated with disease relapse, were identified in initial diagnostic samples that were frequently under the limit of detection of bulk NGS.

Conclusions:Taken together, our results suggest a greater degree of heterogeneity in de novo AML samples than suggested with bulk sequencing methods alone and shows the utility of single-cell sequencing for longitudinal monitoring and identification of resistant clones prior to therapy initiation in select patients. We show here that this approach is a feasible and effective way to identify and track heterogeneous populations of cells in AML and may be valuable for MRD identification.

Disclosures

Aleshin: Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Durruthy-Durruthy:Mission Bio, Inc.: Employment, Equity Ownership. Liedtke:Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; Amgen/Onyx: Consultancy, Honoraria, Research Funding; BlueBirdBio: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.