Single-Cell DNA Sequencing Reveals Clonal Dynamics in FLT3-Mutated Acute Myeloid Leukemia during Crenolanib Combination Therapy

Background
Single cell RNA (scRNA) sequencing analysis suggests that the presence of FLT3-ITD mutation is associated with a progenitor-like phenotype (1van Galen, et al. Cell, 2019: 176, 1265) while activating mutations in the FLT3 kinase domain are associated with monocytic differentiation. One of the patients analyzed by scRNA was a 54-year-old patient with cytogenetically normal AML. NGS showed multiple mutations: DNMT3A (41.9%), NPM1 (37.9%), CEBPA (42.9%), FLT3-ITD (13.5%) and two FLT3-TKD mutations (N841K 16.2% and A680V 29%).

Crenolanib is a FLT3 inhibitor with in vitro activity against FLT3-ITD and various FLT3-TKD mutations being evaluated for clinical activity. This patient was treated with cytarabine/daunorubicin/crenolanib induction, four cycles of HiDAC/crenolanib consolidation and one year of crenolanib maintenance as part a Phase II clinical trial (NCT02283177). We hypothesized that single-cell DNA (scDNA) sequencing technology can review the clearance of distinct clones during treatment by analyzing longitudinal samples.

Aims

To evaluate the clearance of distinct AML clones during treatment with crenolanib combination therapy.

Methods
Mononuclear cells were purified from bone marrow aspirates collected at diagnosis, at achievement of CR after induction (day 35), after four cycles of HIDAC/crenolanib consolidation, and during month two and six of maintenance crenolanib. The Tapestri (Mission Bio) platform was used to prepare single cell genetic libraries for 19 commonly mutated AML genes. scDNA libraries were sequenced using MiSeq, data analysis was performed via Tapestri Insight software.

Results

scDNA analysis of 2,920 cells, revealed 4 distinct FLT3 subclones at diagnosis, including FLT3-ITD, A680V, N841K, and D839G. All of the mutations in FLT3 were found with concurrent NPM1 and DNMT3A-R882C mutations. FLT3-ITD was primarily found in leukemic populations that also had an activating FLT3-A680V mutation; however, the FLT3 mutations N841K and D839G were found to be in clones separate from the A680V and FLT3-ITD clones. We also detected wild type FLT3 clones that contained either NRAS (1%) or KRAS (1%) mutations, with concurrent NPM1 and DNMT3A mutations.