Mutations affecting the tumor suppressor gene, WT1 transcription factor (WT1) are relatively common in adults with acute myeloid leukemia (AML) and have been reported to associate with a poor prognosis.  However, there is limited data describing additional mutations co-occurring with WT1 as well as clonal architecture and complexity.  We performed targeted DNA sequencing on 96 pretreatment samples from adult patients with de novo AML who harbored WT1 mutations and subsequently performed single-cell DNA sequencing on a subset of these samples. FLT3-ITD mutations were the most common co-occurring mutations detected in 47% of WT1-mutated patients (WT1^mut/FLT3-ITD).  Among WT1^mut/FLT3-ITD patients, NPM1 mutations were observed in 58% of patients. In contrast, patients with WT1-mutated AML but no FLT3-ITD (WT1^mut/no FLT3-ITD) had significantly more frequent mutations in alternative signal activating pathways (FLT3-TKD: 25% vs 11%; and NRAS: 37% vs 11%) compared with WT1^mut/FLT3-ITD patients, and, notably, fewer mutations in NPM1 (16% vs 58%). Given the difference in NRAS mutation frequency in WT1^mut/FLT3-ITD compared to WT1^mut/no FLT3-ITD patients, we then compared the variant allele frequencies (VAF) of NRAS mutations in the presence or absence of FLT3 mutations. There was a trend for higher NRAS VAF in WT1-mutated patients with concurrent NRAS mutations but no FLT3 mutations (FLT3-WT), suggesting this may be due to WT1 being the founding mutation with two or more distinct sub-clones. To further delineate this observation, we performed single-cell DNA sequencing on seven samples with co-occurring WT1 and NRAS mutations with or without FLT3 mutations.  Single-cell sequencing revealed that WT1 mutations were more frequently observed in the founder clone (6 out of 7 samples), with or without NPM1 mutations. Five of the seven samples included in the single-cell sequencing harbored both a FLT3 (ITD or TKD) and an NRAS mutation. The analysis shows these mutations arose in mutually exclusive sub-clones from a WT1 mutated founder clone in all 5 cases. These data, to our knowledge, represent the largest series of adults with WT1-mutated AML, and for the first time describe clonal architecture and the combination of mutations co-occurring in single cells. These data provide a deeper understanding of the genomic complexity and biology of WT1-mutated AML and may provide insight into the future development of novel therapeutic strategies.

For single-cell DNA sequencing, the MissionBio (San Francisco, CA) acute myeloid panel was utilized. Total cells were diluted to a concentration 3,500 cells/μL in Cell Buffer and the Tapestri® single-cell DNA sequencing user guide was followed. The final normalized libraries were sequenced using 2x300bp paired-end multiplexed runs on the Illumina MiSeq platform. Tapestri® Analysis pipeline was utilized to process and analyze the sequencing fastq files to generate .loom files. LOOM files were then visualized in Tapestri® Insight to call variants and visualize clones.

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