DOI: 10.5061/dryad.qnk98sfnb
title: Framework of clonal mutations concurrent with WT1 mutations in adults with acute myeloid leukemia (Alliance)

description: 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
 (WT1mut/FLT3-ITD).  Among WT1mut/FLT3-ITD patients, NPM1
 mutations were observed in 58% of patients. In contrast, patients
 with WT1-mutated AML but no FLT3-ITD (WT1mut/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
 WT1mut/FLT3-ITD patients, and, notably, fewer mutations in NPM1 (16% vs
 58%). Given the difference in NRAS mutation frequency in
 WT1mut/FLT3-ITD compared to WT1mut/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.
description: 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.
description: Tapestri Insights
 https://support.missionbio.com/hc/en-us/categories/360002505454-Tapestri-Insights