Framework of clonal mutations concurrent with WT1 mutations in adults with acute myeloid leukemia (Alliance)

<p>Mutations affecting the tumor suppressor gene, WT1 transcription factor (<em>WT1</em>) 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 <em>WT1</em> as well as clonal architecture and complexity.  We performed targeted DNA sequencing on 96 pretreatment samples from adult patients with <em>de novo</em> AML who harbored <em>WT1</em> mutations and subsequently performed single-cell DNA sequencing on a subset of these samples. <em>FLT3</em>-ITD mutations were the most common co-occurring mutations detected in 47% of <em>WT1</em>-mutated patients (<em>WT1^mut</em>/<em>FLT3</em>-ITD).  Among <em>WT1^mut</em>/<em>FLT3</em>-ITD patients, <em>NPM1</em> mutations were observed in 58% of patients. In contrast, patients with <em>WT1</em>-mutated AML but no <em>FLT3</em>-ITD (<em>WT1^mut</em>/no <em>FLT3</em>-ITD) had significantly more frequent mutations in alternative signal activating pathways (<em>FLT3</em>-TKD: 25% vs 11%; and NRAS: 37% vs 11%) compared with <em>WT1^mut</em>/<em>FLT3</em>-ITD patients, and, notably, fewer mutations in <em>NPM1</em> (16% vs 58%). Given the difference in NRAS mutation frequency in <em>WT1^mut</em>/<em>FLT3</em>-ITD compared to <em>WT1^mut</em>/no <em>FLT3</em>-ITD patients, we then compared the variant allele frequencies (VAF) of <em>NRAS</em> mutations in the presence or absence of FLT3 mutations. There was a trend for higher NRAS VAF in <em>WT1</em>-mutated patients with concurrent <em>NRAS</em> mutations but no <em>FLT3</em> mutations (<em>FLT3</em>-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 <em>WT1</em> and <em>NRAS</em> mutations with or without <em>FLT3</em> mutations.  Single-cell sequencing revealed that <em>WT1</em> mutations were more frequently observed in the founder clone (6 out of 7 samples), with or without <em>NPM1</em> mutations. Five of the seven samples included in the single-cell sequencing harbored both a <em>FLT3</em> (ITD or TKD) and an <em>NRAS</em> mutation. The analysis shows these mutations arose in mutually exclusive sub-clones from a <em>WT1</em> mutated founder clone in all 5 cases. These data, to our knowledge, represent the largest series of adults with <em>WT1</em>-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 <em>WT1</em>-mutated AML and may provide insight into the future development of novel therapeutic strategies.</p>