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Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

The European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML) have become an important tool to assess patients’ prognosis and guide treatment. We tested the prognostic impact of the 2017 ELN classification in a large cohort of 863 AML patients aged?<60 years similarly treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. Based on multivariable models within each ELN genetic-risk group, we identified additional gene mutations that may refine the 2017 ELN risk classification. BCOR-or SETBP1-mutated favorable-risk patients with non-core-binding factor AML and IDH-mutated adverse-risk patients had intermediate-risk outcomes. Outcomes of NPM1/WT1co-mutated patients and those of ZRSR2-mutated patients resembled outcome of adverse-risk patients. Moreover, FLT3-ITDhighallelic ratio conferred adverse rather than intermediate-risk irrespective of the NPM1mutation status, and DNMT3Amutations associated with very poor survival. Application of these refinements reclassified 9% of current favorable-risk patients and 53% of current intermediate-risk patients to the adverse-risk group, with similar poor survival as current adverse-risk patients. Furthermore, 4% of current favorable-risk patients and 9% of adverse-risk patients were reclassified to the intermediate-risk group.

Targeted mutation assessment of 81 genes in 1021 adults with de novo acute myeloid leukemia (AML) identified recurrent mutations in the neurofibromin 1(NF1) gene in 52 (5.1%) patients, including 36 (5.2%) younger and 16 (4.8%) older patients, which suggests NF1belongs to the 20 most frequently mutated genes in adult AML. NF1mutations were found throughout the gene, and comprised missense, frameshift, and nonsense mutations. One mutation hotspot, at amino acid threonine 676 (Thr676), was found in 27% of AML patients with NF1mutations. NF1-mutated patients belonged more often to the adverse European LeukemiaNet (ELN) risk category than NF1wild-type patients. Among patients aged <60 years, the presence of NF1Thr676 mutations was associated with lower complete remission (CR) rates (P= 0.04) and shorter overall survival (OS; P= 0.01), as was the presence of any NF1mutation in patients in the adverse ELN risk category (CR, P= 0.05; OS, P< 0.001). CR rates were also lower in NF1-mutated patients aged ≥60 years compared with NF1wild-type patients (P= 0.001). In summary, our findings provide novel insights into the frequency of NF1mutations in AML, and are suggestive of an adverse prognostic impact in patients treated with standard chemotherapy.