AML-Fated Clones Arise in Stem and Progenitor Cells in Myelofibrosis Patients Several Years Prior to AML Diagnosis

In myeloproliferative neoplasms (MPN), somatic mutations in recurrently mutated myeloid leukemia genes, apart from MPN driver genes, are common and can increase transformation risk to acute myeloid leukemia (AML). However, the temporal acquisition of these mutations, the properties of the cells bearing these mutations, and how each mutation cooperates to promote leukemic transformation remains largely unknown. We therefore examined blood and bone marrow samples collected serially from patients with myelofibrosis (MF) who developed AML to further elucidate the genetic basis of leukemic transformation. Specific aims include: 1) determining the timeline of acquisition of mutations that contribute to AML in chronic phase and 2) defining the cell type that harbours the clone fated to initiate AML. Ten patients who transformed to AML following a diagnosis of MF (6 PMF, 3 post-PV MF, 1 post-ET MF) were studied. The time interval from the first MPN collection to AML diagnosis ranged between 1.5 to 6.6 years; and in 4 patients additional MPN samples collected between these time points were available for study. Somatic mutations were identified from whole genome sequencing (WGS) data of AML blasts (CD45dim cells), the MPN clone (CD45highCD33+ and CD34+ cells), and germline references (T-cells or Buccal DNA); and from targeted sequencing results of 54 recurrently mutated myeloid leukemia genes performed on AML samples as part of another study. Stem and progenitor populations (HSC, MPP, LMPP, CMP, MEP, GMP), as well as, mature cell populations (myeloid, erythroid, T-lymphoid, B-lymphoid and NK) were sorted from MPN time points and whole genome amplified using the REPLI-g Single Cell Kit (Qiagen). Somatic mutations were tracked by droplet digital PCR (ddPCR) and/or targeted sequencing. We identified an average of 5 (range 1-8) recurrently mutated myeloid leukemia genes that were somatically mutated in each patient at AML diagnosis, with an average of 5.5 (range 1-11) different mutations affecting these genes. Many of these mutations were present at ≥5% variant allele frequency (VAF) in the MPN clone at the earliest chronic phase time point. In 8 patients, the MPN driver mutation was detected in the MPN clone and AML blasts (MPNdriver-concordant), while in 2 other patients there was discordance as JAK2V617F was present in the MPN clone, but not the AML blasts. Myeloid leukemia genes that were mutated in 2 or more patients included SRSF2 (n=5), ASXL1 (n=4), TET2 (n=4), I