Molecular Profiling of Myeloid Progenitor Cells in Multi-Mutated Advanced Systemic Mastocytosis Identifies KIT D816V As a Distinct and Late Event

Systemic mastocytosis (SM) is characterized by abnormal proliferation and accumulation of mast cells (MC) in various tissues and organs, predominantly skin, bone marrow (BM) and visceral organs. The extent of organ infiltration and subsequent organ damage is the basis for the classification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated clonal hematologic non-MC lineage disease (SM-AHNMD), aggressive SM (ASM) and MC leukemia (MCL). Depending on the subtype of SM, cell source (BM or peripheral blood) and assay sensitivity, an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V, is detectable in 80-90% of patients. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes at known mutational hotspot regions as previously described. Additional mutations in genes encoding for signaling molecules (JAK2, CBL, KRAS, NRAS), transcription factors (RUNX1), epigenetic regulators (ASXL1, DNMT3A, EZH2, TET2) or splicing factors (SRSF2, SF3B1, U2AF1) are detectable in the vast majority of patients KIT D816V+ advanced SM. In order to gain more insight into clonal evolution of myeloid progenitors in indolent and advanced SM with or without AHNMD, we explored the mutation profile of single-cell-derived CFU-GM colonies - by using Sanger sequencing - in 19 KIT D816V+ SM patients (investigated colonies, n=285; median per patient, n=15; range 10-30). The study included 7 patients with ISM/SSM/ASM (0 additional mutations), 4 patients with SM-AHNMD (median 1 additional mutation, range 1-4) and 8 patients with ASM-AHNMD (median 3 additional mutations, range 1-4). KIT D816V+ CFU-GM could be identified in all 8 patients with ASM ± AHNMD but in only 20% (1/5) of patients with SM-AHNMD, while all CFU-GM colonies derived from ISM patients were completely KIT D816V negative. On the other hand, CFU-GM colonies from individual ASM ± AHNMD patients were never entirely KIT D816V+ (median 60%, range 0-95). In contrast to KIT D816V, additional mutations were identified in CFU-GM colonies from all 12 multi-mutated (A)SM-AHNMD patients and many of these mutations were present in 100% of the CFU-GM-derived colonies analyzed. In 8 patients, different subclones with variable proportions of the number of mutated genes were identified and allowed to generate putative evolutionary trees. These mutations included TET2 and SRSF2 mutations in 6/6 and 4/4 patients with (A)SM-AHNMD. In contrast, ASXL1 mutations were not identified in all TET2/SRSF2 positive CFU-GM colonies suggesting that they are likely to occur later than TET2 and SRSF2. When additional mutations and KIT D816V were detected concomitantly in individual single-cell-derived CFU-GM colonies, the overall frequency of CFU-GM colonies positive for additional mutations was always higher than those that were KIT D816V positive, indicating that the KIT D816V mutation was a secondary event. In contrast to advanced SM, all ISM patients were negative for additional mutations. CFU-GM colonies and also microdissected CD15+ cells derived from BM biopsies were entirely KIT D816V negative in these patients, highlighting that the KIT D816V mutation may be restricted to other (probably later) stages of stem cell development and possibly only to the MC lineage. In general, the relative proportion of KIT D816V+ progenitors correlated well with established parameters for quantification of disease burden, e.g. BM MC infiltration, serum tryptase levels and KIT D816V allele burden in individual patients. In conclusion, the presence of multi-mutated myeloid non-MC lineage progenitors of the CFU-GM-type suggests an initial clonal expansion at an early stage of hematopoietic development due to mutations other than KIT D816V with a subsequent phenotype modification towards SM due to a later acquisition of KIT D816V. In contrast, ISM/SSM is not affected by mutations at the CFU-GM level which may at least in part explain its excellent prognosis.