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Introduction:

In systemic mastocytosis (SM), clinical phenotype, response to treatment and prognosis are influenced by the KITD816V mutation (80-90% of patients positive), additional mutations in SRSF2, ASXL1or RUNX1(S/A/Rpos) and chromosomal aberrations. In core-binding factor acute myeloid leukemias (CBF-AML), KITmutations are identified in about 30% of cases, conferring an adverse prognostic impact on survival. However, patients with AML are not routinely checked for the contemporaneous presence or absence of SM. We sought to evaluate a) clinical and molecular characteristics, b) responses to treatment and survival, and c) prognostic factors in 44 patients with SM and associated AML (SM-AML). The median age was 64 years (range 28-83), 64% were male. In the majority of patients (30/44, 68%), AML evolved a median of 24 months (range 2-153) after diagnosis of SM without (6/30, 20%) or with (24/30, 80%) an associated hematologic neoplasm (AHN) such as myelodysplastic syndrome (SM-MDS), myeloproliferative neoplasm (SM-MPN) or MDS/MPN (SM-MDS/MPN). Based on histopathology, SM was contemporaneously diagnosed with AML in 14/44 (32%) patients. At diagnosis of SM-AML, the median bone marrow infiltration by mast cells and myeloid blasts was 15% (range 5-65; ≥20% in 45% of patients) and 39% (range 20-95), respectively, and the median serum tryptase level was 92 μg/l (range 2-885; ≥150 in 39% of patients). In the majority of patients, material was available for molecular analyses and revealed the following markers at diagnosis of SM-AML: a) KITD816V in 36/41 (88%) patients, b) additional mutations by targeted next-generation sequencing (NGS, 28 myeloid gene panel) in 32/33 (97%) patients, the most frequent mutations were: RUNX1(n=12, 36%), TET2(n=11, 33%), SRSF2(n=11, 33%), ASXL1(n=9, 27%), IDH1/ 2(n=6, 18%), NPM1(n=6, 18%), BCOR(n=5, 15%), DNMT3A(n=4, 12%), and, c) an aberrant karyotype in 25/35 (71%) patients (in analogy to related myeloid neoplasms, e.g. MDS or AML, patients were classified according to their karyotype into three groups: favorable-, intermediate- and poor-risk; only 1 patient with a CBF translocation). Patients with a known history of SM were frequently S/A/Rpos(19/22, 86%) and acquisition of additional mutations (NPM1, n=2; IDH1, n=2; BCOR/ JAK2/ TP53/ PHF6/ RUNX1, n=1) or karyotype evolution at time of SM-AML was observed in 8/15 (53%) or 9/13 (69%) patients, respectively. NGS on DNA derived from CD34+ myeloid blasts of 6 KITD816V positive patients revealed the presence of KITD816V positive blasts in only 1/6 (17%) patients while additional mutations were found in blast cells of all 6 patients. Eight patients received supportive care due to age and/or comorbidity. In 36 patients, disparate combinations of AML-type chemotherapy ± hypomethylating agents (HMA) ± cladribine (n=16) were administered. Cytarabine-based consolidation treatment was administered in 4/36 (11%) patients in complete remission (CR). Allogeneic stem cell transplantation (SCT) was performed in 15/36 (42%) patients, only 6/15 (40%) patients were in CR prior to allogeneic SCT. Overall, 5/10 (50%) patients in CR of AML showed persistence of SM. From diagnosis of SM-AML, the median observation was 10 months (range 0-200); 32/44 (73%) patients died during observation. The median overall survival (OS) was 11 months (95% confidence interval [CI], 4-17) with median OS of 2 (range 0-5), 4 (range 0-12) and 74 (range 0-149) months for patient cohorts who were treated with supportive care, intensive chemotherapy/HMA and intensive chemotherapy followed by allogeneic SCT, respectively (P=0.0002). On multivariate analysis, the only independent prognostic marker for OS was favorable-risk karyotype (n=14) vs. intermediate-/poor-risk karyotype (n=21; median OS 21 vs. 7 months; HR 3.2 [1.3-7.8], P=0.007). We conclude that a) SM-AML seems to be more frequent than commonly believed, b) SM-AML should be regarded as secondary AML evolving from multimutated SM-AHN, c) KITmutations are not restricted to CBF-AML, d) progression to SM-AML is often triggered by acquisition of new mutations and/or karyotype evolution, e) SM-AML has an aggressive phenotype and a poor prognosis, f) treatment of SM-AML should include a combination of intensive chemotherapy and allogeneic SCT for eligible patients and g) the disease frequency and poor prognosis warrant routine screening of all AML cases for serum tryptase and KITD816V.

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 KITD816V, 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 KITD816V+ 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 KITD816V+ 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). KITD816V+ 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 KITD816V negative. On the other hand, CFU-GM colonies from individual ASM ± AHNMD patients were never entirely KITD816V+ (median 60%, range 0-95). In contrast to KITD816V, 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 TET2and SRSF2mutations in 6/6 and 4/4 patients with (A)SM-AHNMD. In contrast, ASXL1mutations were not identified in all TET2/SRSF2positive CFU-GM colonies suggesting that they are likely to occur later than TET2and SRSF2. When additional mutations and KITD816V 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 KITD816V positive, indicating that the KITD816V 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 KITD816V negative in these patients, highlighting that the KITD816V 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 KITD816V+ progenitors correlated well with established parameters for quantification of disease burden, e.g. BM MC infiltration, serum tryptase levels and KITD816V 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 KITD816V with a subsequent phenotype modification towards SM due to a later acquisition of KITD816V. In contrast, ISM/SSM is not affected by mutations at the CFU-GM level which may at least in part explain its excellent prognosis.

Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q).