Leukemia (2015) 29, 1115–1122 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Molecular profiling of myeloid progenitor cells in multi-mutated advanced systemic mastocytosis identifies KIT D816V as a distinct and late event M Jawhar1,8, J Schwaab1,8, S Schnittger2, K Sotlar3, H-P Horny3, G Metzgeroth1, N Müller1, S Schneider4, N Naumann1, C Walz3, T Haferlach2, P Valent5, W-K Hofmann1, NCP Cross6,7, A Fabarius1 and A Reiter1 To explore the molecular profile and its prognostic implication in systemic mastocytosis (SM), we analyzed the mutation status of granulocyte–macrophage colony-forming progenitor cells (CFU-GM) in patients with KIT D816V+ indolent SM (ISM, n = 4), smoldering SM (SSM, n = 2), aggressive SM (ASM, n = 1), SM with associated clonal hematologic non-mast cell lineage disorder (SM-AHNMD, n = 5) and ASM-AHNMD (n = 7). All patients with (A)SM-AHNMD (n = 12) carried 1–4 (median 3) additional mutations in 11 genes tested, most frequently TET2, SRSF2, ASXL1, CBL and EZH2. In multi-mutated (A)SM-AHNMD, KIT D816V+ single-cell-derived CFU-GM colonies were identified in 8/12 patients (median 60%, range 0–95). Additional mutations were identified in CFU-GM colonies in all patients, and logical hierarchy analysis indicated that mutations in TET2, SRSF2 and ASXL1 preceded KIT D816V. In ISM/SSM, no additional mutations were detected and CFU-GM colonies were exclusively KIT D816V−. These data indicate that (a) (A)SM-AHNMD is a multi-mutated neoplasm, (b) mutations in TET2, SRSF2 or ASXL1 precede KIT D816V in ASM-AHNMD, (c) KIT D816V is thus a phenotype modifier toward SM and (d) KIT D816V or other mutations are rare in CFU-GM colonies of ISM/SSM patients, which might explain at least in part their better prognosis. Leukemia (2015) 29, 1115–1122; doi:10.1038/leu.2015.4 INTRODUCTION syndromes.11–14 The current understanding of their molecular 11,13,15,16 Systemic mastocytosis (SM) is characterized by abnormal proli- significance has been thoroughly reviewed elsewhere. feration and accumulation of mast cells (MCs) in various tissues, Besides the classification of SM, the newly identified molecular predominantly skin, bone marrow (BM) and visceral organs. The lesions and aberration profiles may play a role in disease severity, 8–10,17,18 extent of organ infiltration and subsequent organ damage is prognosis and treatment responses in SM patients. the basis for the classification of SM into indolent SM (ISM), Despite such significant progress in our knowledge on the smoldering SM (SSM), SM with associated clonal hematologic non- molecular pathogenesis of myeloid neoplasms, it remains to be MC lineage disease (SM-AHNMD), aggressive SM (ASM) and MC elucidated how these mutations develop and accumulate in leukemia (MCL).1–3 In patients with SM-AHNMD, the SM compo- individual patients over time and finally contribute to disease nent can resemble ISM, ASM or even MCL. Depending on the evolution, phenotype, progression and prognosis. In multi- subtype of SM, cell source (BM or peripheral blood, PB) and assay mutated hematologic neoplasms, single-cell assays on colony- sensitivity, an acquired mutation in the receptor tyrosine kinase forming hematopoietic progenitor cells have recently revealed a KIT, usually KIT D816V, is detectable. Using PCR-based assays with complex clonal architecture and heterogeneous evolution includ- high sensitivity, this mutation is detectable in more than 80–90% ing acute myeloid leukemia (AML), MPN, MDS and MDS/ – of all SM patients.4–6 The multilineage involvement by KIT D816V MPN.14,19 24 In SM, several lines of evidence suggest that multi- and the KIT D816V allele burden have an important impact on mutated and thus potentially more aggressive subclones develop disease phenotype and prognosis.6,7 within the non-MC lineage compartment, which also explains why The presence of additional mutations in genes encoding for many of these patients develop an AHNMD or at least marked signaling molecules (CBL, JAK2, KRAS, NRAS), transcription factors myelodysplasia or signs of myeloproliferation. The aim of the (RUNX1), epigenetic regulators (ASXL1, DNMT3A, EZH2, TET2)or present study was to examine the mutational profile of colonies splicing factors (SRSF2, SF3B1, U2AF1) has recently been reported grown from granulocyte–macrophage colony-forming progenitor in KIT D816V+ SM patients in advanced disease.8–10 These cells (CFU-GM) and microdissected mature cells (CD117+, CD3+ or mutations are not specific for SM as they were also identified in CD15+) obtained from patients with various subtypes of SM. The other myeloid neoplasms, including myelodysplastic syndromes results of our study show that advanced SM with AHNMD is a (MDS), myeloproliferative neoplasms (MPN) or MDS/MPN overlap multi-mutated stem cell neoplasm with a phenotype modification 1Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany; 2Munich Leukemia Laboratory, Munich, Germany; 3Department of Pathology, Ludwig-Maximilians-University, Munich, Germany; 4Department of Clinical Chemistry, University Hospital Mannheim, Mannheim, Germany; 5Division of Hematology and Ludwig Boltzmann Cluster Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria; 6Wessex Regional Genetics Laboratory, Salisbury, UK and 7Faculty of Medicine, University of Southampton, Southampton, UK. Correspondence: Professor A Reiter, Department of Hematology and Oncology, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail: [email protected] 8These authors contributed equally to this work. Received 24 July 2014; revised 17 October 2014; accepted 7 November 2014; accepted article preview online 8 January 2015; advance online publication, 30 January 2015 Molecular profiling in systemic mastocytosis M Jawhar et al 1116 toward SM because of a late acquisition of KIT D816V, whereas Mannheim, University of Heidelberg) as part of the ‘German Registry on ISM/SSM seems to be not or only rarely affected by mutations at Disorders of Eosinophils and Mast Cells’. All patients gave written informed the CFU-GM level. consent. PATIENTS AND METHODS Qualitative and quantitative assessment of KIT D816V in BM and PB fi Diagnosis and classi cation of SM Qualitative and quantitative assessments of KIT D816V and KIT D816V allele Diagnosis of SM requires the presence of one major (multifocal dense burden at the RNA level (expressed allele burden) and DNA level in BM infiltrates of MC in BM biopsies and/or in sections of other extracutaneous (n = 16) and PB (n = 3) samples were performed using allele-specific organs) and at least one minor or the presence of at least three minor quantitative real-time PCR analyses as previously described.6,7 A previously criteria (425% atypical cells on BM smears or spindle-shaped MC published CMML cohort was additionally screened for the presence or infiltrates, KIT D816V point mutation in BM or other extracutaneous absence of KIT D816V mutations.13 organs, expression of CD2 and/or CD25 by MC in BM, PB or another extracutaneous organ and baseline serum tryptase concentration 420 μg/l).1,25–27 All BM biopsies were evaluated by two reference Targeted next-generation sequencing analysis pathologists of the ‘European Competence Network on Mastocytosis' Next-Generation Deep Amplicon Sequencing by 454 FLX amplicon (H-PH, KS). Diagnosis of ASM was based on the presence of one or more chemistry (Roche, Penzberg, Germany)11 was performed to investigate C-findings (cytopenia with an absolute neutrophil count o1×109 /l, 18 candidate genes at known mutational hotspot regions as previously hemoglobin o10.0 g/dl or platelets o100 × 109 /l, hepatomegaly with described. Detailed mutation analysis of patients #1, #6, #8 and #14 were impaired liver function, palpable splenomegaly with signs of hypersplenism, reported previously (Table 1).10 malabsorption with significant hypoalbuminemia and/or significant weight loss 410% over the last 6 months).1,25,27 In SM-AHNMD, BM morphology and Colony-forming unit granulocyte–macrophage (CFU-GM) assay PB counts revealed an associated myeloid neoplasm, for example, chronic myelomonocytic leukemia (SM-CMML), MDS/MPN unclassified (SM-MDS/ Methylcellulose (0.9%) was used as semi-solid matrix and was supple- MPNu) or chronic eosinophilic leukemia (SM-CEL).1,2,28 Diagnosis of SSM was mented with 30% fetal bovine serum albumin (FBS), 1% BS albumin, 0.1 M based on the presence of 2/3 diagnostic B-findings ((a) BM MCs 430% and 2-mercaptoethanol and recombinant human GM-CSF (100 ng/ml; Metho- serum tryptase 4200 μg/l, (b) organomegaly, (c) myeloproliferative or Cult, StemCell Technologies, Cologne, Germany). BM mononuclear cells + 5 myelodysplastic features in the BM), in the absence of C-findings.1,25 (MNC) and/or CD34 cells were seeded in the culture mixture (1 × 10 cells/ml or 5 × 103 cells/ml MethoCult) in 35-mm Petri dishes (10 per group) and were incubated at 37 °C in a humidified atmosphere with 5% CO2 Patients´ characteristics for 14 days. Colony counting was performed according to standard + Nineteen KIT D816V patients (male, n = 10; female, n = 9, median age 65 methodology under an inverted microscope. Single-cell-derived CFU-GM years, range 47–76 years) with World Health Organization-based colonies (100–300 cells per colony) were detached from the dishes and SM were evaluated. Clinical characteristics are summarized in Table 1. diluted in phosphate-buffered saline. Classification revealed ISM (n = 4), SSM (n = 2), ASM (n = 1) and (A)SM- AHNMD (n = 12; in detail: ASM-CMML, n = 4; ASM-MDS/MPNu, n = 2; ASM- MDS, n = 1; SM-CMML, n = 2; SM-MDS, n = 2; SM-polycythemia vera (n = 1). Genotyping of CFU-GM The study design adhered to the tenets of the Declaration of Helsinki and DNA extracted from single-cell-derived CFU-GM colonies was submitted was approved by the relevant institutional review board (Medical Faculty to whole-genome amplification (Repli-G, Qiagen, Hilden, Germany).