title: Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing.
abstract: BACKGROUND
  Systemic mastocytosis (SM) is a heterogenous, clonal mast cell (MC) proliferation, rarely associated with clonal hematologic non-mast cell lineage disease (SM-AHNMD). KIT (D816V) is regarded as driver-mutation in SM-AHNMD.
  
  
  METHODS
  DNA isolated from peripheral blood (PB) of an SM-CMML patient was investigated with targeted next generation sequencing. Variants were verified by Sanger sequencing and further characterized in the SM part of the bone marrow trephine (BMT), normal tissue, and FACS sorted PB cell subpopulations.
  
  
  RESULTS
  Low coverage deep-sequencing (mean 10x) on a GS 454 Junior revealed two as yet unreported SNVs (CBFA2T3 and CLTCL1), both germ-line mutations. High coverage (mean 1674x) targeted re-sequencing on an Ion Proton revealed 177 variants in coding regions. Excluding SNPs, the final list comprised 11 variants. Among these, TET2 (p.Thr1027fs, p.Cys1263Ser) and RUNX1 (p.Asn109Ser) were identified in in the peripheral blood and the SM part of BMT, but not in normal tissue. Furthermore, Sanger sequencing of PB cells revealed similar signal intensities for both TET2 mutations in FACS sorted CD34+ precursor cells and CD16+ granulocytes comparable to signals in the SM part of BMT. In contrast, RUNX1 exhibited a double intensity in CD34+ cells compared to the SM part of BMT and a homozygous variant signal in granulocytes. Both TET2 and RUNX1 mutations were not detectable in B- and T-cells.
  
  
  CONCLUSIONS
  We present a heterozygous triple-mutation pattern (KIT, TET2, RUNX1) in mast cells (SM disease part) with additional LOH of RUNX1 in granulocytes (CMML disease part). These identified mutations allow a more detailed insight into a multistep pathogenesis which suggests a common tumor progenitor in SM-CMML.
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Rechsteiner
authors.affiliation: Clinics for Hematology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Rouven
authors.initials: R
authors.lastname: Müller
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Reineke
authors.affiliation: Clinics for Hematology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Goede
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Annette
authors.initials: A
authors.lastname: Bohnert
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhong
authors.affiliation: Clinics for Hematology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Markus G
authors.initials: MG
authors.lastname: Manz
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Holger
authors.initials: H
authors.lastname: Moch
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Wild
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Dieter R
authors.initials: DR
authors.lastname: Zimmermann
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland ; Kempf and Pfaltz, Histological Diagnostics, Seminarstr. 1, 8042 Zurich, Switzerland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Tinguely
keywords: Chronic myelomonocytic leukemia; Next Generation Sequencing; RUNX1 mutation; SM-CMML; Systemic mastocytosis; TET-2 mutation; c-KIT mutation