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. 2014 Jul 11:3:18.
doi: 10.1186/2162-3619-3-18. eCollection 2014.

Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing

Markus Rechsteiner  1 , Rouven Müller  2 , Tanja Reineke  1 , Jeroen Goede  2 , Annette Bohnert  1 , Qing Zhong  1 , Markus G Manz  2 , Holger Moch  1 , Peter J Wild  1 , Dieter R Zimmermann  1 , Marianne Tinguely  3
Affiliations

Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing

Markus Rechsteiner et al. Exp Hematol Oncol. .

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.

Findings: 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.

Conclusion: 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.

Keywords: Chronic myelomonocytic leukemia; Next Generation Sequencing; RUNX1 mutation; SM-CMML; Systemic mastocytosis; TET-2 mutation; c-KIT mutation.

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Figures

Figure 1
Figure 1
Assessment of the KITD816V mutation on the bone marrow trephine. Aupper line: HE stain of the bone marrow trephine showing both, CMML and SM part in distinct areas in this patient (left). A CD117 immunostain (right) highlights the mast cells in the SM compartment, however cells within the CMML compartment are negative. Lower line: HE control before and after punching out the SM area (containing around 90% of MC in the CD117 staining) submitted to PCR and Sanger Sequencing. B Forward and reverse sequences indicating the KITD816V mutation of microdissected SM area of the bone marrow trephine. C FACS sorted cells as indicated with a purity of around 90% for CD34+ stem cells and granulocytes. D Shows the sequencing results of the PCR amplification product for KITD816V, and forward strands for isolated cell-subpopulations respectively.
Figure 2
Figure 2
Verification of identified variants by Sanger sequencing. A: Representative sequences of CBFA2T3, CLTCL1, and BLM using DNA derived from the SM part of BMT, PB, and the SM part of BMT, respectively. B: Sequencing of TET2 and RUNX1 using DNA derived from PB, the SM part of BMT, and normal tissue. C: Sequencing of TET2 and RUNX1 using DNA derived from FACS sorted PB cell populations.
Figure 3
Figure 3
Hypothetical disease model of SM-CMML pathogenesis.
See this image and copyright information in PMC

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