Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing
© Rechsteiner et al.; licensee BioMed Central Ltd. 2014
Received: 9 April 2014
Accepted: 1 July 2014
Published: 11 July 2014
Systemic mastocytosis (SM) is a heterogenous, clonal mast cell (MC) proliferation, rarely associated with clonal hematologic non-mast cell lineage disease (SM-AHNMD). KITD816V is regarded as driver-mutation in SM-AHNMD.
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.
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.
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.
KeywordsChronic myelomonocytic leukemia Systemic mastocytosis SM-CMML Next Generation Sequencing c-KIT mutation TET-2 mutation RUNX1 mutation
Systemic mastocytosis (SM) rarely occurs in combination with so called “associated clonal haematological non-mast cell lineage disease” (AHNMD) [1–5]. In most instances, the AHNMD component is of myeloid origin, including acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML) or primary myelofibrosis (PMF) [1, 6–8]. Recently, KITD816V mutations were identified in both disease components of SM-AHNMD with the highest frequency in SM-CMML , suggesting a common precursor.
Methods and findings
To achieve a higher resolution we performed a high coverage targeted re-sequencing with identical blood derived DNA using the Ion AmpliSeq™ Comprehensive Cancer Panel comprising all exons of 409 known cancer genes (Life Technologies). Forty ng DNA was used as input and the target regions were 13-fold PCR amplified. After ligation of Torrent specific adapters, the amplicons were subjected to clonal expansion on spheres using the Ion One Touch 2 system (Life Technologies). Subsequently, the amplicons were dispersed on a PI chip and sequenced on the Ion Proton platform . Mean coverage was 1674-fold whereas more than 93% of the amplicons were covered at least 100-fold and more than 81% at least 500-fold allowing sensitive variant detection. Alignment (hg19), local re-alignment (2-fold), and probabilistic variant detection (90% probability and 10-fold coverage) were performed using CLC Genomics Workbench version 5.5. Additional filtering resulted in variants with following characteristics: i) non-synonymous variants and INDELs resulting in frame-shifts, ii) phred-score > = 20 and variant coverage > = 50x, and iii) forward/reverse read ratio > = 0.25. These filter criteria resulted in identifying 177 variants (Additional file 4: Table S3). From these, 144 were present in the 1000 Genome database and dbSNP and were therefore excluded in the following analysis. Under additional more stringent filtering criteria for 1-bp INDELs in homopolymeric regions (CLC default settings for 454/Ion) a final list of 11 variants emerged (Additional file 5: Table S4) including KITD816V. Interestingly, variants of two genes previously described in SM-AHNMD  TET2 (p.Thr1027fs, p.Cys1263Ser) and RUNX1 (p.Asn109Ser) were detected. Although detectable by the 454-Junior, their coverage was too low to be called by the GS Reference Mapper. Of note, 44 variants of the 64 variants identified by the 454-Junior/GS Reference Mapper platform lay in target regions of the CCP (Additional file 2: Table S2). From these, 35 were verified by the Ion Torrent/CLC platform, one was differently aligned, and eight were not detected.
Thus, the variants in TET2 and RUNX1 represented most probably true positive calls potentially involved in SM-CMML pathogenesis. Although, they have not yet been reported in the Cosmic database, the mutations highly likely influence the protein function: i) the frame-shift mutation in TET2 occurring in front of the two conserved regions of TET2 (1104–1478, 1845–2002) and the SNV lying within the first region , ii) the mutation in RUNX1 located in the RUNT domain which is responsible for proper DNA binding and heterodimerization .
The 2-bp insertion (p.Thr1027fs) and the SNV (p.Cys1263Ser) in TET2, and the SNV (p.Asn109Ser) in RUNX1 were confirmed in PB and the SM part of BMT (Figure 2B). Very low signals of the variants were detected in normal tissue, most probably due to infiltrating mast cells. Whereas no signals were found in B- and T-lymphocytes, similar signal intensities for both TET2 mutations were seen in CD34+ precursor cells and CD16+ granulocytes comparable to the signals in the SM part of BMT (Figure 2C). In contrast, RUNX1 exhibited a double intensity of the variant in CD34+ cells compared to the SM part of BMT and a homozygous variant signal in granulocytes.
In summary, the current SM-CMML derived from the same mutated tumour progenitor cells (KIT SNV, TET2 frame-shift, TET2 SNV, RUNX1 SNV) which acquired additional mutations (i.e. RUNX1 wild-type loss) in different sub-clones as hypothesised in our model to finally manifest in the pathological state of SM-CMML.
The study was approved by the official authorities of the ethical committee of the County of Zurich (StV2-2007) and a written consent was obtained by the patient.
Associated clonal hematologic non-mast cell lineage disease
Bone marrow trephine
Chronic Myelomonocytic Leukemia
Single nucleotide polymorphism
Single nucleotide variation.
This work has been funded by the Novartis Foundation for Medicine and Biology (MT: No 04C58). We thank Dr. M. Umbricht, Sonja Brun-Schmid, Norbert Wey and the laboratory of in situ techniques, Institute of Surgical Pathology for their excellent support.
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