Clonal analysis of NRAS activating mutations in KIT-D816V
by Todd M. Wilson, Irina Maric, Olga Simakova, Yun Bai, Eunice Ching Chan,
Nicolas Olivares, Melody Carter, Dragan Maric, Jamie Robyn, and Dean D. Metcalfe
Haematologica 2010 [Epub ahead of print]
Citation: Wilson TM, Maric I, Simakova O, Bai Y, Chan E, Olivares N, Carter M,
Maric D, Robyn J, and Metcalfe DD. Clonal analysis of NRAS activating mutations
in KIT-D816V systemic mastocytosis. Haematologica. 2010; 95:xxx
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Published Ahead of Print on December 6, 2010, as doi:10.3324/haematol.2010.031690.
Clonal analysis of NRAS activating mutations in KIT-D816V systemic
Todd M. Wilson1, Irina Maric2, Olga Simakova2, Yun Bai1, Eunice Ching Chan1,
Nicolas Olivares2, Melody Carter1, Dragan Maric3, Jamie Robyn4, and Dean D. Metcalfe1
1Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases,
2Department of Laboratory Medicine, Clinical Center, 3Flow Cytometry Core Facility,
National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland, USA, and 4Department of Veterans Affairs, Springfield, Ohio,
Todd M. Wilson, Building 10, Room 12S235A National Institutes of Health
Bethesda, MD 20892-1881 USA. Phone: international +301.4968891.
Fax: international +301.480-384. E-mail: email@example.com
This study was funded by the Intramural Research Program of the NIAID, NIH.
Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-
D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic
mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations.
Activating NRAS mutations were identified in 2 of 8 patients with advanced disease.
NRAS mutations were not found in patients with indolent systemic mastocytosis. To
better understand the clonal evolution of mastocytosis, we evaluated the cell
compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored
both mutations. KIT-D816V was not detected in bone marrow CD34+ progenitors,
whereas the NRAS mutation was present. These findings suggest that NRAS mutations
may have the potential to precede KIT-D816V in clonal development. Unlike other
mature lineages, mast cell survival is dependent on KIT and the presence of these two
activating mutations may have a greater impact on the expansion of this cell compartment
and in resultant disease severity.
(clinicaltrials.gov identifier: NCT00044122, NCT00001756).
Systemic mastocytosis (SM) is a heterogeneous disorder characterized by the pathologic
accumulation of mast cells within tissues. The majority of adult patients are classified
with indolent systemic mastocytosis (ISM) which generally carries a low risk of
transformation to an aggressive state and is not thought to affect lifespan. However, a
subset of patients with aggressive systemic mastocytosis (ASM) have a poor prognosis
and will require cytoreductive therapy. Somatic activating mutations in c-kit, most
notably KIT-D816V, are detected in the majority of adult patients.(1) Although
multilineage involvement by KIT-D816V clearly has an impact on disease severity and
progression (2); additional unidentified genetic abnormalities are likely to contribute to
more advanced forms of the disease.
RAS proteins are small membrane associated GTPases that play a pivotal role in signal
transduction events regulating cell proliferation, differentiation and survival. Somatic
mutations which disrupt this intrinsic GTPase activity and lock RAS in an active GTP-
bound state are frequent among myeloid malignancies; predominantly involving KRAS
and NRAS. In murine models, oncogenic NRAS has not only produced CMML and
AML-like diseases, but also increased mast cells in the blood, bone marrow, liver and
spleen; a phenotype consistent with aggressive SM. (3-4)
In this study, we demonstrate that RAS gene expression increases with mast cell
maturation and that activating mutations, specifically in NRAS, are found exclusively in
advanced forms of SM and may precede the KIT-D816V mutation in clonal development.
DESIGN AND METHODS
Forty-four patients with systemic mastocytosis were evaluated at the National Institutes
of Health (NIH, Bethesda, MD, USA) between 2006 and 2009 as part of an Institutional
Review Board-approved research protocol designed to study the pathogenesis and natural
history of systemic mastocytosis (NCT00044122). This included 27 patients with
indolent SM (ISM), 9 patients with smoldering SM (SSM), 4 patients with SM with an
associated clonal hematologic non mast cell lineage disease (SM-AHNMD) and 4
patients with aggressive SM (ASM). All patients were diagnosed according to the World
Health Organization (WHO) criteria (5) and carried the KIT-D816V mutation.
RNA/cDNA was prepared from bone marrow mononuclear cells and cell lines as
described.(6) Buccal gDNA was isolated using the Gentra Puregene DNA Purification
Kit (Qiagen) followed by amplification using a Qiagen REPLI-g Mini kit. HMC1, LAD2
and CD34+ derived human mast cells (NCT00001756) were cultured as described.(7)
Immunophenotypic analysis of mast cells and flow cytometry cell sorting
Bone marrow mast cells were analyzed as described(6) using CD45 PerCP, CD117 APC
and CD25 FITC (BD Biosciences) antibodies and FACSCanto II flow cytometer (BD
To obtain mast cells, CD34+ cells, monocytes, granulocytes, eosinophils, B and T cells
fractions; a CD45+ enriched population (Whole Blood CD45 MicroBeads; Miltenyi
Biotec) was stained using CD45 Tri Color, CD3 PE-TR, CD19 PE-TR (Invitrogen);
CD14 FITC, CD49d PE, CD34 FITC (BD Biosciences), CD117 PE (Dako), DAPI and
sorted using FACSVantage SE flow cytometer (BD Biosciences). Sort purity routinely
The KIT-D816V mutation was detected by RT-PCR/RFLP as described.(6) Two round
PCR followed by RFLP was used for flow sorted cells. NRAS, HRAS, KRAS and MRAS
open reading frames were amplified from cDNA either directly or by nested PCR (flow-
sorted cells). PCR products were gel purified and directly sequenced in both sense and
antisense directions using BigDye terminator v3.1 chemistry and an ABI-3100 genetic
analyzer according to standard protocols. Sequencing data was analyzed by Sequencher
(Version 4.5, Softgenetics). Primers and conditions used for all PCR reactions are found
in Online Supplementary Table 1.
RAS real-time PCR
Real-time PCR was performed using RT? SYBR® Green qPCR Master Mixes
(SABiosciences) and the ABI7500 real-time PCR system (Applied Biosystems). The 2–
Ct method was used to calculate the relative expression level of each gene to GAPDH.
RESULTS AND DISCUSSION
The phenotypic diversity displayed among myeloproliferative disorders is thought to be
the result of multiple and complex molecular events. KIT-D816V SM shares this
phenotypic heterogeneity and coexisting mutations are increasingly being identified. The
JAK2 V617F mutation was detected in a rare subset of patients with KIT D816V systemic
mastocytosis associated with chronic idiopathic myelofibrosis.(8) More recently, loss of
function mutations in the putative tumor suppressor gene, TET2, were frequently found in
patients with systemic mastocytosis although did not appear to alter prognosis.(9) We
now report coexisting NRAS activating mutations which potentially collaborate with KIT-
D816V in disease pathogenesis.
Two of 44 patients (4.5%) harbored an NRAS activating mutation. NRAS-G12D and
NRAS-G13D mutations were identified in one patient with SM-CMML and one patient
with ASM respectively (Figure 1A). Bone marrow histology supported these
classifications and although a hypercellular marrow was observed in the patient with
ASM, the overall findings did not meet 2008 WHO criteria for any myeloproliferative or
myelodysplastic disorder (Figure 2). Together, 25% (2/8) of patients with advanced
forms of SM harbored activating NRAS mutations, although no associated phenotype was
observed within this subset (Table 1). These findings parallel observations made in other
myeloproliferative disorders such as AML, where RAS mutation frequency does not vary
with gender, age, leukocytosis, or WHO performance status.(10) Of similar importance
is the absence of NRAS mutations in 36 patients with ISM (27) or SSM (9). This
observation supports the current premise that more benign forms of mastocytosis are
mainly KIT-D816V driven and additional mutations may be required for more severe
forms of the disease. Indeed, NRAS mutations associated with progression from MDS to
AML are described.(11-12)
Efforts to molecularly dissect the cell compartments impacted by these two mutations in
ASM (Patient 2) revealed that bone marrow mast cells harbored both mutations and
uniformly expressed the aberrant CD25 marker, indicating a clonal population (Figure 1B,
1C). KIT-D816V and NRAS-G12D were also detected in both myeloid and lymphoid
lineages (Figure 1B). This is consistent with previous observations that aggressive forms
of mastocytosis display multilineage involvement, likely the result of a common
progenitor.(2, 13) However, detection of KIT-D816V in CD34+ bone marrow progenitors
has varied between studies. Akin, et al. did not detect KIT-D816V in the CD34+ cells of
3/3 patients with SM displaying multilineage involvement.(13) In contrast, Garcia-
Montero, et al. observed that 3/4 of patients with ASM harbored KIT-D816V in CD34+
progenitors.(2) In our study we detected only NRAS-G12D in the CD34+ progenitors;
despite the KIT-D816V RT-PCR/RFLP assay having greater sensitivity than cDNA
sequencing. This observation suggests that NRAS-G12D may have preceded KIT-D816V
in clonal development. According to the clonal expansion model, early mutations should
be more prevalent in the clonal population than late mutations.(14) Consistent with this
model, NRAS-G12D penetrated more cell populations than KIT-D816V (Figure 1B).
Cooperating Class I (enhanced proliferation and/or survival) and Class II (impaired
differentiation) mutations are thought necessary for leukemogenesis.(15) NRAS and KIT
mutations both represent Class I mutations. Although likely that an unidentified Class II
mutation may exist, this “exception” has been observed in other studies.(16-17) A model
utilizing information from the Cancer Genome Atlas recently predicted that NRAS
activating mutations would coexist with KIT mutations in hematopoietic malignancies;
therefore were strong candidates for cosequencing.(14) Mutations in other RAS genes
were not predicted to coexist with KIT mutations. In support of this prediction, we did
not detect KRAS, HRAS or MRAS mutations in our KIT-D816V population. Arguably, this
may be a reflection of the sampling size or alternatively NRAS may have a significant
role in mast cell homeostasis.
The RAS gene family appears to play a role in mast cell development, as their relative
expression levels uniformly increased as mast cells matured in vitro (Figure 1D). Peak
RAS expression was observed at 8 weeks; at which time mature mast cells were the only
cell type present. NRAS and KRAS, but not HRAS are reported to be the dominant
isoforms in LAD2 and HMC1 cell lines.(18) We observed that the message for all
isoforms was detectable, including MRAS, albeit expression was relatively low and
comparable to that of an immature mast cell. This may reflect the maturity state of the
cell lines and/or the cell division rate. Cell lines and cultured CD34+ cells at 1-2 weeks
are rapidly dividing and may not require significant RAS expression, whereas the
relatively quiescent mature mast cell may be more dependent. RAS mutations were not
observed in the cell lines.
Clinical trials targeting KIT-D816V have demonstrated only modest efficacy in SM.(19-
20) This study contributes to growing evidence that additional genetic alterations are
present in KIT-D816V SM and effective treatment will likely require a multi-targeted
approach.(21-22) Specifically identifying RAS mutations may influence the choice and
dosing of cytoreductive therapy, as AML patients carrying activated forms of RAS appear
to benefit from higher cytosine arabinoside doses in response rate and overall
survival.(23-24) As patients with advanced SM are relatively rare; large multicenter
studies will be required to support not only our findings, but future studies investigating
the molecular pathogenesis of SM.
AUTHORSHIP AND DISCLOSURES
TMW and JR designed the research. TMW, MC, JR and DDM recruited and cared for
the patients. TMW, OS, YB, ECC, and NO performed the research. TMW, IM, DM, JR
and DDM analyzed and interpreted the data. TMW wrote the paper with edits from all
authors. The authors reported no potential conflicts of interest.
We wish to thank the patients for their participation; as well as Alasdair Gilfillan and
Kimberly Dyer and the clinical research staff for their scientific and medical assistance.
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Table 1. Characteristics of the study population with advanced forms of KIT-D816V systemic mastocytosis. Shading indicates
patients with additional NRAS mutations.
Age 68 54
Gender M F
#3 #4 #5 #6 #7 #8
Bone marrow biopsy mast cell %
Aspirate mast cell %
Peripheral blood mast cell %
Serum tryptase (ng/mL)
SM-CMML, systemic mastocytosis with chronic myelomonocytic leukemia; SM-MDS, systemic mastocytosis with myelodysplastic syndrome; SM-ET,
systemic mastocytosis with essential thrombocytosis; ASM, aggressive systemic mastocytosis; NA, not available.
Figure 1. A. Activating NRAS mutations in two patients with severe forms of
KIT-D816V systemic mastocytosis. KIT-D816V RT-PCR/RFLP: Detection of KIT-
D816V (arrow) in the bone marrow mononuclear cells (BMMC) of the two patients.
HMC1.2 cells were used as positive control. NRAS sequencing: Patient 1: Compared
to control, a heterozygous missense mutation GGT?GAT at codon 13 was identified
resulting in a Glycine to Aspartic Acid substitution (NRAS-G13D). Patient 2: A
similar heterozygous missense mutation GGT?GAT at codon 12 was identified
resulting in a Glycine to Aspartic Acid substitution (NRAS G12D). A wild type
sequence in the buccal germline DNA confirmed a somatic event. B. Patient 2:
Segregation of KIT-D816V and NRAS-G12D in flow sorted bone marrow cell
populations. KIT-D816V is present in mast cells (arrow), but absent in the CD34+
cells. NRAS-G12D is present in both populations (asterisks). Results are
representative of three separate flow sorting experiments and summarized in the table.
C. Mast Cell Immunophenotyping. Bone marrow mast cells were initially identified
as CD117 bright positive, CD45 positive cells with characteristic forward and side
scatter properties (circled). Compared to the isotype control, gated mast cells
uniformly expressed the aberrant CD25 marker. D. RAS gene expression in cultured
human mast cells. HRAS, NRAS, KRAS and MRAS gene expression standardized to
GAPDH is plotted on the Y axis. Mast cells on the X axis include CD34+ derived
human mast cells (HuMC) harvested at weeks 1, 2, 4 and 8; HMC1.1, HMC1.2 and
LAD2 mast cell lines. Error bars are the Standard Error Mean (SEM) of three
separate experiments each performed in triplicate. Different CD34+ donors were used
for each HuMC experiment.
*Note: KIT-D816V RT-PCR/RFLP gel lanes are cropped from the same gel without
Figure 2. Histopathological changes in bone marrow biopsies from two NRAS
positive patients. Both patients have hypercellular marrow biopsies, but with
markedly different mast cell burden, as assessed by % mast cell involvement of the
biopsy sections. Representative H&E (A) and CD117 (B) immunostained sections
show that Patient 1 has minimal involvement by mast cell aggregates, while Patient 2
has extensive marrow replacement by mast cells (C H&E; D CD117). In addition,
Patient 1 has myeloid hyperplasia with increase in monocytic precursors and blasts,
while Patient 2 has normal trilineage maturation (Magnification 40x for all