Novel, activating KIT-N822I mutation in familial cutaneous mastocytosis
Bartosz Wasaga,b, Marek Niedoszytkoc, Anna Piskorza, Magdalena Langed, Joanna Renkee,
Ewa Jassemc, Wojciech Biernatf, Maria Debiec-Rychterb,*, and Janusz Limona,*
aDepartment of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland;bDepartment of Human Genetics, K.U. Leuven, Leuven,
Belgium;cDepartment of Allergology, Medical University of Gdansk, Gdansk, Poland;dDepartment of Dermatology, Medical University of Gdansk,
Gdansk, Poland;eChildren’s Hospital Polanki, Gdansk, Poland;fDepartment of Pathology, Medical University of Gdansk, Gdansk, Poland
(Received 18 March 2011; revised 18 May 2011; accepted 20 May 2011)
Objective. We report the rare family in which cutaneous mastocytosis was diagnosed in the
father and two children, with urticaria pigmentosa as the only manifestation of the disease.
The diagnosis of mastocytosis in the father included bone marrow histopathological and cyto-
logical examinations and flow cytometry, and histopathological examination of the skin. In the
children, tryptase measurement and skin histopathological examination were performed.
Materials and Methods. Blood, urine, and buccal swab specimens were collected from
the family members. HEK293T cells were transiently transfected with plasmids expressing
KIT-WT and KIT-N882I. In addition, Ba/F3 cell lines expressing KIT-N822I, KIT-D816V,
and KIT-V559D mutants were treated with imatinib and dasatinib. The effect of treatment
on proliferation, survival, and signaling was determined.
Results. Germ-line KIT-N822I missense mutation was detected in the affected members of the
family. Western blot analysis using HEK293T and Ba/F3 cells expressing KIT-N822I isoform
showed that KIT-N822I constitutively activated KIT tyrosine phosphorylation. In vitro assays
on KIT-N822I-expressing Ba/F3 cells confirmed that the N822I mutant is resistant to imatinib
mesylate. In contrast, a high efficacy of dasatinib toward the KIT-N822I–expressing Ba/F3
cells was observed.
Conclusions. We provided evidence that KIT p.N822I mutation has transforming potential and
can cause a constitutive activation of KIT. In addition, we demonstrated that KIT-N822I is
resistant to imatinib and sensitive to dasatinib. Finally, our findings support the hypothesis
that not only KIT mutations but other additional genetic abnormalities are contributing
to more advanced forms of the disease.
? 2011 ISEH - Society for Hematology and Stem
Cells. Published by Elsevier Inc.
Mastocytosis is a rare group of diseases characterized by
abnormal proliferation and infiltration of mast cells within
the tissues. The skin is most frequently involved but mast
cells also accumulate in the bone marrow, gastrointestinal
tract, lymph nodes, spleen, and liver. Oncogenic mutations
within KIT gene are the most common causal genetic
abnormalities in sporadic mastocytosis, therefore, KIT
tyrosine kinase forms a promising therapeutic target .
Multilineage involvement by KIT-D816V has an impact
on disease severity and progression . However, it is
widely accepted that other cooperating genetic events are
contributing to the phenotypic diversity of KIT-positive
In contrast to mastocytosis associated with somatic KIT
mutations, the accounts of familial forms of mastocytosis
with KIT germline mutations are extremely rare . To
date, KIT germline mutations have been reported in four
families with mastocytosis [4–7]. Identified mutations
were located within extracellular domain or in transmem-
brane domain. More recently, Bodemer et al. found KIT-
D816V mutation in two children with a familial form of
the disease . The presence and type of the KIT mutation
is important because it is known that sensitivity to imatinib
depends on the location of the alteration. Previous studies
showed that a subset of mutations in the KIT second
*Drs. Debiec-Rychter and Limon contributed equally as senior authors.
Offprint requests to: Bartosz Wasag, Ph.D., Department of Biology and
Genetics, Medical University of Gdansk, Debinki 1, 80-210 Gdansk,
Poland; E-mail: firstname.lastname@example.org
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.exphem.2011.05.009.
0301-472X/$ - see front matter. Copyright ? 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
Experimental Hematology 2011;39:859–865
tyrosine kinase domain (activation loop domain) does not
respond to imatinib treatment .
Dasatinib, formerly known as BMS-354825, is an aden-
osine triphosphate–competitive, multitarget inhibitor of
BCR-ABL and SRC family kinases . Inhibitory activity
of dasatinib was also recorded for other protein kinases,
such as KIT, platelet-derived growth factor receptors,
c-FMS, and EPHA2 receptor . More recently, dasatinib
was shown to inhibit the kinase activity of KIT mutants
resistant to imatinib . Therefore, use of dasatinib could
be considered as a new therapeutic approach to the treatment
of patients whose cancer cells carry imatinib-resistant KIT
In the present study, we describe a family with urticaria
pigmentosa as the only manifestation of the disease. A
novel germline KIT mutation in exon 17 (p.N822I) was
detected in all affected family members. We have explored
the transforming potential of that mutation and tested
the efficacy of tyrosine kinase inhibitors, imatinib and
dasatinib, for the inhibition of Ba/F3 cell line expressing
Materials and methods
A 33-year-old male was referred to the Department of Allergol-
ogy, Medical University of Gdansk, with an initial diagnosis of
mastocytosis for further examination. The patient suffered from
skin lesions (urticaria pigmentosa) and mild itching since early
childhood. The changes were present mainly on the trunk and
lower extremities and did not affect the patient’s quality of life.
The skin biopsy confirmed the clinical diagnosis. Mast cell infil-
trate was present in the subepidermal location and presented
KIT (CD117) and tryptase expression. CD25 and CD2 antigens
were not disclosed in these mast cells. Histological and immuno-
histochemical findings are presented in Figure 1 (CD25 not
shown). There were no symptoms of systemic or aggressive mas-
tocytosis; the patient did not suffer from anaphylaxis and did not
present any symptoms of food intolerance or allergy. No abnor-
malities were found on the chest x-ray and abdomen ultrasound
images. The patient has two children. The family anamnesis
revealed similar skin lesions (urticaria pigmentosa) in both chil-
dren of the patient, a 5-year-old daughter and 1-year-old son.
The children had no systemic symptoms nor any food intolerance
or allergy. No complications were present during pregnancy and
delivery. The skin lesions were present since infancy. The diag-
nosis of mastocytosis was confirmed by histopathology.
The bone marrow analysis was performed in the adult patient,
including trephine bone marrow biopsy, aspiration cytology,
and cytophotometry with CD2/CD25 evaluation. None of the
procedures revealed bone marrow involvement by mastocytosis.
Serum tryptase level was 11.6 ng/mL (normal serum tryptase
levels: 0–11.4 ng/mL). None of the other members of the family
suffered from urticaria pigmentosa or systemic mastocytosis and
the family history did not disclose any neoplastic disease in the
first-line family members. Lung cancer was present in the distant
family, in a heavy smoker, male.
Treatment with H1 antihistamine and rescue kit containing
adrenaline, glucocorticosteroids, and H1 blockers was started.
During 3-year follow-up, the patient and his children did not
show any symptoms of mastocytosis other than urticaria
Figure 1. Histological biopsy of the skin lesion discloses the nodular infiltrate of the mast cell below the epidermis (A, hematoxylyn and eosin, 200?). The
mast cell infiltrate expresses tryptase (B) and CD117 (C), while CD2 is absent (D).
860B. Wasag et al./ Experimental Hematology 2011;39:859–865
Buccal swabs, peripheral blood, and urine samples from the
father, mother, and two children were collected after obtaining
informed consent. The study was approved by the ethical
committee of the Medical University of Gdansk. Genomic
DNA was extracted using a standard procedure based on ionic
detergent lysis and proteinase K digestion, phenol/chloroform
extraction, and isopropanol precipitation. The coding sequence
of KIT was analyzed as described previously . Polymerase
chain reaction products were sequenced using ABI3130 (Applied
Biosystems, Foster City, CA, USA) and data were analyzed by
Sequencher v.4.7 DNA Software (Gene Codes Corporation,
Ann Arbor, MI, USA). The presence of a mutation was
confirmed by independent polymerase chain reaction amplifica-
tion followed by bidirectional sequencing. In addition, to assess
the presence of a KIT-D816V mutation, allele-specific poly-
merase chain reaction assay was applied.
The inhibitors, imatinib mesylate (Glivec/Gleevec; Novartis, New
York, NY, USA) and dasatinib (BMS-354825; Bristol-Meyers-
Squibb, New York, NY, USA), were purchased from Selleck
Chemicals. 10 mM stock solutions of the inhibitors, dissolved in
dimethyl sulfoxide, were stored at ?80?C.
Detailed protocols for generation of the construct, cell culture,
retroviral transduction, proliferation, apoptotic, and Western
blotting assays were described previously [10,13]. Briefly, the KIT
was amplified from proband peripheral blood leukocytes comple-
mentary DNA (cDNA) and cloned into the pcDNA3.1 (Invitrogen,
Carlsbad, CA, USA) or retroviral pMSCVpuro vector (Clontech,
Mountain View, CA, USA). All constructs used in this study
were encoding the GNNK isoforms of KIT. HEK293T and
Ba/F3 cells were grown in Dulbecco’s modified Eagle’s medium
and RPMI-1640, supplemented with 10% fetal bovine serum.
After 24-hour serum starvation, HEK293T cells were stimulated
or not with 200 mg/mL stem cell factor (SCF; Sigma-Aldrich,
St Louis, MO, USA) at þ37?C for 10 minutes. The Celltiter
AQueousOne Solution (Promega, Madison, WI, USA) was applied
to obtain dose-response curves of transduced Ba/F3 cells, treated
with vehicle alone or with varying concentrations of imatinib and
dasatinib for 48 hours. Induction of apoptosis of transduced Ba/F3
cells was evaluated by flow cytometry using Annexin-V-FLUOS
Staining Kit (Roche Applied Science, Indianapolis, IN, USA).
Finally, Western immunoblotting was performed using thefollowing
antibodies: anti–a-tubulin (Sigma-Aldrich), anti-KIT (DAKO,
Carpinteria, CA, USA), anti–phospho-KIT (pY703) (BioSource,
Carlsbad, CA, USA), anti-AKT, anti–phospho-AKT (Ser473),
anti-p44/42 mitogen-activated protein kinase, anti–phospho-p44/42
mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204), anti-
RPS6K, and anti–phospho-RPS6K (Ser235/236) (Cell Signaling
Technology, Danvers, MA, USA). Quantitative analysis of protein
blot images was performed using Scion software.
Results and discussion
KIT mutations within codon p.N822 were detected in
a number of pediatric and adult patients with acute myeloid
leukemia, sporadic and familial gastrointestinal stromal
tumors (GIST), and seminomas. The vast majority of the
identified alterations were c.2563TOA point mutations,
resulting in p.N822K substitution. In addition, a variety
of other amino acid substitutions affecting codon 822 have
been reported previously. Point mutation resulting in
p.N822H was detected in patients with seminomas and
GISTs [14,15]. Shimada et al. identified p.N822T substitu-
tion in a pediatric acute myeloid leukemia patient .
More recently, a novel KIT germline mutation resulting in
the replacement of asparagine by tyrosine (p.N822Y) was
found in kindred with familial GISTs .
In the present report, we describe a novel germline KIT
mutation in codon 822 in the family with cutaneous masto-
cytosis. In all affected family members, the kinase domain
mutant p.N822I was detected in buccal swabs, peripheral
blood, and urine samples (Supplementary Figure E1). No
other KIT mutation was detected. Recently, such a KIT
mutation was reported by McDonnell et al. in a patient
with melanoma . In this individual, p.N822I substitution
Figure 2. Western blot of HEK293T cells transfected with plasmids
encoding KIT-N822I construct (lanes 1, 2), empty pcDNA3.1 vector
(lanes 3, 4) and KIT-WT construct (lanes 5, 6). After 24 hours, cells
were treated with 200 ng/mL SCF for 15 minutes. Immunoblots with total
lysates were probed with anti-KIT and anti–phospho-KIT[Y703] anti-
bodies. Bottom: P-KIT/KIT ratios were normalized to 1 for the KIT-WT
(stimulated). Student’s t-test was used for statistical analysis. *p ! 0.001.
Error bars represent standard deviation (n 5 2).
861 B. Wasag et al./ Experimental Hematology 2011;39:859–865
coexisted with p.V559A mutation. In our family, the
affected members had urticaria pigmentosa as the only
manifestation of the disease. Urticaria pigmentosa has
already been reported as an additional symptom in two fami-
lies with GISTs and a syndrome of diffuse leiomyomatosis-
associated achalasia, in which KIT germline mutations were
Given the lack of enough primary neoplastic cells of the
patient to establish a number of primary cultures dishes, the
expression vector containing KIT-N822I cDNA was trans-
fected into HEK293T cells to investigate the biological
consequences of asparagine for isoleucine substitution.
The empty expression vector and vector containing the
KIT-WT cDNA were also transfected into the cells and
the status of tyrosine phosphorylation of KIT was assessed
by Western blot. As shown in Figure 2, autophosphoryla-
tion of KIT-WT without SCF stimulation was very weak,
but became significant in response to SCF addition. In
contrast, high constitutive tyrosine phosphorylation was
detected in KIT-N822I–expressing cells in the absence of
SCF. However, stimulation of HEK293T cells with SCF
resulted in further phosphorylation of KIT-N822I mutant.
It is of note that the same phenomena have already been
observed for the KIT p.A502_Y503dup and p.K509I muta-
tions . Another characteristic feature of KIT mutant
isoforms is greater or equal expression of immature forms
(145 kDa) when compared with mature forms (160 kDa),
while in the KIT-WT expressing cells, the mature hypergly-
cosylated form predominates . In line with previously
published results, KIT-N822I mutant also showed greater
expression of immature form as compared with mature form.
To investigate KIT-N822I oncogenic potential, we de-
signed another construct and expressed it in the interleukin-
3–dependent Ba/F3 cells. As controls, imatinib-sensitive
KIT-V559D and imatinib-resistant KIT-D816V–transduced
Ba/F3 cells were used. Oncogenic activity of the studied
KIT mutant isoforms was confirmed because expression of
KIT-V559D, KIT-D816V, and KIT-N822I transformed the
Ba/F3 cells to interleukin-3–independent growth (Fig. 3A).
During 3-year follow-up, the patient and his children did
not suffer from any other symptoms of mastocytosis than
urticaria pigmentosa, therefore, they have not required
Figure 3. In vitro assays of KIT-V559D, KIT-N822I, and KIT-D816V–expressing Ba/F3 cells. (A) Interleukin-3 (IL-3)–deprivation of KIT-V559D,
KIT-N822I, and KIT-D816V–transduced Ba/F3 cells resulted in transformation to IL-3–independent growth. The mean growth 6 standard error of mean
of three separate measurements over 4 consecutive days are shown. (B) In vitro effect of dasatinib on KIT-V559D, KIT-N822I, and KIT-D816V-
expressing Ba/F3 cells. The dose-response proliferation curves of transduced Ba/F3 cells, treated with dasatinib for 48 hours are shown. Points represent
the average results of experiment done in triplicate; bars: standard deviation (SD). The calculated IC50for each cell line is indicated. Graph was plotted
with the curve-fitting GraphPad Prism 5 software. (C) In vitro effect of imatinib on apoptosis of KIT-V559D, KIT-N822I, and KIT-D816V–expressing
Ba/F3 cells treated for 48 hours. The percentage of apoptotic and necrotic transduced Ba/F3 cells are indicated. (D) Induction of apoptosis in KIT-V559D,
KIT-N822I, and KIT-D816V-expressing Ba/F3 cells upon dasatinib treatment.
862B. Wasag et al./ Experimental Hematology 2011;39:859–865
treatment with tyrosine kinase inhibitors. However, because
KIT-N822 mutations have been reported in other tumors,
we decided to define the tyrosine kinase inhibitors sensi-
tivity of the KIT-N822I.
We examined the inhibitory effect of imatinib and
dasatinib on the ligand-independent KIT phosphorylation in
Ba/F3 cells. By proliferation assay, imatinib exhibited
a high efficacy only toward the imatinib-sensitive KIT-
V559D mutant, with an IC50of 82 nM (data not shown).
Furthermore, significant induction of apoptosis of these
cells was recorded at 100 nM imatinib (Fig. 3C). KIT-
D816V and KIT-N822I–expressing Ba/F3 cells were resis-
tant to imatinib and no induction of apoptosis was recorded
(Fig. 3C). In contrast, dasatinib effectively inhibited the
KIT-V559D, KIT-D816V, and KIT-N822I isoforms with
an IC50value of 6 nM, 77 nM, and 68 nM, respectively
(Fig. 3B). An apoptosis assay showed the induction of
apoptosis in KIT-D816V and KIT-N822I Ba/F3 transform-
ants 48 hours after treatment with 100 nM dasatinib
(Fig. 3D). Massive apoptosis of KIT-V559D was recorded
already at 50 nM dasatinib (data not shown). Addition of
interleukin-3, the normal growth factor for Ba/F3 cells,
resulted in near complete rescue, suggesting selective inhi-
bition of transduced Ba/F3 by dasatinib (Supplementary
The effect of the inhibitors on signaling in KIT-V559D,
KIT-D816V, and KIT-N822I-expressing Ba/F3 cells was
also investigated by Western immunoassays in wide dose
range. Imatinib was very efficient in reducing the tyrosine
phosphorylation of the KIT-V559D and downstream proteins
because low nanomolar concentrations were required to
inhibit KIT kinase activity (Fig. 3B). Decrease in phosphor-
ylation status of AKT, ERK1/2, and RPS6K were observed
already at 50 nM imatinib. As expected, KIT-D816V and
KIT-N822I-expressing Ba/F3 cells were resistant to imatinib
Western blot analysis for KIT-V559D, KIT-D816V, and
KIT-N882I–expressing Ba/F3 cells confirmed a decrease
in KIT activation with an increasing dose of dasatinib,
while total protein expression was unaffected (Fig. 3F).
Western immunoassays showed that dasatinib inhibited the
phospho-KIT already at 50 nM in KIT-V559D–expressing
Ba/F3. In KIT-D816V and KIT-N822I–expressing Ba/F3
cells, inhibition of phosphorylation of KIT was recorded at
100 nM. Downstream effectors ERK1/2, AKT, and RPS6K
also showed decreased phosphorylation with increasing
inhibitor concentrations, while total protein expression re-
mained unaffected (Fig. 3F).
Some of the previously published in vitro experiments
have shown that cells carrying p.N822K mutations or other
Figure 3. (Continued). (E) Western blot analyses of KIT-V559D, KIT-N822I, and KIT-D816V-transduced Ba/F3 cells after treatment with imatinib. Western
blot analysis showing that only KIT-V559D–expressing Ba/F3 cells were sensitive for imatinib. Bottom: P-RPS6K/RPS6K ratios were normalized to 1 for the
KIT-V559D, KIT-N822I, and KIT-D816V vehicles. Student’s t-test was used for statistical analysis. *p ! 0.05. Error bars represent SD (n 5 2). (F) Western
immunoassays showing the effect of dasatinib on Ba/F3 cells transduced with KIT-V559D, KIT-N822I, and KIT-D816V. All expressing cells were sensitive
for low nanomolar concentrations of dasatinib. Bottom: P-RPS6K/RPS6K ratios were normalized to 1 for the KIT-V559D, KIT-N822I, and KIT-D816V
vehicles. Student’s t-test was used for statistical analysis. *p ! 0.05. Error bars represent SD (n 5 2).
863 B. Wasag et al./ Experimental Hematology 2011;39:859–865
mutations in the receptor A-loop are sensitive to imatinib
. Contrary data illustrating the minimal response of
such mutations to imatinib have been presented by other
groups . In the current study, we showed that KIT-
N822I mutation is resistant to imatinib. Our in vitro obser-
vations are consistent with the findings of McDonnell et al.
. The patient with melanoma and double KIT mutation,
including p.N822I, failed to respond to imatinib.
More recently, Guo et al. reported that KIT exon Ba/F3
cells expressing KIT exon 13 or exon 17 single or double
mutants were sensitive to dasatinib inhibition . Our
studies were in line with this report and we showed that
dasatinib exhibited a high efficacy toward the KIT-N822I
mutant on Ba/F3 cells.
The results of phase II study in systemic mastocytosis
by Verstovsek et al. showed that dasatinib does not elimi-
nate the disease in systemic mastocytosis patients with
KIT-D816V mutation . However, contrary data were
presented by Ustun et al. . They presented in vitro
and in vivo efficacy of dasatinib in systemic mastocytosis
acute myeloid leukemia patient with KIT(D816V) muta-
tion. In addition, authors claimed that no significant adverse
effects of dasatinib occurred.
In the current study, functional studies revealed that
KIT-N822I mutation is oncogenic and results in a high
constitutive tyrosine phosphorylation of KIT. However,
we demonstrated that urticaria pigmentosa was the only
manifestation of mastocytosis in affected family members.
Therefore, our results support previous findings that pheno-
typic diversity among patients with mastocytosis is due to
multiple and complex molecular events.
This work was partially supported by research grants from the Life
Raft Group (M.D.-R.) and from the Fonds voor Wetenschappelijk
Onderzoek Vlaanderen (G.0589.09, M.D.-R.).
The authors would like to thank Barbara Dewaele and Jan
Cools from the Department of Human Genetics, K.U. Leuven,
Leuven, Belgium, who kindly provided KIT-V559D and KIT-
D816V–expressing Ba/F3 cells. We are indebted to Dr. Dorota
Kupnicka from the Department of Tumour Pathology, Medical
University of Lodz, Poland, for histopathological analysis of
CD2 expression in the skin biopsy.
Conflict of interest disclosure
No financial interest/relationships with financial interest relating
to the topic of this article have been declared.
B.W. designed and performed experiments, analysed the data and
wrote the report; M.N. provided clinical data and contributed to
the paper; A.P. performed and analyzed molecular data of
patients’ samples; M.L., J.R. provided patients’ samples; E.J.
revised the article for intellectual content; W.B. performed
histopathological examination; M.D.R., J.L. analyzed the data
and wrote the report.
1. Valent P, Akin C, Escribano L, et al. Standards and standardization
in mastocytosis: consensus statements on diagnostics, treatment recom-
mendations and response criteria. Eur J Clin Invest. 2007;37:435–453.
2. Garcia-Montero AC, Jara-Acevedo M, Teodosio C, et al. KIT mutation
in mast cells and other bone marrow hematopoietic cell lineages in
systemic mast cell disorders: a prospective study of the Spanish
Network on Mastocytosis (REMA) in a series of 113 patients. Blood.
3. Wilson TM, Maric I, Simakova O, et al. Clonal analysis of NRAS acti-
vating mutations in KIT-D816V systemic mastocytosis. Haematologica.
4. Beghini A, Tibiletti MG, Roversi G, et al. Germline mutation in the
juxtamembrane domain of the kit gene in a family with gastrointestinal
stromal tumors and urticaria pigmentosa. Cancer. 2001;92:657–662.
5. Hartmann K, Wardelmann E, Ma Y, et al. Novel germline mutation of
KIT associated with familial gastrointestinal stromal tumors and mas-
tocytosis. Gastroenterology. 2005;129:1042–1046.
6. Tang X, Boxer M, Drummond A, Ogston P, Hodgins M, Burden AD.
A germline mutation in KIT in familial diffuse cutaneous mastocyto-
sis. J Med Genet. 2004;41:e88.
7. Zhang LY, Smith ML, Schultheis B, et al. A novel K509I mutation of
KIT identified in familial mastocytosisdin vitro and in vivo respon-
siveness to imatinib therapy. Leuk Res. 2006;30:373–378.
8. Bodemer C, Hermine O, Palm? erini F, et al. Pediatric mastocytosis is
a clonal disease associated with D816V and other activating c-KIT
mutations. J Invest Dermatol. 2010;130:804–815.
9. Lombardo LJ, Lee FY, Chen P, et al. Discovery of N-(2-
a dual Src/Abl kinase inhibitor with potent antitumor activity in
preclinical assays. J Med Chem. 2004;47:6658–6661.
10. Dewaele B, Wasag B, Cools J, et al. Activity of dasatinib, a dual
SRC/ABL kinase inhibitor, and IPI-504, a heat shock protein
90inhibitor, against gastrointestinal
PDGFRAD842V mutation. Clin Cancer Res. 2008;14:5749–5758.
11. Shah NP, Lee FY, Luo R, Jiang Y, Donker M, Akin C. Dasatinib
(BMS-354825) inhibits KITD816V, an imatinib-resistant activating
mutation that triggers neoplastic growth in most patients with systemic
mastocytosis. Blood. 2006;108:286–291.
12. Mital A, Piskorz A, Lewandowski K, Wasa ˛g B, Limon J, Hellmann A.
A case of mast cell leukemia with exon 9 KIT mutation and good
response to imatinib. Eur J Haematol. 2011;86:531–535.
13. Debiec-Rychter M, Cools J, Dumez H, et al. Mechanisms of resistance
to imatinib mesylate in gastrointestinal stromal tumors and activity of
the PKC412 inhibitor against imatinib-resistant mutants. Gastroenter-
14. Kemmer K, Corless CL, Fletcher JA, et al. KIT mutations are common
in testicular seminomas. Am J Pathol. 2004;164:305–313.
15. Lux ML, Rubin BP, Biase TL, et al. KIT extracellular and kinase
domain mutations in gastrointestinal stromal tumors. Am J Pathol.
16. Shimada A, Taki T, Kubota C, et al. N822 mutation of KIT gene was
frequent in pediatric acute myeloid leukemia patients with t(8;21) in
Japan: a study of the Japanese childhood AML cooperative study
group. Leukemia. 2007;21:2218–2219.
17. Thalheimer A, Schlemmer M, Bueter M, et al. Familial gastrointes-
tinal stromal tumors caused by the novel KIT exon 17 germline muta-
tion N822Y. Am J Surg Pathol. 2008;32:1560–1565.
18. McDonnell K, Betz B, Fullen D, Lao CD. V559A and N822I double
KIT mutant melanoma with predictable response to imatinib? Pigment
Cell Melanoma Res. 2011;24:390–392.
864B. Wasag et al./ Experimental Hematology 2011;39:859–865
19. Carballo M, Roig I, Aguilar F, et al. Novel c-KIT germline mutation in
a family with gastrointestinal stromal tumors and cutaneous hyperpig-
mentation. Am J Med Genet A. 2005;132:361–364.
20. Yang Y, L? etard S, Borge L, et al. Pediatric mastocytosis-associated
KIT extracellular domain mutations exhibit different functional and
signaling properties compared with KIT-phosphotransferase domain
mutations. Blood. 2010;116:1114–1123.
21. Beghini A, Bellini M, Magnani I, et al. STI 571 inhibition effect on
KITAsn822Lys-mediated signal transduction cascade. Exp Hematol.
22. Guo T, Agaram NP, Wong GC, et al. Sorafenib inhibits the imatinib-
resistant KITT670I gatekeeper mutation in gastrointestinal stromal
tumor. Clin Cancer Res. 2007;13:4874–4881.
23. Verstovsek S, Tefferi A, Cortes J, et al. Phase II study of dasatinib in
Philadelphia chromosome-negative acute and chronic myeloid diseases,
including systemic mastocytosis. Clin Cancer Res. 2008;14:3906–3915.
24. Ustun C, Corless CL, Savage N, et al. Chemotherapy and dasatinib
induce long-term hematologic and molecular remission in systemic
mastocytosis with acute myeloid leukemia with KIT D816V. Leuk
865 B. Wasag et al./ Experimental Hematology 2011;39:859–865
Supplementary Figure E1. Electropherograms with the fragments of KIT exon 17 sequences of the PCR products. (A) No KIT mutation was detected in
proband’s parents and his sister. (B) The heterozygous KIT-N822I mutation was detected in the proband and his children. Alteration is indicated by the arrow.
865.e1B. Wasag et al./ Experimental Hematology 2011;39:859–865
Supplementary Download full-text
KIT-N822I-, and KIT-D816V–transduced Ba/F3 cells, incubated with
dasatinib for 48 hours in the presence of 10 ng/mL interleukin-3 (IL-3).
Proliferation of the cells became independent of the KIT pathway
and was not influenced by dasatinib. In KIT-V559D, KIT-N822I, and
KIT-D816V–transduced Ba/F3 cells, dasatinib was not able to overcome
FigureE2. Proliferationassayfor KIT-V559D-,
865.e2 B. Wasag et al./ Experimental Hematology 2011;39:859–865