Mutations in c-KIT in Canine Cutaneous Mast Cell Tumors

1
Mutations in c-KIT in Canine Cutaneous Mast Cell
Tumors J. Webster1,2, M. Kiupel1,2, J.
Kaneene1,3, R. Miller3, V. Yuzbasiyan-Gurkan1,4,5
1Comparative Medicine and Integrative Biology
Program, 2Department of Pathobiology and
Diagnostic Investigation, 3Center for Population
Medicine, 4Department of Small Animal Clinical
Sciences, 5 Department of Microbiology and
Molecular Genetics, College of Veterinary
Medicine, Michigan State University, East
Lansing, MI
ABSTRACT Spontaneous tumors in dogs provide
a rich resource for studies in comparative tumor
biology. Canine cutaneous mast cell tumors
(MCTs) are one of the most common neoplasms in
dogs, and have an extremely variable biologic
behavior. Recent work in our laboratory has
focused on understanding the und erlying
biology of canine MCTs. Our studies have
identified activating mutations in the
juxtamembrane domain of c-KIT in 15 of canine
MCTs. Furthermore, c-KIT mutations are
significantly associated with higher histologic
grade MCTs and with decreased survival as
compared with MCTs that lack c-KIT mutations.
In order to determine the association between
c-KIT mutations and cellular proliferation in
canine MCTs, immunohistochemical staining with
anti-KI-67 antibodies and AgNOR staining of
canine MCTs was performed. Canine MCTs with
c-KIT mutations were significantly associated
with an increased rate of cellular proliferation
and a significantly increased proliferation index
as compared to MCTs without c-KIT mutations. In
human mastocytosis patients c-KIT mutations
commonly occur in the exon 17 of c-KITs kinase
domain, however, no kinase domain mutations have
been described in canine MCTs. In order to
screen canine MCTs for kinase domain c-KIT
mutations exons 16-20 were amplified from 35
canine MCTs using PCR amplification with primers
that flank each exon, and each exon was
subsequently sequenced in order to identify any
mutations. No mutations were identified in exons
16-20 of canine MCTs. In conclusion,
juxtamembrane domain c-KIT mutations appear to
play a critical role in the progression of canine
MCTs, as evidenced by their association to
decreased survival intervals and increased
cellular proliferation, whereas the kinase domain
c-KIT mutations do not appear to play a critical
role in the progression of canine MCTs.
A
B
Figure 4 Exon structure of the functional
domains of c-KIT, and locations of known human
and canine c-KIT mutations. In order to screen
for kinase domain c-KIT mutations in canine MCTs,
exons 16-20 from 35 MCTs were amplified using PCR
amplification with primer pairs that flank each
exon (Fig 9). Each amplified product was
subsequently sequenced using 33P
dideoxy-terminator sequencing.
C
D
AgNOR Count/ Cell
Figure 3 Diagram of the receptor tyrosine kinase
KIT.
KIT Staining Pattern
Figure 10 Mean KI-67 count/ grid area (A and B)
and mean AgNOR count/ cell (C and D) in canine
MCTs with and without ITD c-KIT mutations (A and
C) and with KIT staining patterns 1, 2, and 3.
Canine MCTs with ITD c-KIT mutations have a
significantly increased proliferation index (p
0.002) and rate of cell cycle progression
(p0.003), as measured by KI-67 immuno-staining
and AgNOR staining, respectively, as compared to
canine MCTs without c-KIT mutations. Canine MCTs
with increased cytoplasmic KIT protein
localization had a significantly higher
proliferation index (p0.045), and a
significantly higher rate of cell cycle
progression (p0.048) as measured by KI-67
immuno-staining and AgNOR staining, respectively.
RESULTS
INTRODUCTION The c-KIT proto-oncogene
encodes the receptor tyrosine kinase KIT, which
has been shown to play important roles in the
cellular maturation, survival, proliferation, and
migration of several cell types including
melanocytes, germ cells, interstitial cells of
Cajal, and mast cells. Mutations in c-KIT and
over-expression of KIT have been associated with
several neoplastic diseases including
gastrointestinal stromal tumors, mastocytosis,
and germ cell tumors in humans, and cutaneous
mast cell tumors (MCTs) in canines. Canine
cutaneous mast cell tumors are one of the most
common neoplasms in dogs and have a variable
biologic behavior, ranging from a single solitary
mass to a potentially fatal metastatic disease.
Recently, c-KIT mutations and aberrant KIT
expression have been identified in canine MCTs.
Unlike human mastocytosis patients, in which
point mutations primarily occur in the kinase
domain of c-KIT, internal tandem duplications and
deletions have been identified in the
juxtamembrane domain of c-KIT in 15-20 of canine
MCTs. Due to the high prevalence of canine MCTs,
their variable biologic behavior, and their poor
response to current chemotherapeutic agents a
better understanding of the biology of canine
MCTs is critically needed. The goal of the
studies described below was to better understand
the role c-KIT plays in the progression of canine
cutaneous MCTs, and the potential for c-KIT and
KIT as prognostic markers and therapeutic targets
for canine cutaneous MCTs.

• SUMMARY OF RESULTS
• 57 of canine MCTs examined in this study had
  aberrant cytoplasmic KIT localization.
• 15 of canine MCTs examined in this study had ITD
  c-KIT mutations.
• Aberrant KIT localization is significantly
  associated with a decrease in survival duration
  in canine MCT patients.
• ITD c-KIT mutations are significantly associated
  with a decrease in survival duration in canine
  MCT patients.
• Kinase domain c-KIT mutations were not found in
  any canine MCTs examined.
• ITD c-KIT mutations are significantly associated
  with an increased rate of cell cycle progression
  and an increased proliferation index in canine
  MCTs.
• Aberrant KIT localization is significantly
  associated with an increased rate of cell cycle
  progression and an increased proliferation index
  in canine MCTs.

Figure 6 Immunohistochemical staining of canine
MCTs with anti-KI-67 antibodies. In order to
evaluate the proliferation index of canine MCTs,
56 MCTs were immuno-histochemical stained with
anti-KI-67 antibodies, using an alkaline
phosphatase technique in order to identify cells
in any phase of the cell cycle (pink nuclei).
The average number of proliferating cells per 10
x10mm reticle grid area at 40X magnification was
used to estimate the proliferation index. Five
microscopic fields were evaluated for each tumor.
Figure 5 AgNOR staining of canine MCTs. In
order to evaluate the rate of cellular
proliferation of canine MCTs, agyrophilic
nucleolar organizing region (AgNOR) silver
staining was performed on 56 canine MCTs. The
number of AgNOR bodies (black precipitate) per
nucleus has been shown to be directly correlated
with the rate of cell cycle progression. The
average AgNOR count/cell was determined based on
the evaluation of 100 cells per tumor in order to
estimate the rate of cellular proliferation in
canine MCTs.
MATERIALS AND METHODS

• CONCLUSIONS
• Aberrant KIT localization plays a significant
  role in the progression of canine MCTs.
• ITD c-KIT mutations play a significant role in
  the progression of canine MCTs.
• Kinase domain c-KIT mutations do not play a
  significant role in the progression of canine
  MCTs.
• c-KIT mutations and aberrant KIT localization may
  be responsible for increased cellular
  proliferation in canine MCTs by
• Increasing the growth fraction of the neoplastic
  cell population.
• Increasing the cell cycle progression of the
  neoplastic cells.

1.0 .75 .50 .25 0.0
Figure 1 Immunohistochemical staining of canine
MCTs with anti-KIT antibodies. KIT protein
expression was evaluated in 98 canine cutaneous
MCTs using immunohistochemical staining of
archival formalin-fixed paraffin-embedded tissue
sections with polyclonal anti-KIT antibodies.
Immuno-reactions were visualized with
3,3-diaminobenzidine (brown precipitate). Three
patterns of KIT protein localization were
identified. A. KIT staining pattern 1 KIT
protein is localized to the cytoplasmic membrane
only (as seen in normal mast cells). B. KIT
staining pattern 2 KIT protein is localized to
the cytoplasm, either as a focal peri-nuclear
aggregate or stippled throughout the cytoplasm.
C. KIT staining pattern 3 KIT protein is
localized diffusely throughout the cytoplasm.
Aberrant KIT localization (patterns 2 and 3) was
identified in 56/98 (57) of canine MCTs
evaluated.
10 20 30
40 Time (months)
Figure 8 Kaplan-Meier survival curve Percent
survival vs. time in months for canine MCT
patients with and without ITD c-KIT mutations.
Canine MCT patients with ITD c-KIT mutations had
a significantly decreased survival duration as
compared to patients without c-KIT mutations.
Figure 7 Kaplan-Meier survival curve Percent
survival vs. time in months for canine MCT
patients with KIT staining patterns 1, 2, and 3.
Patients with aberrant KIT localization in
neoplastic mast cells (KIT staining patterns 2
and 3) had a significantly decreased survival
duration as compared to patients that had the
normal peri-membrane KIT localization (KIT
staining pattern 1).

• REFERENCES
• Downing, et al. Am J Vet Res 631718-1723, 2002
• London CA, et al. J Comp Path 113399-414, 1996
• London CA, et al. Exp Hematol 27689-697, 1999
• Reguera MJ, et al. Am J Dermatopathol 2249-54,
  2000
• Webster JD, et al. Vet Pathol 41371-377, 2004
• Zemke D, et al. Vet Pathol 39529-535, 2002

Figure 9 Diagram of intron-exon structure of the
phospho-transferase region of the kinase domain
of c-KIT, and primer design (blue arrows) for
amplification and sequencing. No mutations were
identified in c-KIT exons 16-20 of canine MCTs.

• ACKNOWLEDGEMENTS
• Joshua Webster is funded by the Department of
  Pathobiology and Diagnostic Investigations NIH
  T-32 post-doctoral training grant number RR17189.
• Funding for this study was provided in part by
  the Companion Animal Fund of the College of
  Veterinary Medicine, Michigan State University.

Figure 2 PCR detection of internal tandem
duplication (ITD) c-KIT mutations. Sixty canine
cutaneous MCTs were evaluated for the presence of
ITD c-KIT mutations using PCR amplification.
Mutations were identified on a 2 agarose gel of
amplified c-KIT exon 11 and intron 11 products
from DNA extracted from canine MCTs. White
arrow 191bp normal allele. Blue arrow
Approximately 250bp mutant allele. Red arrow
Heterodimerization of normal and mutant allele.
L 100bp ladder M heterozygous mutant N
homozygous normal NC negative control.
Mutations were identified in 9/60 (15) canine
MCTs examined.