article

1. An Immunohistochemical Analysis of Canine
Haemangioma and Haemangiosarcoma
S. Sabattini and G. Bettini
Department of Veterinary Public Health and Animal Pathology, Faculty of Veterinary Medicine, Alma Mater Studiorum,
University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy
Summary
The aim of the present study was to investigate immunohistochemically aspects of the biology of canine endo-
thelial neoplasia. Forty samples of canine cutaneous and visceral haemangiosarcoma (HSA), 29 samples of
cutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelled
with antisera specific for vimentin, smooth muscle actin, von Willebrand factor (vWF), CD117 (KIT), vascu-
lar endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGFC) and
CD44. Further antisera were employed to determine the level of cellular proliferation (MIB-1 index) and to-
luidine blue staining was used to detect populations of tumour-infiltrating mast cells (MCs). There was greater
expression of CD117, VEGFR-3 and CD44 in HSA than in HA, suggesting that these proteins might be suit-
able targets for the future development of novel therapeutic approaches to canine HSA. Marked infiltration of
MC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neo-
plasia in the dog.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: dog; haemangioma; haemangiosarcoma; immunohistochemistry; mast cells
Introduction
Haemangioma (HA) and haemangiosarcoma (HSA)
are benign and malignant neoplasms of vascular en-
dothelial cells (EC) that are common in the dog.
HA generally arises in the skin, with a particular pre-
dilection for dogs with short hair coats and lightly pig-
mented skin (Hargis et al., 1992). HSA most often
arises in the spleen, right atrium or skin (Pearson
and Head, 1976). Canine HSA is locally infiltrative
and readily metastasizes, particularly to the lung
and liver (Oksanen, 1978; Brown et al., 1985). Most
affected dogs die from acute internal haemorrhage
secondary to rupture of the tumour. Despite surgical
and chemotherapeutic management, the median sur-
vival time for dogs diagnosed with HSA is little more
than 6 months (Hammer et al., 1991; Clifford et al.,
2000; Sorenmo et al., 2000).
Canine HSA resembles human angiosarcoma, a tu-
mour that also carries an unfavourable prognosis
(Timaran et al., 2000; Budd, 2002). For both tumours
there have been relatively few studies of the cellular
and molecular features, which means that there are
few markers that can be applied to the detection of
early lesions or identification of potential targets for
the development of novel immunotherapeutic ap-
proaches (Hoover et al., 1993; Masuzawa et al.,
1999; Krump-Konvalinkova et al., 2003). Previous
studies have described the expression of von Wille-
brand factor (vWF) (von Beust et al., 1988) and
CD31 (Ferrer et al., 1995) by canine HSA. More re-
cently, the expression of CD117 (Fosmire et al.,
2004), cyclooxygenase-2 (Heller et al., 2005), vascular
endothelial growth factor-A and its receptors, basic fi-
broblastic growth factor and its receptor (Yonemaru
et al., 2006), bcl-2 and survivin (Murakami et al.,
2008) has also been investigated. The aim of the pres-
ent study was to examine the expression of an ex-
panded panel of immunohistochemical markers in
canine HA and HSA in order to further define the bi-
ological characteristics of these tumours.
Materials and Methods
Cases of HA (n ¼ 29) and HSA (n ¼ 40) were selected
from the archives of the Pathology Unit of theCorrespondence to: G. Bettini (e-mail: giuliano.bettini@unibo.it).
0021-9975/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcpa.2008.10.006
J. Comp. Path. 2009, Vol. 140, 158e168 Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa

2. Department of Veterinary Public Health and Animal
Pathology, Faculty of Veterinary Medicine, Univer-
sity of Bologna, Italy. These samples had been sub-
mitted over a 10 year period (1998e2008).
Additionally, 10 samples of cutaneous granulation tis-
sue (GT) were selected for comparative analysis.
Samples had been fixed in 10% neutral buffered for-
malin, embedded in paraffin wax, sectioned (4 mm)
and stained with haematoxylin and eosin (HE).
HSAs (29 visceral and 11 cutaneous) were graded
for overall differentiation and nuclear pleomorphism
as described in Table 1, whereas HAs (4 visceral
and 25 cutaneous) were classified by dominant histo-
logical pattern as cavernous, capillary or mixed. The
level of angiogenesis was evaluated in the samples of
GT and scored as mild, moderate or marked accord-
ing to the number and thickness of the wall of newly
formed blood vessels and the immaturity of the lining
endothelial cells.
A tissue array technique was employed in order to
limit the duration of the experiment, reduce reagent
waste and minimize tissue damage. The most repre-
sentative areas in each section were defined by study
of the HE-stained sections, and then 3 mm tissue cores
were punched from the associated tissue blocks. These
cores were transferred to a recipient block with pre-
formed holes to form a composite block containing
up to 20 samples in the same section (Nocito et al.,
2001; Hidalgo et al., 2003). Replicate sections
(4 mm) of tissue arrays were stained with HE and to-
luidine blue and processed for immunohistochemistry
(IHC). The panel of antibodies used in IHC is de-
scribed in Table 2.
IHC was performed with a streptavidin-biotin-per-
oxidase technique. Slides were initially incubated
with hydrogen peroxide 0.3% in methanol for
20 min to block endogenous peroxidase activity and
were then microwaved in citrate buffer (pH 6.0), for
two cycles of 5 min, in a 750 Watt microwave oven
for antigen retrieval (for all antibodies except that
specific for VEGFC). The sections were incubated
overnight at 4
C in a humid chamber with the pri-
mary antibody diluted in phosphate-buffered saline
(PBS; pH 7.4, 0.01 M). Following washing in PBS,
sections were then incubated with secondary biotiny-
lated anti-rabbit IgG, anti-mouse IgG and anti-goat
IgG (LSAB, Dako Cytomation, Glostrup, Denmark)
for 30 min at room temperature, and then subse-
quently with streptavidin-peroxidase complex for
25 min at room temperature. After incubation in
DAB chromogenic substrate solution (diaminobenzi-
dine 0.02% and H2O2 0.001% in PBS) for 12 min,
sections were immediately rinsed in PBS and in run-
ning tap water, counterstained with haematoxylin,
dehydrated and mounted under DPX (Fluka, Rie-
del-de Hae¨ n, Germany). Appropriate positive con-
trols were used throughout to assess the specificity of
the reactions. As negative control, an isotype-
matched antibody of irrelevant specificity (Neo-
Markers, Fremont, CA) was used in place of the
primary antibody.
The intensity of immunolabelling for each marker
was graded as absent (0), weak (1), moderate (2) or
strong (3). Cellular proliferation was assessed using
the MIB-1 antibody, which recognizes the Ki67 anti-
gen, a nuclear protein expressed in all of the active
phases of the cell cycle (G1, S, G2, M), but not in rest-
ing cells (G0). The labelling index (MIB-1 index) was
subjectively estimated as the percentage of positively
labelled nuclei and scored as 0 (less than 5% of nuclei
labelled), 1 (5e15% of nuclei labelled), 2 (15e30%
of nuclei labelled) or 3 (more than 30% of nuclei la-
belled). The number of tumour-infiltrating mast cells
(MCs) was graded in toluidine blue-stained sections
as 0 (no mast cells), 1 (occasional mast cells), 2 (mod-
erate number of scattered MCs or clusters of at least
three cells) or 3 (MCs present in a high number). Cor-
relations between considered variables were evalu-
ated by Spearman’s rank analysis and Kruskale
Wallis nonparametric analysis of variance.
Results
Haemangiosarcomas
The mean age of dogs with HSA was 9.3 years
(n ¼ 40, range 3e15 years). Breed was recorded in
37 cases and there were 12 German shepherd dogs.
The ratio of male to female dogs was 2.1:1 with
67.6% of dogs with HSA being male. It was not pos-
sible to assess accurately the proportion of animals
that had been neutered.
Table 1
Histological scoring system for canine HSA
Score Overall
differentiation
Nuclear variation
1 Well-differentiated tumour
with numerous irregular
vascular channels
Minimal variation in nuclear
size and shape
2 Moderately differentiated
neoplasm with at least
50% of the tumour
showing well-defined
vascular channels
Moderate variation in
nuclear size and shape
3 Poorly differentiated,
solid tumour with few
vascular channels
Marked variation in nuclear
size and shape. Nuclear
size often differs by
twofold or more between
tumour cells
Immunohistochemistry of Canine Vascular Tumours 159

3. Of the 29 visceral tumours, the primary site of ori-
gin could not be determined in 7 cases due to the pres-
ence of multicentric lesions. The remaining 22 cases
had primary origin in the spleen (n ¼ 16), right
atrium (n ¼ 3), liver (n ¼ 1), omentum (n ¼ 1), retro-
peritoneal area and the urinary bladder (n ¼ 1).
Microscopically, the HSA cells had highly heteroge-
neous morphology, ranging from spindle-shaped to po-
lygonal to ovoid, with vasoformative to solid growth
patterns. Eleven of the 40 HSAs (27.5%) were catego-
rized as well-differentiated, with numerous, irregular
vascular channels and large areas of haemorrhage with
few cells, mimicking haematomas (Fig. 1D). Twenty-
four HSAs (60%) were characterized by areas of moder-
ate differentiation, where neoplastic tissue was more
compact but still showing, at least focally, a vasoforma-
tive component. Five samples (12.5%) were poorly dif-
ferentiated and were difficult to distinguish from
fibrosarcoma or other poorly differentiated sarcoma.
These tumours comprised solid sheets of cells with few
vascular channels. Nuclear variation was minimal in
12.5% (n ¼ 5) of specimens, moderate in 40%
(n ¼ 16) and marked in 47.5% (n ¼ 19). There was no
relationship between these different morphological pat-
terns and the anatomical location of the tumour.
The immunohistochemical expression of the panel
of markers by the HSAs is summarized in Table 3.
All HSA tumour cells exhibited strong and diffuse cy-
toplasmic expression of vimentin and actin, whereas
there was more variable expression of vWF, this rang-
ing from weak and inconsistent (n ¼ 15, 60%) to
strong (n ¼ 3, 12%). CD117 expression was detected
in the cytoplasm of 76.3% of HSAs, and the intensity
of expression ranged from low (n ¼ 16, 42.1%), to
moderate (n ¼ 9, 23.7%) to strong (n ¼ 4, 10.5%)
(Fig. 2C, D). Only 7 cases of HSA displayed cytoplas-
mic expression of VEGFC, while 36% (n ¼ 14) of
samples showed mild to intense cytoplasmic expres-
sion of VEGFR-3. Fifteen tumours had cytoplasmic
expression of CD44 with moderate (34.6%) to strong
(23.1%) intensity. Between 5 and 40% of nuclei ex-
pressed MIB-1 and the mean MIB-1 index was
16.7%.
Ten of the HSAs had a low to moderate number of
infiltrating MCs and in one tumour there were nu-
merous MCs. No MCs were detected in the remainder
of the samples (72.5%).
Haemangiomas
The mean age of dogs with HA was 9.3 years (range
4e15 years). There were almost equal numbers of
males and females and the German shepherd dog
was the most represented breed (n ¼ 8, 27.6%).
Twenty-four HAs arose within the skin and five
were splenic in origin.
Microscopically, most of the samples were of the
cavernous subtype, showing large, regular and well
defined vascular spaces filled with erythrocytes, com-
pletely enclosed by a single layer of endothelial cells
aligned on thin collagenous septa (Fig. 1B). Five spec-
imens (17.2%) were mixed capillary-cavernous in
which, focally, the vascular structures were smaller
and lined by slightly plump endothelial cells protrud-
ing into vessel lumens (Fig. 1C). Cellularity and nu-
clear pleomorphism in these areas were usually
higher than in cavernous HA.
The immunohistochemical expression of the panel
of markers by HAs is summarized in Table 3. Neo-
plastic cells were strongly labelled by antibodies spe-
cific for vimentin and actin in all samples. Low to
moderate expression of vWF and VEGFR-3 was con-
sistently detected, whilst CD44, CD117 and VEGFC
were not expressed in the majority of samples
(Fig. 2B). Conversely, there was strong cytoplasmic
labelling for CD117 and VEGFC in the areas of cap-
illary differentiation of two mixed HAs. MIB-1 ex-
pression was detected in only occasional endothelial
nuclei and the MIB-1 index was less than 5% in all
cases.
Infiltrating MCs were seen in the stromal compart-
ment of all HAs with the single exception of one
splenic HA in which no MCs were detected. The
Table 2
Primary antibodies used in IHC
Marker Type of antibody Source Dilution
Vimentin Mouse monoclonal Dako, Glostrup, Denmark 1 in 100
Actin Mouse monoclonal Dako 1 in 100
von Willebrand factor Rabbit polyclonal Dako 1 in 1500
CD117 Rabbit polyclonal Dako 1 in 100
VEGFR-3 Rabbit polyclonal Alpha Diagnostic International, San Antonio, USA 1 in 350
VEGFC Rabbit polyclonal Zymed laboratories Inc., San Francisco, USA 1 in 40
CD44 Mouse monoclonal Bender MedSystems, Vienna, Austria 1 in 12.5
MIB-1 Mouse monoclonal Dako 1 in 30
VEGFR-3, vascular endothelial growth factor receptor-3; VEGFC, vascular endothelial growth factor-C.
160 S. Sabattini and G. Bettini

4. number of MCs ranged from low (n ¼ 8, 33.3%) to
moderate (n ¼ 9, 37.5%) to high (n ¼ 6, 25%)
(Fig. 3AeF).
Granulation Tissue
Five of ten samples of GT had evidence of fibroplasia
and intense angiogenesis; three had fibrosis and mild
angiogenesis, one had non-recent angiogenesis and
one had low angiogenic activity (Fig. 1A). The immu-
nohistochemical expression of the panel of markers by
GT is summarized in Table 3. In all cases endothelial
cells expressed vimentin, actin and vWF but did not
label for VEGFC. Reactive endothelial cells had
mild expression of CD44 and VEGFR-3 and low,
yet consistent, expression of CD117 (Fig. 2A). Con-
versely, in the sample with a low level of angiogenesis,
the ECs did not express CD44, VEGFR-3 or CD117.
Five to 40% of nuclei were MIB-1-positive with
a mean MIB-1 index of 11.4%. A low to moderate
number of infiltrating MCs was detected in 50% of
samples.
Statistical Analysis
In HSA there was significant correlation (Spearman’s
method) between overall differentiation and the de-
gree of nuclear variation (P ¼ 0.0042), but there
was no significant correlation between MIB-1 index
and nuclear variation, between MIB-1 index and
overall differentiation, or between MIB-1 index and
CD117 expression.
There was significantly greater expression of
CD117 by HSA (average score 1.2) than HA (average
score 0.1) (P ¼ 0.0000) and GT (average score 0.9)
(P ¼ 0.037). The same trend was noted for CD44,
with a significantly greater expression in HSA (aver-
age score 1.7) than in HA (average score 0.1)
Fig. 1. Microscopical features of canine vascular lesions. (A) Granulation tissue showing fibroplasia and endothelial cells of an immature
phenotype engaged in angiogenesis. HE. Bar, 50 mm. (B) Cavernous haemangioma showing large, uniform blood-filled vascular
spaces lined by a single layer of endothelial cells aligned on thin collagenous septa. HE. Bar, 100 mm. (C) Haemangioma with cap-
illary differentiation showing narrow and irregular vascular structures lined by slightly plump endothelial cells with minimal stro-
mal interposition. HE. Bar, 50 mm. (D) Well-differentiated haemangiosarcoma showing plump endothelial cells aligned on delicate
collagen trabeculae creating an anastomosing meshwork of blood-filled channels of varying size. HE. Bar, 50 mm.
Immunohistochemistry of Canine Vascular Tumours 161

5. Table 3
Summary of immunohistochemical labelling of canine vascular tumours and granulation tissue
Marker Hemangiosarcoma Mean score Hemangioma Mean score Granulation tissue Mean score
Number positive
(% total positive)
Number positive
(% total positive)
Number positive
(% total positive)
0 1 2 3 0 1 2 3 0 1 2 3
Vimentin 0/39 (0%) 0/39 (0%) 17/39
(43.6%)
22/39
(56.4%)
2.5 0/29 (0%) 0/29 (0%) 16/29
(55.2%)
13/29
(44.8%)
2.4 0/10 (0%) 0/10 (0%) 3/10
(30%)
7/10
(70%)
2.7
Actin 1/37
(2.7%)
11/37
(29.7%)
11/37
(29.7%)
14/37
(37.8%)
2.6 0/29 (0%) 4/29
(13.8%)
18/29
(62.1%)
7/29
(24.1%)
2.1 0/10 (0%) 1/10
(10%)
2/10
(20%)
7/10
(70%)
2.6
vWF 3/25
(12%)
15/25
(60%)
4/25
(16%)
3/25
(12%)
1.3 22/25
(88%)
3/25
(12%)
0/25 (0%) 0/25 (0%) 1.1 0/10 (0%) 2/10
(20%)
5/10
(50%)
3/10
(30%)
2.1
CD117 9/38
(23.7%)
16/38
(42.1%)
9/38
(23.7%)
4/38
(10.5%)
1.2 27/29
(92.1%)
2/29
(6.9%)
0/29 (0%) 0/29 (0%) 0.1 2/10
(20%)
7/10
(70%)
1/10
(10%)
0/10 (0%) 0.9
VEGFR-3 10/39
(25.6%)
15/39
(38.5%)
12/39
(30.8%)
2/39
(5.1%)
1.6 2/29
(6.9%)
27/29
(93.1%)
0/29 (0%) 0/29 (0%) 0.9 1/10
(10%)
4/10
(40%)
5/10
(50%)
0/10 (0%) 1.4
VEGFC 33/40
(82.5%)
4/40
(10%)
2/40 (5%) 1/40
(2.5%)
0.3 27/29
(93.1%)
0/29 (0%) 0/29 (0%) 2/29
(6.9%)
0.2 10/10
(100%)
0/10 (0%) 0/10 (0%) 0/10 (0%) 0
CD44 4/26
(15.4%)
7/26
(26.9%)
9/26
(34.6%)
6/26
(23.1%)
1.7 25/28
(89.3%)
3/28
(10.7%)
0/28 (0%) 0/28 (0%) 0.1 1/10
(10%)
5/10
(50%)
3/10
(30%)
1/10
(10%)
1.4
MIB-1 12/40
(30%)
9/40
(22.5%)
13/40
(32.5%)
6/40
(15%)
16.7% 29/29
(100%)
0/29 (0%) 0/29 (0%) 0/29 (0%) 5% 3/10
(30%)
5/10
(50%)
2/10
(20%)
0/10 (0%) 11.4%
Mast cells 29/40
(72.5%)
5/40
(12.5%)
5/40
(12.5%)
1/40
(2.5%)
0.4 1/24
(4.2%)
8/24
(33.3%)
9/24
(37.5%)
6/24
(25%)
1.9 5/10
(50%)
3/10
(30%)
2/10
(20%)
0/10 (0%) 0.7
vWF, von Willebrand Factor; VEGFR-3, vascular endothelial growth factor receptor-3; VEGFC, vascular endothelial growth factor-C; MIB-1 mean score is the MIB-1 index expressed as a percentage.
For each lesion type (haemangiosarcoma, haemangioma, granulation tissue) the number of samples (and % of total) expressing a particular marker with intensity 0e3 is given. MIB-1 expression and
the number of mast cells are also assigned to a four point scale.
162S.SabattiniandG.Bettini

6. (P ¼ 0.0000), although there was no significant differ-
ence between HSA and granulation tissue (average
score 1.4).
Cellular proliferation was significantly lower
(P ¼ 0.0000) in HA (mean MIB-1 index 5%)
than in HSA (mean MIB-1 index 16.7%), whilst
the difference between HSA and GT (mean MIB-1
index 11.4%) was not significant (P ¼ 0.3).
MCs were more prominent in HA (average score 1.9)
than in HSA (average score 0.4) (P ¼ 0.0001), but the
number of mast cells was not related to the anatomical
location of the tumour (i.e. cutaneous or visceral).
Discussion
The distribution of age, breed and sex in this population
of 69 dogs with vascular tumours is consistent with that
described previously (Prymak et al., 1988; Srebernik
and Appleby, 1991). Dogs with both HA and HSA
hadameanageofover9yearsandtheGermanshepherd
dog was the breed most represented for both tumour
types.Althoughnocleargenderpredilectionhasbeenre-
portedforcanineHSA,inourstudy,asinothers(Sreber-
nik and Appleby, 1991; Bettini et al., 2001), males were
overrepresented. Nevertheless, these findings should be
interpreted cautiously due to the relatively small size of
the population examined.
There is continued controversy as to whether
multi-organ involvement in canine HSA represents
true multicentric origin or reflects the development
of one primary tumour with metastasis (Waters
et al., 1988; Ward et al., 1994; Goldschmidt and Hen-
drick, 2002). In 26.9% of the cases in the present se-
ries, two or more sites were involved in the same
animal and, based on knowledge of the common
metastatic patterns in sarcomas, neither would be
considered as a likely metastatic site. However, we
found no evidence that this subset of cases had dis-
tinct morphological or immunohistochemical
features.
Fig. 2. CD117 expression in canine vascular lesions. (A) Immature ECs in active GT showing consistent cytoplasmic labelling. (B) CD117-
negative HA with infiltrating MCs that exhibit strong labelling. (C) HSA cells with moderate expression of CD117. (D) Subcuta-
neous HSA with strong cytoplasmic immunoreactivity for CD117. IHC. Bars, 100 mm.
Immunohistochemistry of Canine Vascular Tumours 163

7. Unlike for other canine tumour types, a histological
grading system has not been widely applied to HSA.
In the present study, overall differentiation and nu-
clear variation were scored for each sample in order
to define the correlation between morphological fea-
tures and proliferative activity or immunophenotypic
characteristics. Other parameters tested in previous
studies were not estimated (Ogilvie et al., 1996). These
have included parameters such as number of mitoses
and amount of necrosis; however, we believe that pro-
liferative activity is more accurately assessed by eval-
uating the expression of the nuclear antigen Ki67, and
Fig. 3. Cutaneous haemangiomas showing interstitial infiltration of mast cells at low (A and B), moderate (C and D) and high (E and F)
number. Toluidine blue. Bars in A, C and E 200 mm, Bars in B, D and F 50 mm.
164 S. Sabattini and G. Bettini

8. the presence of necrosis is entirely dependent on the
sampling site. A positive correlation was found be-
tween differentiation and nuclear variation, whilst
there was no correlation between these parameters
and MIB-1 index or the expression of immunohisto-
chemical markers, with the exception of lower vWF
expression and higher VEGFC expression in poorly
differentiated HSA.
Previous immunohistochemical studies of canine
HSA have been restricted to the use of endothelial
cell-specific markers such as vWF and CD31 to distin-
guish poorly differentiated examples from other mes-
enchymal tumours. In the present study we applied
an extended panel of antibodies in order to test their
diagnostic utility and contribute to the understanding
of the biological characteristics of these tumours.
Vimentin is an intermediate filament protein that is
part of the cytoskeleton of mesenchymal cells and was
widely expressed in the cytoplasm of both neoplastic
and non-neoplastic ECs. Immunohistochemical la-
belling for vimentin could therefore be of value in
the diagnosis of atypical variants of these tumours
such as epithelioid HSA.
The strong expression of smooth muscle actin by the
endothelial cells from all of the vascular lesions studied
here reflects the contractile ability of these cells. There
was, however, no significant difference in expression be-
tween GT, HA and HSA, suggesting that actin expres-
sion holds no prognostic value for this group of lesions.
Similar to earlier observations (Ferrer et al., 1995),
all HA and GT samples were consistently positive for
the vascular marker vWF, whilst labelling was often
focal and weak in malignant ECs. This pattern of ex-
pression limits the utility of this antibody in the diag-
nosis of HSA, especially where the tumours are poorly
differentiated.
C-kit proto-oncogene product (KIT, CD117)is a ty-
rosine kinase growth factor receptor for stem cell fac-
tor (SCF, mast cell growth factor) involved in the
development and maintenance of haematopoietic
stem cells, mast cells, germ cells, melanocytes and in-
terstitial cells of Cajal. Mutations in the tyrosine ki-
nase or juxtamembrane domains of the c-kit gene
have been detected in mastocytoma, seminoma and
gastrointestinal stromal tumours (Miettinen et al.,
2000). Greater than half of human angiosarcomas ex-
press CD117, but KIT was not detected in the major-
ity of benign vascular tumours. KIT-immunoreactive
HSAs do not have mutations of c-kit, so such positivity
is more likely related to an immature phenotype of the
neoplastic cells (Miettinen et al., 2000). CD117 ex-
pression has also been reported in 7 canine splenic
HSAs (Fosmire et al., 2004).
These findings are consistent with those of the pres-
ent study in which there was low to intense cytoplas-
mic expression of CD117 in most HSAs. In contrast,
the benign vascular tumours were consistently
CD117-negative, except for two HAs with mixed cap-
illary-cavernous pattern. These observations may be
taken to support the theory whereby canine HSA
and human angiosarcoma originate from primitive,
poorly differentiated endothelial cells identified by
the expression of CD117 and other surface proteins re-
stricted to bone marrow precursor cells (EPC),
whereas HA would originate from mature, well differ-
entiated endothelial cells. This may provide a theoret-
ical explanation for multicentric involvement and
represents not only a major diagnostic advancement,
but also an appealing target for the development of
novel therapy based on kinase-inhibitors (Fosmire
et al., 2004; Lamerato-Kozicki et al., 2006). CD117
was also expressed, albeit weakly, by endothelial cells
from all sections of granulation tissue, except for the
sample with a low level of angiogenesis. This finding
is consistent with the immature phenotype of angio-
blasts, and precludes the use of KIT as a marker to
distinguish HSA from GT.
The VEGFR family comprises a group of three ty-
rosine kinase receptors for vascular endothelial
growth factors. VEGFR-3 (the receptor for VEGFC
and VEGFD) has been found in most endothelia dur-
ing embryogenesis, whilst later in development ex-
pression of this molecule is restricted to lymphatic
endothelium in most tissues. Recently, VEGFR-3 ex-
pression has been shown to be up-regulated in the en-
dothelial cells of areas of tumour neovascularization,
and this molecule is expressed by human vascular
neoplasms (Lymboussaki et al., 1999; Partanen et al.,
1999; Neuhauser et al., 2000; Laakkonen et al., 2007;
Petrova et al., 2008). This pattern of expression sug-
gests that antagonists or inhibitors of this protein
may be promising candidates for use in molecular-tar-
geted therapies.
In the present study, the majority of canineHSAs ex-
pressed VEGFR-3, suggesting that this marker would
not be suitable for the immunohistochemical discrimi-
nation between lymphangiosarcoma and HSA (Neu-
hauser et al., 2000). In contrast, expression of the
VEGFR-3 ligand (VEGFC) was limited to foci within
two HAs with capillary differentiation and seven
poorly differentiated HSAs. The greater expression of
VEGFR-3 in HSA compared with HA suggests that
HSA may be more influenced by VEGFC. As VEGFC
was not over-expressed in HSA, other growth promo-
tion mechanisms rather than autocrine may be impor-
tant in this tumour. No correlation was found between
VEGFR-3 expression and cell proliferation activity.
CD44 is a ubiquitous multi-structural and multi-
functional cell surface adhesion molecule involved in
cell-to-cell and cell-to-matrix interactions, cell traffic,
Immunohistochemistry of Canine Vascular Tumours 165

9. lymph node homing, transmission of growth signals
and signals mediating haematopoiesis and apoptosis.
Hyaluronic acid, an important component of the ex-
tracellular matrix (ECM), is the principal ligand for
CD44; others include collagen, fibronectin, laminin
and chondroitin sulphate (Naor et al., 1997; Hidalgo
et al., 2002). Many malignant cell types express high
levels of CD44 and it has been shown in animal models
that injection of reagents interfering with CD44-li-
gand interaction can inhibit local tumour growth
and metastatic spread (Kajita et al., 2001). These ob-
servations suggest that CD44 may confer a growth ad-
vantage on some neoplastic cells and, therefore, that
this molecule may be used as a target for cancer ther-
apy (Naor et al., 1997). The results of the present study
are consistent with these concepts as all HAs failed to
express this marker, whilst more than half of HSAs
were immunoreactive. CD44 was also expressed by
the proliferating ECs of GT, confirming their molecu-
lar similarity with malignant ECs.
Proliferative activity, as determined by the MIB-1 in-
dex, was markedly variable in HSA and not correlated
to differentiation and nuclear pleomorphism, suggesting
thattumourswithsimilarmorphologymayhaveadiffer-
entkineticbehaviour. TheMIB-1indexofHSAsdidnot
differ significantly from that of granulation tissue,
whereas almost no cycling cells were identified in HAs.
During the course of these immunohistochemical
studies, we often identified isolated VEGFC,
VEGFR-3 and CD117-positive cells in the interstitial
stroma of HAs. Morphologically, these were oblong to
round cells with round nuclei that were consistent
with either angioblasts or infiltrating mast cells. In or-
der to further identify these cells, toluidine blue stain-
ing was performed on replicate sections of the tissues
examined. This staining confirmed that these were
mast cells and that these were numerous in the major-
ity of HAs but less commonly found in GT and HSA.
There is evidence that several mast cell mediators
may have angiogenic activity by regulating EC prolif-
eration (e.g. vascular endothelial growth factor, fibro-
blast growth factor, histamine, heparin, interleukin-8,
tumour necrosis factor-a, platelet-derived growth fac-
tor, and hepatocyte growth factor), inducing vasodi-
latation, increasing vascular permeability and
degrading the extracellular matrix (e.g. chymase,
tryptase, matrix metalloproteinases, urokinase, inter-
leukins-3, -4 and -8). It has also been postulated that
MCs may play both pro-angiogenic and anti-angio-
genic roles in the proliferative and involuting phases
of infantile haemangioma (Marks et al., 1986; Yama-
moto et al., 2000; Sun et al., 2007).
Mast cells and their precursors are known to ex-
press the KIT receptor. The ligand for this receptor
is SCF, which acts to promote the proliferation, differ-
entiation, migration and secretory activity of these
cells. Endothelial cells in murine are known to secrete
SCF, which may act to recruit MCs into the neoplas-
tic microenvironment (Meininger et al., 1995). Yama-
moto et al. (2000) detected a significantly increased
number of MCs in the lesions of human angiosarcoma
compared with normal skin, additionally demonstrat-
ing the presence of SCF-positive cells in these neoplas-
tic tissues.
The above findings may be interpreted to suggest
that tumour cell-derived SCF might recruit infiltrat-
ing MCs, which may further contribute to the prolif-
eration and progression of tumour cells (Yamamoto
et al., 2000). The fact that fewer MCs were detected
in canine HSA compared with HA is at odds with
this hypothesis, but could be explained if there were
alternative proliferative mechanisms of an autocrine
nature that meant that canine HSA cells were inde-
pendent of external stimuli (Yonemaru et al., 2006).
In conclusion, the present study has shown that the
panel of markers employed here is able to distinguish be-
nign from malignant vascular tumours, but not discrim-
inate between neoplastic ECs and normal ECs
proliferating in GT. This finding is consistent with the
ontogenetic hypothesis that states that HSA originates
from incompletely differentiated, bone marrow-derived
stemcellsthatarenearoratthestageofendothelialcom-
mitment (haemangioblasts) (Schatteman and Awad,
2004). The significantly greater expression of CD117,
VEGFR-3 and CD44 by HSAs suggests that these mol-
ecules might be further explored as potential targets for
molecular interventional therapy for this tumour. Fi-
nally, the high number of mastcells infiltrating HAs sug-
geststhatthesecellsmighthavearoleinthepathogenesis
of benign vascular tumours.
References
Bettini, G., Mandrioli, L., Brunetti, B. and Marcato, P. S.
(2001). Canine splenic pathology: a retrospective study
of 109 surgical samples, with special emphasis on fibro-
histiocytic nodules. European Journal of Veterinary Patho-
logy, 7, 101e109.
von Beust, B. R., Suter, M. M. and Summers, B. A. (1988).
Factor VIII-related antigen in canine endothelial neo-
plasms: an immunohistochemical study. Veterinary Pa-
thology, 25, 251e255.
Brown, N. O., Patnaik, A. K. and MacEwen, E. G. (1985).
Canine hemangiosarcoma: retrospective analysis of 104
cases. Journal of the American Veterinary Medical Association,
186, 56e58.
Budd, G. T. (2002). Management of angiosarcoma. Current
Oncology Report, 4, 515e519.
Clifford, C. A., Mackin, A. J. and Henry, C. J. (2000).
Treatment of canine hemangiosarcoma: 2000 and be-
yond. Journal of Veterinary Internal Medicine, 14, 479e485.
166 S. Sabattini and G. Bettini

10. Ferrer, L., Fondevila, D., Rabanal, R. M. and
Vilafranca, M. (1995). Immunohistochemical assess-
ment of CD31 antigen in normal and neoplastic canine
endothelial cells. Journal of Comparative Pathology, 112,
319e326.
Fosmire, S. P., Dickerson, E. B., Scott, A. M., Bianco, S. R.,
Pettengill, M. J., Meylemans, H., Padilla, M., Frazer-
Abel, A. A., Akhtar, N., Getzy, D. M. et al. (2004).
Canine malignant hemangiosarcoma as a model of
primitive angiogenic endothelium. Laboratory Investiga-
tion, 84, 562e572.
Goldschmidt, M. H. and Hendrick, M. J. (2002). Tumors
of the skin and soft tissues. In: Tumors in Domestic Animals,
4th Edit., D. J. Meuten, Ed., Iowa State Press, Ames,
pp. 99e101.
Hammer, A. S., Couto, C. G., Filppi, J., Jetzy, D. and
Shank, K. (1991). Efficacy and toxicity of VAC chemo-
therapy (vincristine, doxorubicin, and cyclophospha-
mide) in dogs with hemangiosarcoma. Journal of
Veterinary Internal Medicine, 5, 160e166.
Hargis, A. M., Ihrke, P. J., Spangler, W. L. and
Stannard, A. A. (1992). A retrospective clinicopatho-
logic study of 212 dogs with cutaneous hemangiomas
and hemangiosarcomas. Veterinary Pathology, 29,
316e328.
Heller, D. A., Clifford, C. A., Goldschmidt, M. H.,
Holt, D. E., Manfredi, M. J. and Sorenmo, K. U.
(2005). Assessment of cyclooxygenase-2 expression in ca-
nine hemangiosarcoma, histiocytic sarcoma, and mast
cell tumor. Veterinary Pathology, 42, 350e353.
Hidalgo, A., Robledo, M. M. and Teixido, J. (2002).
CD44-mediated hematopoietic progenitor cell adhesion
and its complex role in myelopoiesis. Journal of Hemato-
therapy and Stem Cell Research, 11, 539e547.
Hidalgo, A., Pi~na, P., Guerrero, G., Lazos, M. and
Salcedo, M. (2003). A simple method for the construc-
tion of small format tissue arrays. Journal of Clinical Pa-
thology, 56, 144e146.
Hoover, M. L., Vetvicka, V., Hoffpauir, J. M. and
Tamburro, C. H. (1993). Human endothelial cell line
from an angiosarcoma. In Vitro Cellular Developmental
Biology, 29A(3 Pt 1), 199e202.
Kajita, M., Itoh, Y., Chiba, T., Mori, H., Okada, A.,
Kinoh, H. and Seiki, M. (2001). Membrane-type 1 ma-
trix metalloproteinase cleaves CD44 and promotes cell
migration. Journal of Cell Biology, 153, 893e904.
Krump-Konvalinkova, V., Kleideiter, E., Friedrich, U.,
Klotz, U. and Kirkpatrick, C. J. (2003). Tumorigenic
conversion of endothelial cells. Experimental and Molecular
Pathology, 75, 154e159.
Laakkonen, P., Waltari, M., Holopainen, T.,
Takahashi, T., Pytowski, B., Steiner, P., Hicklin, D.,
Persaud, K., Tonra, J. R., Witte, L. and Alitalo, K.
(2007). Vascular endothelial growth factor receptor 3
(VEGFR-3) is involved in tumor angiogenesis and
growth. Cancer Research, 67, 593e599.
Lamerato-Kozicki, A. R., Helm, K. M., Jubala, C. M.,
Cutter, G. C. and Modiano, J. F. (2006). Canine he-
mangiosarcoma originates from hematopoietic precur-
sors with potential for endothelial differentiation.
Experimental Hematology, 34, 870e878.
Lymboussaki, A., Olofsson, B., Eriksson, U. and Alitalo, K.
(1999). Vascular endothelial growth factor (VEGF) and
VEGF-C show overlapping binding sites in embryonic
endothelia and distinct sites in differentiated adult endo-
thelia. Journal of Circulation Research, 85, 992e999.
Marks, R. M., Roche, W. R. and Czerniecki, M. (1986).
Mast cell granules cause proliferation of human micro-
vascular endothelial cells. Laboratory Investigation, 55,
289e294.
Masuzawa, M., Fujimura, T., Hamada, Y., Fujita, Y.,
Hara, H., Nishiyama, S., Katsuoka, K., Tamauchi, H.
and Sakurai, Y. (1999). Establishment of a human he-
mangiosarcoma cell line (ISOHAS). International Journal
of Cancer, 81, 305e308.
Meininger, C. J., Brightamn, S. E., Kelly, K. A. and
Zetter, B. R. (1995). Increased stem cell factor release
by hemangioma-derived endothelial cells. Laboratory In-
vestigation, 72, 166e173.
Miettinen, M., Sarlomo-Rikala, M. and Lasota, J. (2000).
KIT expression in angiosarcomas and fetal endothelial
cells: lack of mutations of exon 11 and exon 17 of c-kit.
Modern Pathology, 13, 536e541.
Murakami, M., Sakai, H., Kodama, A., Mori, T.,
Maruo, K., Yanai, T. and Masegi, T. (2008). Expres-
sion of the anti-apoptotic factors bcl-2 and survivin in
canine vascular tumours. Journal of Comparative Pathology,
139, 1e7.
Naor, D., Sionov, R. V. and Ish-Shalom, D. (1997). CD44:
structure, function, and association with the malignant
process. Advances in Cancer Research, 71, 241e319.
Neuhauser, T. S., Derringer, G. A., Thompson, L. D., Fan-
burg-Smith, J. C., Miettinen, M., Saaristo, A. and
Abbondanzo, S. L. (2000). Splenic angiosarcoma: a clin-
icopathologic and immunophenotypic study of 28 cases.
Modern Pathology, 13, 978e987.
Nocito, A., Kononen, J., Kallioniemi, O. P. and Sauter, G.
(2001). Tissue microarrays (TMAs) for high-through-
put molecular pathology research. International Journal
of Cancer, 94, 1e5.
Ogilvie, G. K., Powers, B. E., Mallinckrodt, C. H. and
Withrow, S. J. (1996). Surgery and doxorubicin in
dogs with hemangiosarcoma. Journal of Veterinary Internal
Medicine, 10, 379e384.
Oksanen, A. (1978). Haemangiosarcoma in dogs. Journal of
Comparative Pathology, 88, 585e595.
Partanen, T. A., Alitalo, K. and Miettinen, M. (1999).
Lack of lymphatic vascular specificity of vascular endo-
thelial growth factor receptor 3 in 185 vascular tumors.
Cancer, 86, 2406e2412.
Pearson, G. R. and Head, K. W. (1976). Malignant hae-
mangioendothelioma (angiosarcoma) in the dog. Jour-
nal of Small Animal Practice, 17, 737e745.
Petrova, T. V., Bono, P., Holnthoner, W., Chesnes, J.,
Pytowski, B., Sihto, H., Laakkonen, P., Heikkila¨ , P.,
Joensuu, H. and Alitalo, K. (2008). VEGFR-3 expres-
sion is restricted to blood and lymphatic vessels in solid
tumors. Cancer Cell, 13, 554e556.
Immunohistochemistry of Canine Vascular Tumours 167

11. Prymak, C., McKee, L. J., Goldschmidt, M. H. and
Glickman, L. T. (1988). Epidemiologic, clinical, patho-
logic, and prognostic characteristics of splenic heman-
giosarcoma and splenic hematoma in dogs: 217 cases.
Journal of the American Veterinary Medical Association,
193, 706e712.
Schatteman, G. C. and Awad, O. (2004). Hemangioblasts,
angioblasts, and adult endothelial cell progenitors. The
Anatomical Record. Part A, Discoveries in Molecular, Cellular
and Evolutionary Biology, 276, 13e21.
Sorenmo, K., Duda, L., Barber, L., Cronin, K.,
Sammarco, C., Usborne, A., Goldschmidt, M. and
Shofer, F. (2000). Canine hemangiosarcoma treated
with standard chemotherapy and minocycline. Journal
of Veterinary Internal Medicine, 14, 395e398.
Srebernik, N. and Appleby, E. C. (1991). Breed prevalence
and sites of haemangioma and haemangiosarcoma in
dogs. Veterinary Record, 129, 408e409.
Sun, Z. J., Zhao, Y. F. and Zhao, J. H. (2007). Mast cells in
hemangioma: a double-edged sword. Medical Hypotheses,
68, 805e807.
Timaran, C. H., Grandas, O. H. and Bell, J. L. (2000). He-
patic angiosarcoma: long-term survival after complete
surgical removal. American Surgeon, 66, 1153e1157.
Ward, H., Fox, L. E., Calderwood-Mays, M. B.,
Hammers, A. S. and Couto, C. G. (1994). Cutane-
ous hemangiosarcoma in 25 dogs: a retrospective
study. Journal of Veterinary Internal Medicine, 8,
345e348.
Waters, D. J., Caywood, D. D., Hayden, D. W. and
Klausner, J. S. (1988). Metastatic pattern in dogs with
splenic hemangiosarcomas: clinical implications. Journal
of Small Animal Practice, 29, 805e814.
Yamamoto, T., Umeda, T. and Nishioka, K. (2000). Im-
munohistological distribution of stem cell factor and
KIT receptor in angiosarcoma. Acta Dermato-Venereolog-
ica, 80, 443e445.
Yonemaru, K., Sakai, H., Murakami, M., Yanai, T. and
Masegi, T. (2006). Expression of vascular endothelial
growth factor, basic fibroblast growth factor, and their
receptors (flt-1, flk-1, and flg-1) in canine vascular tu-
mors. Veterinary Pathology, 43, 971e980.
½ Received, August 8th, 2008
Accepted, October 31st, 2008 Š
168 S. Sabattini and G. Bettini