Human Pathology (2013) 44, 23022311

www.elsevier.com/locate/humpath

Original contribution

Pathogenesis of human hemangiosarcomas and

hemangiomas
Liping Liu MD, PhD a , Satoko Kakiuchi-Kiyota PhD b , Lora L. Arnold MS a ,
Sonny L. Johansson MD, PhD a , David Wert BS a , Samuel M. Cohen MD, PhD a,
a

Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center,
Omaha, NE 68198-3135, USA
b
Compound Safety Prediction, Pfizer, Inc, Groton, CT 06340, USA
Received 5 March 2013; revised 16 May 2013; accepted 17 May 2013

Keywords:
Hemangiosarcoma;
Hemangioma;
Hematopoietic stem cells;
Endothelial progenitor
cells;
Immunohistochemistry

Summary Hemangiosarcomas are uncommon aggressive vascular tumors that have recently become
the focus of attention because several chemicals and pharmaceuticals increase their incidence in mice.
The relevance of these mouse vascular tumors to humans is unclear. In the present study, we semiquantitatively evaluated the expression profiles of hematopoietic stem cell markers (CD117 [c-kit],
CD133, CD34, and CD45), endothelial cell markers (vascular endothelial growth factor receptor 2,
CD31, and factor VIIIrelated antigen), and a myeloid lineage cell marker (CD14) in human hemangiosarcoma (n = 12) and hemangioma (n = 10) specimens using immunohistochemistry. CD133 was
completely negative in almost all cases of hemangiosarcomas and hemangiomas. Most hemangiosarcomas, but not hemangiomas, stained for CD117 and CD45. Both groups diffusely expressed CD34,
vascular endothelial growth factor receptor 2, and factor VIIIrelated antigen; however, hemangiomas
had more intense and diffuse CD34 and factor VIIIrelated antigen expression compared with hemangiosarcomas, whereas CD31 was positive in all hemangiosarcomas but only half of the hemangiomas.
CD14 staining was negative in most hemangiosarcoma and hemangioma cases. Our results indicate that
multipotential bone marrowderived hematopoietic stem cells or early endothelial progenitor cells
(EPCs) expressing CD117, CD34, and CD45 are involved in hemangiosarcoma formation, whereas
hemangiomas originate from late EPCs or differentiated endothelial cells, which have lost the expression of most hematopoietic stem cell markers. This contrasts with our previous results that demonstrated
that both hemangiosarcomas and hemangiomas in mice may be derived from early EPCs that are not
completely differentiated.
2013 Elsevier Inc. All rights reserved.

Abbreviations: EC, endothelial cell; EPC, endothelial progenitor cell;

H&E, hematoxylin and eosin; PPAR, peroxisome proliferatoractivated
receptor; RTU, ready to use; UEA-1, Ulex europaeus agglutinin-1;
VEGFR2, vascular endothelial growth factor receptor 2; vWF, von
Willebrand factor.

Statement of interest: Funding for the immunostains was provided, in

part, by a grant from Pfizer, Inc, Groton, CT. Dr Cohen has previously
consulted for Pfizer, Inc, but on matters unrelated to this report.
Corresponding author.
E-mail address: scohen@unmc.edu (S. M. Cohen).
0046-8177/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.humpath.2013.05.012

1. Introduction
Hemangiosarcomas are rare aggressive malignant tumors
in humans and usually have no known cause [1,2]. The only
known causes of hemangiosarcomas in humans include a
few rare genetic disorders, previous irradiation, and exposure
to certain genotoxic agents such as vinyl chloride and thorotrast [3,4]. Recently, it was reported that a wide range of

Pathogenesis of human hemangiosarcomas and hemangiomas

nongenotoxic chemical and pharmaceutical agents such as
peroxisome proliferatoractivated receptor agonists increased the incidence of hemangiosarcomas in rodents,
primarily in mice in 2-year carcinogenicity studies [3,5].
Human hemangiomas are benign proliferations of blood
vessels, can occur in several tissues, and are relatively
common tumors [6]. In mice, vascular tumors occur
frequently, with incidences in controls ranging from 2% to
5% [3].
The pathogenesis of hemangiosarcomas is not well
understood in humans or animals. It has been proposed
that the malignant endothelial cells (ECs) are derived either
from differentiated ECs that developed malignant potential
through mutations or from transformed bone marrow
derived endothelial progenitor cells (EPCs) [7]. Numerous
studies show that adult bone marrow and peripheral blood
contain EPCs that are capable of differentiating into mature
ECs [8,9]. Bone marrowderived EPCs are considered
hematopoietic in origin, expressing hematopoietic stem cell
markers including CD117 (c-kit), CD133, CD34, and CD45.
The mobilization of EPCs from the bone marrow is initiated
by the activation of matrix metalloproteinase-9, resulting in
detachment of early stem and progenitor cells expressing
CD117 from the bone marrow stromal niche and their
subsequent movement to the vascular zone of the bone
marrow [810]. CD133 is expressed in hematopoietic stem
cells and EPCs but absent in mature ECs [8]. Beaudry et al
[11] demonstrated that circulating EPCs appear to have lost
CD117 expression while retaining CD133 expression in
mice. Expression of CD34 is not restricted to hematopoietic
stem cells or early EPCs but is also present on late EPCs and
mature ECs at lower levels [8,9]. CD45 is expressed in
hematopoietic stem cells and in both lymphoid and myeloid
cell lineages from progenitor to mature cells [12]. Early
EPCs in the bone marrow or in blood just after migration into
the circulation begin to express the EC marker vascular
endothelial growth factor receptor 2 (VEGFR2) in addition
to hematopoietic stem cell markers [8,9]. During differentiation from EPCs to mature ECs, the circulating EPCs lose
the expression of CD117 and CD133 [810] and begin to
express mature EC markers such as CD31 and factor VIII
related antigen (or von Willebrand factor) [8,9]. Recently,
the contribution of myeloid lineage cells in angiogenesis has
been reported. Yoder et al [13] showed that CD45- and
CD14 (monocyte/macrophage cell surface antigen)-expressing cells also expressed EC markers such as CD31,
VEGFR2, factor VIIIrelated antigen [14], and Ulex
europaeus agglutinin-1.
We demonstrated that mouse spontaneous and chemically
induced hemangiosarcomas and hemangiomas are both
derived from EPCs expressing CD34, VEGFR2, and CD31
but are negative for CD45, factor VIIIrelated antigen, and
CD14. Factor VIIIrelated antigen expression is known to
occur later than CD31 expression in EPCs. Therefore, the
lack of factor VIIIrelated antigen expression may indicate
that these tumor cells are arrested at a stage before complete

2303
differentiation. Different from mouse EC tumors [15], canine
hemangiosarcoma cell lines expressed CD117, CD133,
CD34, and, in some cases, CD45 and CD14 [7]. However,
their expression pattern in canine EC tumors in vivo is not
yet available. In humans, one study showed that CD117
expression was observed in hemangiosarcomas (15%) [4].
Some case reports demonstrated that CD45 expression is
negative in hemangiosarcomas [1619], whereas CD34,
VEGFR2, CD31, and factor VIIIrelated antigen expression
has been demonstrated in hemangiosarcomas and hemangiomas [4,16,17,2022]. However, expression of CD133 and
CD14 in human EC tumors is not well understood.
In the present study, we evaluated the expression profiles
for hematopoietic stem cell markers (CD117, CD133, CD34,
and CD45), EC markers (VEGFR2, CD31, and factor VIII
related antigen), and a myeloid cell marker (CD14) in human
hemangiosarcomas and hemangiomas. These tumors were
also stained with the p53 tumor suppressor protein because
this marker has been present in most hemangiosarcomas in
humans, but not in human hemangiomas or in any mouse
vascular tumor types [3]

2. Materials and methods

2.1. Selection of cases and controls
Pathology files of the University of Nebraska Medical
Center were searched for cases of hemangiosarcoma (from
years 2000 to March 2013) and hemangioma (from years 2011
to 2012). Twelve cases of hemangiosarcomas and 10 cases of
hemangiomas (including 1 epithelial type) were identified,
and formalin-fixed and paraffin-embedded tissue specimens
were retrieved from the tissue archives of the University of
Nebraska Medical Center. Clinical data including age, sex,
and site(s) of involvement were available in all cases
(Table 1). The study was approved by the institutional review
board of the University of Nebraska Medical Center. Representative hematoxylin and eosin (H&E)stained histology
slides from hemangiosarcoma and hemangioma specimens are
shown in Fig. 1A and B, respectively.

2.2. Immunohistochemistry
Immunohistochemical procedures including antibodies
and negative controls for primary antibodies used in our
study are summarized in Table 2. Formalin-fixed and
paraffin-embedded tissues were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase
activity was quenched with 3% hydrogen peroxide, followed
by heat-induced antigen retrieval using Leica Bond Max
(Leica Biosystems, Buffalo Grove, IL) for 20 minutes at
100C in citrate buffer, pH 6.0; ER1 (Leica Biosystems) for
CD117 and VEGFR2 staining or EDTA, pH 9.0; ER2 (Leica
Biosystems) for CD133, CD45, CD34, CD31, CD14, and

2304
Table 1

L. Liu et al.
Summary of patients' clinical information

Tumors
Hemangiosarcomas

Hemangiomas

Patient ID Age

Sex Location

1
2
3
4
5

81
48
48
60
85

F
F
M
M
F

6
7
8
9
10
11
12
13
14
15
16
17

60
65
99
82
77
34
70
86
17
36
66
10
mo
49
62
81
47
41

F
F
M
F
F
M
M
F
M
M
M
F

Skin, chest
Breast
Leg
Kidney
Soft tissue, right
forearm
Breast
Breast
Skin, forehead
Breast
Skin, chest
Pleura
Skin, face
Breast
Muscle
Soft tissue
Skin
Skin

M
M
F
F
M

Liver
Skin
Bone
Sinus
Bone

18
19
20
21
22 b

Abbreviations: F, female; M, male.

a
Age in years except for patient 17, which is in months.
b
Diagnosed as having epithelioid hemangioma.

p53 staining; or for 5 minutes at 37C in ER1 for factor VIII

related antigen staining. Additional blocking of nonspecific
binding was performed using 10% normal serum from the
host species of the secondary antibody (Leica Biosystems)
for 10 minutes at room temperature for CD133, CD34,
VEGFR2, CD31, CD14, CD117, and p53 staining or normal
serum contained in VECTASTAIN Elite ABC Kit (Rabbit
IgG; Vector Laboratories, Burlingame, CA) for factor VIII
related antigen staining, as directed by the manufacturer. No

serum block was performed for CD45 staining. Sections

were incubated with primary antibodies or negative controls
for 30 minutes at room temperature and then incubated with
antirabbit polymer or antimouse polymer (Leica Biosystems)
for all antibodies. Refine Detection System (Leica Biosystems) containing 3,3-diaminobenzidine was used for the
detection of target proteins, resulting in brown staining.
Slides were then counterstained with hematoxylin, dehydrated, and coverslipped.

2.3. Immunohistochemical evaluation

Immunohistochemically stained sections were semiquantitatively evaluated by clinical pathologists (L.L., S.L.J., and
S.M.C.) for the presence/absence of staining, the percentage of
tumor cells expressing positive staining, and the overall
staining intensity of positively stained tumor cells and were
subsequently classified into one of the following grades: 0, no
positive staining in any of the tumor cells; 1+, positive staining
in 25% or less of the tumor cells; 2+, positive staining in 26%
to 50% of the tumor cells; 3+, positive staining in 51% to 75%
of the tumor cells; and 4+, positive staining in 76% to 100% of
the tumor cells. The overall staining intensity was graded as
weak (w), moderate (m), or strong (s).

3. Results
For each immunostain, no major differences were observed between males and females or between different
tissues containing the vascular tumors, in terms of staining
pattern and/or intensity (data not shown).

3.1. Hematopoietic stem cell markers

Eight of 12 hemangiosarcoma cases were positive for
CD117 staining with moderate to strong intensity (Fig. 2A

Fig. 1 Hemangiosarcomas (A) and hemangiomas (B) stained by H&E (10). The hemangiosarcomas were hypercellular and composed of
spindle cells with significant atypia, forming anastomosing immature vascular channels. Hemangiomas showed anastomosing vascular
structures lined by bland ECs.

Pathogenesis of human hemangiosarcomas and hemangiomas

Table 2

2305

Antibodies and negative controls used for immunohistochemistry

Description of
antibodies and
blocking reagents

Manufacturer

Stock concentration Antigen Primary Secondary

of primary antibodies retrieval antibody antibodies
dilution

Internal positive
control cells

CD 117
Normal rabbit serum

Cell Marque (117R-18)

Jackson ImmunoResearch
(001-000-001)
Millipore (MAB4399)
Jackson ImmunoResearch
(001-000-001)
Leica (PA0042)
N/A
Leica (PA0212)
Jackson ImmunoResearch
(001-000-001)
Cell Signaling (2479)
Jackson ImmunoResearch
(001-000-001)
Leica (PA0250)
Jackson ImmunoResearch
(001-000-001)
Cell Marque (250A-18)

RTU

ER1

RTU

Leica
Polymer

GIST

1.0 mg/mL

ER2

1:100

Leica
Polymer

Not applicable

RTU

ER2

RTU

Leukocytes

RTU

ER2

RTU

Leica
Polymer
Leica
Polymer

Vascular
endothelium

Unknown

ER1

1:200

Leica
Polymer

Vascular
endothelium

RTU

ER2

RTU

Leica
Polymer

Vascular
endothelium

RTU

ER1

RTU

Leica
Polymer

Vascular
endothelium

Unknown

ER2

1:100

Leica
Polymer

Neutrophils

RTU

ER2

RTU

Leica
Polymer

Tumor cells

PROM1 (CD133)
Normal rabbit serum
CD45
Not used
CD34
Normal rabbit serum
VEGFR2
Normal rabbit serum
CD31
Normal rabbit serum
Factor VIIIrelated
antigen
Normal rabbit serum
CD14
Normal rabbit serum
p53
Normal rabbit serum

Jackson ImmunoResearch
(001-000-001)
Leica (NCL-CD14-223)
Jackson ImmunoResearch
(001-000-001)
DAKO (IR616)
Jackson ImmunoResearch (001000-001)

NOTE. Cell Marque, Rocklin, CA; Millipore, Auburn, CA; DAKO, Carpinteria, CA; Cell Signaling, Danvers, MA; GeneTex, Irvine, CA; Jackson
ImmunoResearch, West Grove, PA.
Abbreviations: ER1, Leica Bond Epitope Retrieval Citrate pH 6.0; ER2, Leica Bond Epitope Retrieval EDTA pH 9.0; GIST, gastrointestinal stromal tumor;
N/A, not applicable; RTU, ready to use.

and Table 3). In contrast, only 3 of 10 hemangioma cases had

weak CD117-positive staining (Fig. 2B and Table 3). We
were unable to find a positive internal control for CD133
because of the absence of expression of the marker in our
specimens. However, we used pancreatic tissue for our
positive control, which showed dot-like staining in the apical
side of the acini. In our study samples, only 1 hemangiosarcoma specimen showed scattered CD133-positive cells
(Table 3). Nine cases of hemangiosarcomas (Fig. 2C and
Table 3) and all cases of hemangiomas (Fig. 2D and Table 3)
were positive for CD34 staining, exhibiting intense membrane staining on most tumor cells. In general, the percentage
of CD34-positive tumor cells in hemangiomas was higher
than that in hemangiosarcomas. The staining intensity of
CD34 in both groups was similar to the internal vascular
controls (Fig. 2C and D). The staining pattern of CD45 was
hard to identify in 3 cases in the solid parts of hemangiosarcomas because of the interference of residual CD45expressing leukocytes (Fig. 2E, inset). However, CD45
tumor cell expression was observed in at least 7 cases of
hemangiosarcoma (Fig. 2E and Table 3). Nine hemangioma

cases were completely negative for CD45 staining (Fig. 2F

and Table 3), despite positive membrane staining of internal
control cells (leukocytes) within these sections.

3.2. EC markers
The percentage of cells staining positive for VEGFR2
was similar between hemangiosarcomas and hemangiomas
(Table 3); more than 75% of cells stained in most cases.
However, the staining intensity for VEGFR2 was stronger in
the hemangiosarcoma cases (Fig. 3A and Table 3) than the
hemangioma cases (Fig. 3B and Table 3). CD31 tumor cell
staining was observed in all cases of hemangiosarcoma
(ranging from moderate to strong; Fig. 3C and Table 3),
although only 5 hemangioma cases were positive for CD31
(ranging from weak to strong; Fig. 3D and Table 3). Factor
VIIIrelated antigen staining was focal and relatively weak
in hemangiosarcoma cases (Fig. 3E and Table 3), whereas it
was strong and diffuse in hemangioma specimens (Fig. 3F
and Table 3).

2306

L. Liu et al.

Fig. 2 Immunostains for hematopoietic stem cell markers showed that hemangiosarcomas were positive for CD117 (A) and CD45 (E) and
patchy positive for CD34 (C); hemangiomas were negative for CD117 (B) and CD45 (F) and positive for CD34 (D). Several cases of
hemangiosarcoma showed scattered positive cells for CD45 in the lesions. However, they were interpreted as indeterminate owing to the lack
of vasculature in the solid lesion and possible contamination of CD45-expressing leukocytes (E, inset).

3.3. Myeloid lineage cell marker and tumor

suppressor protein p53
Membrane/cytoplasmic staining was observed in neutrophils (a positive internal control); however, most hemangiosarcomas (Table 3) and hemangiomas (Fig. 4B and Table 3)
were negative for CD14 staining. Only 4 hemangiosarcomas
showed patchy positivity for CD14 (Fig. 4A). p53 positivity
was observed in all cases of hemangiosarcoma (Fig. 4C),
whereas most hemangiomas were negative (Fig. 4D), except

in 2 cases (Table 3). The intensity ranged from moderate

to strong in all hemangiosarcoma cases, except 2 cases
(Table 3), but was very weak in hemangioma cases (Table 3).

3.4. Staining pattern in an epithelioid hemangioma

One case was diagnosed as epithelioid hemangioma (a
variant of hemagonium). This benign vascular tumor is
composed of well-formed immature vessels that are lined
predominantly with plump epithelioid ECs [23]. Epithelioid

Pathogenesis of human hemangiosarcomas and hemangiomas

Table 3

2307

Staining for markers and p53 in human hemangiosarcomas (HS) and hemangiomas (HA)

Tumors Patient Hematopoietic stem cell markers

EC markers
ID
CD117
CD133 CD34
CD45 VEGFR2 CD31
HS

HA

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
20
22 a

4+
0
3+
0
4+
4+
3+
0
4+
1+
0
1+
0
0
1+
0
0
3+
0
0
0
1+

0
1+ s
m
0
0
s
0
s
0
m
0
0
s
0
m
0
0
m
0
0
0
w
0
0
0
w to m 0
0
0
0
w
0

4+ s
0
2+ s
0
0
ind
0
3+ s
4+ s
2+ s
4+ s
1+ s
4+ m to s ind
0
2+ s
4+ s
ind
3+ s
2+ s
1+ m to s 1+ s
2+ s
2+ s
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
0
4+ s
2+ s

4+ s
1+ w
4+ s
1+ w to m
4+ s
4+ s
4+ s
4+ s
4+ s
4+ m to s
4+ s
4+ m to s
4+ s
1+ w
3+ w to m
4+ m
4+ s
4+ m
4+ m
4+ w to m
2+ w to m
3+ w to m

3+
3+
4+
1+
4+
4+
3+
3+
3+
4+
3+
4+
0
0
0
0
4+
3+
0
2+
4+
1+

s
m
s
m to s
s
s
m to s
s
m
s
s
s

Factor VIII
related antigen

4+ s
1+ w
3+ m
1+ m
4+ s
4+ m to
4+ m to
3+ m
4+ s
3+ m to
3+ m to
3+ s
4+ s
4+ s
4+ s
4+ s
m to s 4+ s
w
4+ s
4+ s
s
4+ s
s
4+ s
w to m 4+ s

s
s

s
s

Myeloid cell Tumor suppressor

marker: CD14 marker: p53

0
0
0
2+ m
0
0
0
ind
0
1+ m
1+ s
1+ s
0
0
0
0
0
0
1+ m
0
0
2+ m

4+
1+
2+
1+
3+
1+
1+
1+
1+
1+
1+
3+
0
0
0
0
1+
0
0
0
0
1+

s
m
m
m
m
w
w
m
s
s
m
s

NOTE. Staining for markers in human HS and HA: 0, no positive staining in any of the tumor cells; 1+, positive staining in 25% or less of the tumor cells;
2+, positive staining in 26% to 50% of the tumor cells; 3+, positive staining in 51% to 75% of the tumor cells; 4+, positive staining in 76% to 100% of the
tumor cells.
Abbreviations: w, weak; m, moderate; s, strong; ind, indeterminate.
a
The patient has epithelioid hemangioma.

hemangiomas are observed in a wide age range, peaking in

the third to fifth decades, and the incidence in female patients
is higher than that in male patients [23]. In the present study,
it was removed from a 41-year-old man who presented with a
painful scapular destructive lesion. The patient had no relevant history and had been disease-free for 15 months after
surgery. An H&E-stained slide revealed a cellular lesion,
with epithelial-like rather than spindle tumor cells forming
anastomosing vasculature structures (Fig. 5A). No readily
identified mitoses were observed. The tumor exhibited a
positive staining pattern similar to hemangiosarcoma for
CD117 (Fig. 5B), CD45 (Fig. 5C), and p53 (Fig. 5D), but its
expression pattern for EC markers was similar to that of
hemangiomas (Table 3).

4. Discussion
In the present study, we showed that expression of CD117
and CD45 was observed in most hemangiosarcoma cases,
whereas they were negative in most of the hemangioma
cases. CD133 was negative and CD34 was positive for both

hemangiosarcomas and hemangiomas. Staining for

VEGFR2 and factor VIIIrelated antigen was positive for
both hemangiosarcomas and hemangiomas. CD31 staining
was observed in all cases of hemangiosarcomas, although
only 5 hemangioma cases were positive for CD31. Most
hemangiosarcoma and hemangioma cases were negative for
CD14 staining.
CD133 and CD117 are expressed in immature cells such
as hematopoietic stem cells and early EPCs located in the
bone marrow or in blood just after migration into the circulation, and their expression is decreased in late progenitor
cells and absent in mature ECs [8,9,11]. Expression of CD34
is not restricted to hematopoietic stem cells or early EPCs,
but also is present on late EPCs and mature ECs at lower
levels [8,9]. CD45 is expressed not only on hematopoietic
stem cells but also on lymphoid and myeloid cell lineages
from progenitor to mature cells as a pan-hematopoietic
marker [9,12]. A previous study found that 15% (5/34 specimens) of hemangiosarcomas were positive for CD117 [4].
No reports are available regarding the expression of CD133
in these tumors in humans. Multiple studies demonstrated
positivity for CD34 on human hemangiosarcomas
[17,20,22]. Some case studies reported that immunostaining

2308

L. Liu et al.

Fig. 3 Immunostains for EC markers demonstrated that hemangiosarcomas were strongly and diffusely positive for these 3 markers,
VEGFR2 (A), CD31 (C), and factor VIIIrelated antigen (E); hemangiomas were weakly positive for VEGFR2 (B), partially positive for
CD31 (D), and strongly positive for factor VIIIrelated antigen (F).

in human hemangiosarcomas for CD45 was negative [16

18]. In the present study, in contrast to previous publications,
most cases of human hemangiosarcomas showed strong and
at least focally positive staining for CD117 and CD45.
CD133 was completely negative in almost all hemangiosarcomas (9/10). That relatively few hemangiosarcomas showed
positivity for CD133 is in accord with the fact that it is an
early marker in progenitors or initiating cells [8,9,11], the
expression level of which is below the level of detection by
immunohistochemistry after differentiation or malignant
tumor heterogeneity. CD34 was positive with intensity

similar to the internal control (nonneoplastic vasculature)

for most hemangiosarcomas (8/12). In contrast, most hemangioma cases were negative for CD45 and CD117. Similar to
hemangiosarcomas, CD133 was negative but CD34 was
positive in all cases of hemangiomas, with intensity similar to
the internal control (nonneoplastic vasculature). These results
suggest that more immature EPCs expressing CD45 and
CD117, in addition to CD34, contribute to hemangiosarcoma
formation compared with hemangiomas in humans. Interestingly, our results demonstrated that tumor cells in hemangiosarcomas express CD117 but not CD133. If the lack of

Pathogenesis of human hemangiosarcomas and hemangiomas

2309

Fig. 4 Immunostains for myeloid lineage cell marker CD14 and tumor suppressor protein p53 revealed that several hemangiosarcomas
showed focal CD14 positivity (A), whereas a majority were negative for CD14 and positive for p53 (C); hemangiomas were largely negative
for CD14 (B) and p53 protein (D).

CD133 expression is caused by loss of expression during

differentiation, this would be opposite from the observation
of mouse circulating EPCs, suggesting that CD133 is retained
longer than CD117 [11]. This provides some evidence that
the mechanism for differentiation from hematopoietic stem
cells to the EC lineage in humans differs from that in mice.
Among the 3 EC markers used in our study, VEGFR2 is
considered to be expressed earliest and can be seen in immature cells, which also express CD133 [24]. During the
differentiation process, these cells lose the expression of
hematopoietic stem cell markers and begin to express other
EC markers, first CD31 and then factor VIIIrelated antigen
[8,25]. In the current study, human hemangiosarcomas
expressed higher levels of VEGFR2 and CD31 but lower
levels of factor VIIIrelated antigen staining compared with
hemangioma cases. These findings are consistent with our
results of hematopoietic stem cell staining, demonstrating
that more immature EPCs are involved in hemangiosarcomas
compared with hemangiomas. Staining results of EC markers
may indicate that expression levels of VEGFR2, CD31, and
factor VIIIrelated antigen are determined by the differentiation of ECs.
CD14 is a monocyte/macrophage cell surface antigen, and
the contribution of myeloid lineage cells to angiogenesis has

recently been demonstrated [13,26]. In addition, coexpression of a dendritic cell marker and EC markers was observed
in human infantile hemangioma [27]. Also, it is possible that
myeloid-derived cells are involved in the hemangiosarcoma
formation in dogs, at least in some cases [7]. A few hemangiosarcomas (4/12) demonstrated CD14 positivity; this supports the possibility of involvement of myeloid-derived cells
in the pathogenesis of the disease.
In contrast to human specimens, murine spontaneous and
chemically induced hemangiosarcomas and hemangiomas
showed similar expression profiles: both entities were negative for CD45, factor VIIIrelated antigen, and CD14, and
positive for CD34 (stronger than internal vasculature control), VEGFR2, and CD31 [15]. Based on the increased
intensity of CD34 and the lack of factor VIIIrelated antigen
in these tumors, we concluded that both types of the mouse
tumors may be derived from early EPCs that are not completely differentiated [15]. The origin of these 2 vascular
tumors appears to be different between mice and humans.
For example, although hematopoietic stem cells and/or
circulating EPCs seem to be involved in both mouse and
human hemangiosarcoma formation, CD45 was positive for
human vascular tumors but not for mice. In addition, human
hemangiosarcoma cases are positive for factor VIIIrelated

2310

L. Liu et al.

Fig. 5 A, One case diagnosed as epithelioid hemangioma showed spindle to epithelioid cells with mild atypia forming anastomosis vascular
channels. The neoplastic cells stained positive for CD117 (weak) (B), CD45 (C), and p53 (D).

antigen, indicating that they retain the ability to further differentiate similar to canines [28], whereas mouse tumor cells
are arrested at a stage before complete differentiation owing
to the lack of factor VIIIrelated antigen. Although mouse
hemangiomas may consist of early EPCs, human hemangiomas showed diminished intensity of VEGFR2 and CD31
and increased expression of factor VIIIrelated antigen, a
staining pattern of late EPCs or differentiated ECs.
We also investigated the expression of the tumor suppressor gene TP53 in human hemangiosarcomas and
hemangiomas. Wild-type p53 protein has a short half-life;
however, the mutated (inactivated) form of p53 protein is
stabilized and can be detected in the nucleus of neoplastic
cells [29]. Studies have reported that in humans, immunostains for p53 were almost entirely negative in hemangiomas
[30], whereas nuclei of malignant cells in hemangiosarcomas
stained positive for p53 [18,31]. Furthermore, TP53 mutations
in exons 5 to 8 were observed in spontaneous and vinyl
chlorideinduced hemangiosarcomas in humans [32,33].
There was moderate to strong nuclear staining for p53 in all
hemangiosarcomas in the current study, whereas only 2
hemangiomas showed weak staining.
Our findings for the expression profile of the epithelial
hemangioma warrant further exploration. This specific entity

exhibited an expression profile similar to hemangiosarcomas, positive for CD45, CD117, and p53, which indicates
these cells might be derived from EPCs and might have
malignant potential.
Our study showed relative heterogeneity of the expression
profile of hemangiosarcomas, although a clear trend of the
expressed markers indicates the different origins of hemangiosarcoma and hemangioma in humans. The heterogeneity might
be caused by intratumor clonal diversity, dysregulation of
different proteins because of genomic or epigenetic modifications, or aberrant expression of transcription factors, microRNAs, and so on, and even sampling issues of the tumor [34].
In summary, our study indicates that human hemangiosarcomas are most likely derived from hematopoietic stem
cell/EPCs with high malignant potential compared with
hemangiomas. Human hemangiomas are formed from late
EPCs or differentiated ECs. Hemangiosarcomas and hemangiomas appear to be 2 different entities with distinct
pathogenesis, and hemangiomas are unlikely to transform
into hemangiosarcomas. There is also no evidence that
human hemangiosarcomas (rare tumors) arise from hemangiomas (common tumors) or any other benign precursor [3].
Thus, our findings raise questions regarding the relevance of
the mouse model to human vascular tumors.

Pathogenesis of human hemangiosarcomas and hemangiomas

Acknowledgments
We gratefully appreciate Cheryl Putnam (University of
Nebraska Medical Center) for her assistance with the preparation of this manuscript. We also thank Drs Jon C. Cook
and Leslie A. Obert (Pfizer, Inc) for advice and comments
regarding this project.

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