Human Pathology (2013) 44, 2302–2311

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:
Summary Hemangiosarcomas are uncommon aggressive vascular tumors that have recently become
Hemangiosarcoma;
the focus of attention because several chemicals and pharmaceuticals increase their incidence in mice.
Hemangioma;
The relevance of these mouse vascular tumors to humans is unclear. In the present study, we semi-
Hematopoietic stem cells;
quantitatively evaluated the expression profiles of hematopoietic stem cell markers (CD117 [c-kit],
Endothelial progenitor
CD133, CD34, and CD45), endothelial cell markers (vascular endothelial growth factor receptor 2,
cells;
CD31, and factor VIII–related antigen), and a myeloid lineage cell marker (CD14) in human heman-
Immunohistochemistry
giosarcoma (n = 12) and hemangioma (n = 10) specimens using immunohistochemistry. CD133 was
completely negative in almost all cases of hemangiosarcomas and hemangiomas. Most hemangio-
sarcomas, but not hemangiomas, stained for CD117 and CD45. Both groups diffusely expressed CD34,
vascular endothelial growth factor receptor 2, and factor VIII–related antigen; however, hemangiomas
had more intense and diffuse CD34 and factor VIII–related antigen expression compared with heman-
giosarcomas, 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 marrow–derived 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 expres-
sion 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; 1. Introduction

H&E, hematoxylin and eosin; PPAR, peroxisome proliferator–activated
receptor; RTU, ready to use; UEA-1, Ulex europaeus agglutinin-1;
VEGFR2, vascular endothelial growth factor receptor 2; vWF, von Hemangiosarcomas are rare aggressive malignant tumors
Willebrand factor. in humans and usually have no known cause [1,2]. The only
☆
Statement of interest: Funding for the immunostains was provided, in known causes of hemangiosarcomas in humans include a
part, by a grant from Pfizer, Inc, Groton, CT. Dr Cohen has previously
consulted for Pfizer, Inc, but on matters unrelated to this report.
few rare genetic disorders, previous irradiation, and exposure
⁎ Corresponding author. to certain genotoxic agents such as vinyl chloride and thoro-
E-mail address: scohen@unmc.edu (S. M. Cohen). trast [3,4]. Recently, it was reported that a wide range of

0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.humpath.2013.05.012
Pathogenesis of human hemangiosarcomas and hemangiomas
nongenotoxic chemical and pharmaceutical agents such as
peroxisome proliferator–activated receptor γ agonists in-
creased 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 marrow–derived 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 [8–10]. 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 differen-
tiation from EPCs to mature ECs, the circulating EPCs lose
the expression of CD117 and CD133 [8–10] 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)-expres-
sing cells also expressed EC markers such as CD31,
VEGFR2, factor VIII–related 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 VIII–related antigen, and
CD14. Factor VIII–related antigen expression is known to
occur later than CD31 expression in EPCs. Therefore, the
lack of factor VIII–related 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 [16–19], whereas CD34,
VEGFR2, CD31, and factor VIII–related antigen expression
has been demonstrated in hemangiosarcomas and hemangi-
omas [4,16,17,20–22]. 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. Repre-
sentative 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 rehy-
drated 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
100°C 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 L. Liu et al.

Table 1 Summary of patients' clinical information serum block was performed for CD45 staining. Sections
Tumors Patient ID Age a
Sex Location were incubated with primary antibodies or negative controls
for 30 minutes at room temperature and then incubated with
Hemangiosarcomas 1 81 F Skin, chest antirabbit polymer or antimouse polymer (Leica Biosystems)
2 48 F Breast
for all antibodies. Refine Detection System (Leica Biosys-
3 48 M Leg
tems) containing 3,3′-diaminobenzidine was used for the
4 60 M Kidney
5 85 F Soft tissue, right detection of target proteins, resulting in brown staining.
forearm Slides were then counterstained with hematoxylin, dehy-
6 60 F Breast drated, and coverslipped.
7 65 F Breast
8 99 M Skin, forehead 2.3. Immunohistochemical evaluation
9 82 F Breast
10 77 F Skin, chest
Immunohistochemically stained sections were semiquanti-
11 34 M Pleura
12 70 M Skin, face
tatively evaluated by clinical pathologists (L.L., S.L.J., and
Hemangiomas 13 86 F Breast S.M.C.) for the presence/absence of staining, the percentage of
14 17 M Muscle tumor cells expressing positive staining, and the overall
15 36 M Soft tissue staining intensity of positively stained tumor cells and were
16 66 M Skin subsequently classified into one of the following grades: 0, no
17 10 F Skin positive staining in any of the tumor cells; 1+, positive staining
mo in 25% or less of the tumor cells; 2+, positive staining in 26%
18 49 M Liver to 50% of the tumor cells; 3+, positive staining in 51% to 75%
19 62 M Skin of the tumor cells; and 4+, positive staining in 76% to 100% of
20 81 F Bone the tumor cells. The overall staining intensity was graded as
21 47 F Sinus
weak (w), moderate (m), or strong (s).
22 b 41 M Bone
Abbreviations: F, female; M, male.
a
Age in years except for patient 17, which is in months.
b
Diagnosed as having epithelioid hemangioma. 3. Results

p53 staining; or for 5 minutes at 37°C 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
For each immunostain, no major differences were ob-
served 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 2305

Table 2 Antibodies and negative controls used for immunohistochemistry

Description of Manufacturer Stock concentration Antigen Primary Secondary Internal positive
antibodies and of primary antibodies retrieval antibody antibodies control cells
blocking reagents dilution
CD 117 Cell Marque (117R-18) RTU ER1 RTU Leica GIST
Normal rabbit serum Jackson ImmunoResearch Polymer
(001-000-001)
PROM1 (CD133) Millipore (MAB4399) 1.0 mg/mL ER2 1:100 Leica Not applicable
Normal rabbit serum Jackson ImmunoResearch Polymer
(001-000-001)
CD45 Leica (PA0042) RTU ER2 RTU Leica Leukocytes
Not used N/A Polymer
CD34 Leica (PA0212) RTU ER2 RTU Leica Vascular
Normal rabbit serum Jackson ImmunoResearch Polymer endothelium
(001-000-001)
VEGFR2 Cell Signaling (2479) Unknown ER1 1:200 Leica Vascular
Normal rabbit serum Jackson ImmunoResearch Polymer endothelium
(001-000-001)
CD31 Leica (PA0250) RTU ER2 RTU Leica Vascular
Normal rabbit serum Jackson ImmunoResearch Polymer endothelium
(001-000-001)
Factor VIII–related Cell Marque (250A-18) RTU ER1 RTU Leica Vascular
antigen Polymer endothelium
Normal rabbit serum Jackson ImmunoResearch
(001-000-001)
CD14 Leica (NCL-CD14-223) Unknown ER2 1:100 Leica Neutrophils
Normal rabbit serum Jackson ImmunoResearch Polymer
(001-000-001)
p53 DAKO (IR616) RTU ER2 RTU Leica Tumor cells
Normal rabbit serum Jackson ImmunoResearch (001- Polymer
000-001)
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 hemangio-
sarcoma 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 mem-
brane 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 hemangio-
sarcomas because of the interference of residual CD45-
expressing 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
VIII–related 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 neutro-
phils (a positive internal control); however, most hemangio-
sarcomas (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 2307

Table 3 Staining for markers and p53 in human hemangiosarcomas (HS) and hemangiomas (HA)
Tumors Patient Hematopoietic stem cell markers EC markers Myeloid cell Tumor suppressor
ID CD117 CD133 CD34 CD45 VEGFR2 CD31 Factor VIII– marker: CD14 marker: p53
related antigen
HS 1 4+ s 0 4+ s 0 4+ s 3+ s 4+ s 0 4+ s
2 0 1+ s 2+ s 0 1+ w 3+ m 1+ w 0 1+ m
3 3+ m 0 0 ind 4+ s 4+ s 3+ m 0 2+ m
4 0 0 0 3+ s 1+ w to m 1+ m to s 1+ m 2+ m 1+ m
5 4+ s 0 4+ s 2+ s 4+ s 4+ s 4+ s 0 3+ m
6 4+ s 0 4+ s 1+ s 4+ s 4+ s 4+ m to s 0 1+ w
7 3+ m 0 4+ m to s ind 4+ s 3+ m to s 4+ m to s 0 1+ w
8 0 0 0 2+ s 4+ s 3+ s 3+ m ind 1+ m
9 4+ s 0 4+ s ind 4+ s 3+ m 4+ s 0 1+ s
10 1+ m 0 3+ s 2+ s 4+ m to s 4+ s 3+ m to s 1+ m 1+ s
11 0 0 1+ m to s 1+ s 4+ s 3+ s 3+ m to s 1+ s 1+ m
12 1+ m 0 2+ s 2+ s 4+ m to s 4+ s 3+ s 1+ s 3+ s
HA 13 0 0 4+ s 0 4+ s 0 4+ s 0 0
14 0 0 4+ s 0 1+ w 0 4+ s 0 0
15 1+ w 0 4+ s 0 3+ w to m 0 4+ s 0 0
16 0 0 4+ s 0 4+ m 0 4+ s 0 0
17 0 0 4+ s 0 4+ s 4+ m to s 4+ s 0 1+ w
18 3+ w to m 0 4+ s 0 4+ m 3+ w 4+ s 0 0
19 0 0 4+ s 0 4+ m 0 4+ s 1+ m 0
20 0 0 4+ s 0 4+ w to m 2+ s 4+ s 0 0
20 0 0 4+ s 0 2+ w to m 4+ s 4+ s 0 0
22 a 1+ w 0 4+ s 2+ s 3+ w to m 1+ w to m 4+ s 2+ m 1+ w
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 rele-
vant 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 VIII–related 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 cir-
culation, 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 spe-
cimens) 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 VIII–related antigen (E); hemangiomas were weakly positive for VEGFR2 (B), partially positive for
CD31 (D), and strongly positive for factor VIII–related 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 hemangiosar-
comas (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 heman-
gioma 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. Interest-
ingly, our results demonstrated that tumor cells in heman-
giosarcomas express CD117 but not CD133. If the lack of
Pathogenesis of human hemangiosarcomas and hemangiomas
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 im-
mature 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 VIII–related antigen
[8,25]. In the current study, human hemangiosarcomas
expressed higher levels of VEGFR2 and CD31 but lower
levels of factor VIII–related 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 VIII–related antigen are determined by the differen-
tiation of ECs.
CD14 is a monocyte/macrophage cell surface antigen, and
the contribution of myeloid lineage cells to angiogenesis has
2309
recently been demonstrated [13,26]. In addition, coexpres-
sion 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 heman-
giosarcomas (4/12) demonstrated CD14 positivity; this sup-
ports 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 nega-
tive for CD45, factor VIII–related antigen, and CD14, and
positive for CD34 (stronger than internal vasculature con-
trol), VEGFR2, and CD31 [15]. Based on the increased
intensity of CD34 and the lack of factor VIII–related antigen
in these tumors, we concluded that both types of the mouse
tumors may be derived from early EPCs that are not com-
pletely 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 VIII–related
2310
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 dif-
ferentiate similar to canines [28], whereas mouse tumor cells
are arrested at a stage before complete differentiation owing
to the lack of factor VIII–related antigen. Although mouse
hemangiomas may consist of early EPCs, human hemangi-
omas showed diminished intensity of VEGFR2 and CD31
and increased expression of factor VIII–related antigen, a
staining pattern of late EPCs or differentiated ECs.
We also investigated the expression of the tumor sup-
pressor 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, immuno-
stains 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
chloride–induced 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
L. Liu et al.
exhibited an expression profile similar to hemangiosarco-
mas, 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 hemangio-
sarcoma and hemangioma in humans. The heterogeneity might
be caused by intratumor clonal diversity, dysregulation of
different proteins because of genomic or epigenetic modifica-
tions, or aberrant expression of transcription factors, micro-
RNAs, and so on, and even sampling issues of the tumor [34].
In summary, our study indicates that human hemangio-
sarcomas 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 hem-
angiomas 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 heman-
giomas (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 pre-
paration 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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