J. Comp. Path. 2009, Vol. 140, 158e168 Available online at www.sciencedirect.com

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An Immunohistochemical Analysis of CanineHaemangioma 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

resp

1-99

:10.1

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 ofcutaneous and visceral haemangioma (HA) and 10 control samples of granulation tissue (GT) were labelledwith 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) andCD44. 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 greaterexpression 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 ofMC was detected in HA, suggesting a possible role for these cells in the pathogenesis of benign vascular neo-plasia in the dog.

� 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 oftenarises in the spleen, right atrium or skin (Pearsonand Head, 1976). Canine HSA is locally infiltrativeand readily metastasizes, particularly to the lungand liver (Oksanen, 1978; Brown et al., 1985). Mostaffected dogs die from acute internal haemorrhagesecondary to rupture of the tumour. Despite surgicaland chemotherapeutic management, the median sur-vival time for dogs diagnosed with HSA is little morethan 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 tumoursthere have been relatively few studies of the cellular

ondence to: G. Bettini (e-mail: giuliano.bettini@unibo.it).

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and molecular features, which means that there arefew markers that can be applied to the detection ofearly lesions or identification of potential targets forthe development of novel immunotherapeutic ap-proaches (Hoover et al., 1993; Masuzawa et al.,1999; Krump-Konvalinkova et al., 2003). Previousstudies have described the expression of von Wille-brand factor (vWF) (von Beust et al., 1988) andCD31 (Ferrer et al., 1995) by canine HSA. More re-cently, the expression of CD117 (Fosmire et al.,2004), cyclooxygenase-2 (Heller et al., 2005), vascularendothelial growth factor-A and its receptors, basic fi-broblastic growth factor and its receptor (Yonemaruet 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 incanine HA and HSA in order to further define the bi-ological characteristics of these tumours.

Materials and Methods

Cases of HA (n ¼ 29) andHSA (n ¼ 40) were selectedfrom the archives of the Pathology Unit of the

� 2008 Elsevier Ltd. All rights reserved.

Immunohistochemistry of Canine Vascular Tumours 159

Department of Veterinary Public Health and AnimalPathology, 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 gradedfor overall differentiation and nuclear pleomorphismas described in Table 1, whereas HAs (4 visceraland 25 cutaneous) were classified by dominant histo-logical pattern as cavernous, capillary or mixed. Thelevel of angiogenesis was evaluated in the samples ofGT and scored as mild, moderate or marked accord-ing to the number and thickness of the wall of newlyformed blood vessels and the immaturity of the liningendothelial cells.

A tissue array technique was employed in order tolimit the duration of the experiment, reduce reagentwaste and minimize tissue damage. The most repre-sentative areas in each section were defined by studyof the HE-stained sections, and then 3 mm tissue coreswere punched from the associated tissue blocks. Thesecores were transferred to a recipient block with pre-formed holes to form a composite block containingup 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.

IHCwas performed with a streptavidin-biotin-per-oxidase technique. Slides were initially incubatedwith hydrogen peroxide 0.3% in methanol for

Table 1

Histological scoring system for canine HSA

Score Overall

differentiation

Nuclear variation

1 Well-differentiated tumour

with numerous irregularvascular channels

Minimal variation in nuclear

size and shape

2 Moderately differentiated

neoplasm with at least

50% of the tumourshowing well-defined

vascular channels

Moderate variation in

nuclear size and shape

3 Poorly differentiated,

solid tumour with fewvascular channels

Marked variation in nuclear

size and shape. Nuclearsize often differs by

twofold or more between

tumour cells

20 min to block endogenous peroxidase activity andwere then microwaved in citrate buffer (pH 6.0), fortwo cycles of 5 min, in a 750 Watt microwave ovenfor antigen retrieval (for all antibodies except thatspecific for VEGFC). The sections were incubatedovernight 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-goatIgG (LSAB, Dako Cytomation, Glostrup, Denmark)for 30 min at room temperature, and then subse-quently with streptavidin-peroxidase complex for25 min at room temperature. After incubation inDAB 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 Haen, Germany). Appropriate positive con-trols were used throughout to assess the specificity ofthe reactions. As negative control, an isotype-matched antibody of irrelevant specificity (Neo-Markers, Fremont, CA) was used in place of theprimary antibody.

The intensity of immunolabelling for each markerwas graded as absent (0), weak (1), moderate (2) orstrong (3). Cellular proliferation was assessed usingthe MIB-1 antibody, which recognizes the Ki67 anti-gen, a nuclear protein expressed in all of the activephases of the cell cycle (G1, S, G2,M), but not in rest-ing cells (G0). The labelling index (MIB-1 index) wassubjectively estimated as the percentage of positivelylabelled nuclei and scored as 0 (less than 5% of nucleilabelled), 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 sectionsas 0 (no mast cells), 1 (occasional mast cells), 2 (mod-erate number of scattered MCs or clusters of at leastthree cells) or 3 (MCs present in a high number). Cor-relations between considered variables were evalu-ated by Spearman’s rank analysis and KruskaleWallis 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 in37 cases and there were 12 German shepherd dogs.The ratio of male to female dogs was 2.1:1 with67.6% of dogs with HSA being male. It was not pos-sible to assess accurately the proportion of animalsthat had been neutered.

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 100von 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 40CD44 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

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 caseshad primary origin in the spleen (n ¼ 16), rightatrium (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 growthpatterns. Eleven of the 40 HSAs (27.5%) were catego-rized as well-differentiated, with numerous, irregularvascular channels and large areas of haemorrhage withfew cells, mimicking haematomas (Fig. 1D). Twenty-fourHSAs (60%)were characterized by areas ofmoder-ate differentiation, where neoplastic tissue was morecompact but still showing, at least focally, a vasoforma-tive component. Five samples (12.5%) were poorly dif-ferentiated and were difficult to distinguish fromfibrosarcoma or other poorly differentiated sarcoma.These tumours comprised solid sheets of cells with fewvascular channels. Nuclear variation was minimal in12.5% (n ¼ 5) of specimens, moderate in 40%(n¼ 16) and marked in 47.5% (n¼ 19). There was norelationship between these different morphological pat-terns and the anatomical location of the tumour.

The immunohistochemical expression of the panelof markers by the HSAs is summarized in Table 3.All HSA tumour cells exhibited strong and diffuse cy-toplasmic expression of vimentin and actin, whereasthere was more variable expression of vWF, this rang-ing from weak and inconsistent (n ¼ 15, 60%) tostrong (n ¼ 3, 12%). CD117 expression was detectedin the cytoplasm of 76.3% of HSAs, and the intensityof expression ranged from low (n ¼ 16, 42.1%), tomoderate (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) ofsamples showed mild to intense cytoplasmic expres-sion of VEGFR-3. Fifteen tumours had cytoplasmicexpression 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 was16.7%.

Ten of the HSAs had a low to moderate number ofinfiltrating MCs and in one tumour there were nu-merousMCs. NoMCswere detected in the remainderof the samples (72.5%).

Haemangiomas

The mean age of dogs with HA was 9.3 years (range4e15 years). There were almost equal numbers ofmales and females and the German shepherd dogwas the most represented breed (n ¼ 8, 27.6%).Twenty-four HAs arose within the skin and fivewere splenic in origin.

Microscopically, most of the samples were of thecavernous subtype, showing large, regular and welldefined vascular spaces filled with erythrocytes, com-pletely enclosed by a single layer of endothelial cellsaligned on thin collagenous septa (Fig. 1B). Five spec-imens (17.2%) were mixed capillary-cavernous inwhich, focally, the vascular structures were smallerand lined by slightly plump endothelial cells protrud-ing into vessel lumens (Fig. 1C). Cellularity and nu-clear pleomorphism in these areas were usuallyhigher than in cavernous HA.

The immunohistochemical expression of the panelof 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 tomoderate expression of vWF and VEGFR-3 was con-sistently detected, whilst CD44, CD117 and VEGFCwere not expressed in the majority of samples(Fig. 2B). Conversely, there was strong cytoplasmiclabelling for CD117 and VEGFC in the areas of cap-illary differentiation of two mixed HAs. MIB-1 ex-pression was detected in only occasional endothelialnuclei and the MIB-1 index was less than 5% in allcases.

InfiltratingMCs were seen in the stromal compart-ment of all HAs with the single exception of onesplenic HA in which no MCs were detected. The

Fig. 1. Microscopical features of canine vascular lesions. (A) Granulation tissue showing fibroplasia and endothelial cells of an immaturephenotype engaged in angiogenesis. HE. Bar, 50 mm. (B) Cavernous haemangioma showing large, uniform blood-filled vascularspaces 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 showingplump endothelial cells aligned ondelicatecollagen trabeculae creating an anastomosing meshwork of blood-filled channels of varying size. HE. Bar, 50 mm.

Immunohistochemistry of Canine Vascular Tumours 161

number of MCs ranged from low (n ¼ 8, 33.3%) tomoderate (n ¼ 9, 37.5%) to high (n ¼ 6, 25%)(Fig. 3AeF).

Granulation Tissue

Five of ten samples of GT had evidence of fibroplasiaand intense angiogenesis; three had fibrosis and mildangiogenesis, one had non-recent angiogenesis andone had low angiogenic activity (Fig. 1A). The immu-nohistochemical expression of the panel of markers byGT is summarized in Table 3. In all cases endothelialcells expressed vimentin, actin and vWF but did notlabel for VEGFC. Reactive endothelial cells hadmild 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 moderatenumber of infiltrating MCs was detected in 50% ofsamples.

Statistical Analysis

InHSA there was significant correlation (Spearman’smethod) between overall differentiation and the de-gree of nuclear variation (P ¼ 0.0042), but therewas no significant correlation between MIB-1 indexand nuclear variation, between MIB-1 index andoverall differentiation, or between MIB-1 index andCD117 expression.

There was significantly greater expression ofCD117 byHSA (average score 1.2) thanHA (averagescore 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)

Table 3

Summary of immunohistochemical labelling of canine vascular tumours and granulation tiss e

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%) 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%)

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, vonWillebrand Factor; VEGFR-3, vascular endothelial growth factor receptor-3;VEGFC, vascular endothelial growth factor-C;MIB-1mean sc e is theMIB-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 w intensity 0e3 is given. MIB-1 expression and

the number of mast cells are also assigned to a four point scale.

162

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ettin

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0/

0/

orith

Fig. 2. CD117 expression in canine vascular lesions. (A) Immature ECs in activeGT 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

(P ¼ 0.0000), although there was no significant differ-ence between HSA and granulation tissue (averagescore 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%), whilstthe difference between HSA and GT (mean MIB-1index 11.4%) was not significant (P ¼ 0.3).

MCsweremore prominent inHA (average score 1.9)than in HSA (average score 0.4) (P¼ 0.0001), but thenumber of mast cells was not related to the anatomicallocation of the tumour (i.e. cutaneous or visceral).

Discussion

The distribution of age, breed and sex in this populationof 69 dogs with vascular tumours is consistent with thatdescribed previously (Prymak et al., 1988; Srebernikand Appleby, 1991). Dogs with both HA and HSAhadameanageofover9yearsandtheGermanshepherd

dog was the breed most represented for both tumourtypes.Althoughnocleargenderpredilectionhasbeenre-ported for canineHSA, inour study, as inothers (Sreber-nik and Appleby, 1991; Bettini et al., 2001), males wereoverrepresented. Nevertheless, these findings should beinterpreted cautiously due to the relatively small size ofthe population examined.

There is continued controversy as to whethermulti-organ involvement in canine HSA representstrue multicentric origin or reflects the developmentof one primary tumour with metastasis (Waterset 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 sameanimal and, based on knowledge of the commonmetastatic patterns in sarcomas, neither would beconsidered as a likely metastatic site. However, wefound no evidence that this subset of cases had dis-tinct morphological or immunohistochemicalfeatures.

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

Unlike for other canine tumour types, a histologicalgrading system has not been widely applied to HSA.In the present study, overall differentiation and nu-clear variation were scored for each sample in orderto define the correlation between morphological fea-tures and proliferative activity or immunophenotypic

characteristics. Other parameters tested in previousstudies were not estimated (Ogilvie et al., 1996). Thesehave included parameters such as number of mitosesand amount of necrosis; however, we believe that pro-liferative activity is more accurately assessed by eval-uating the expression of the nuclear antigenKi67, and

Immunohistochemistry of Canine Vascular Tumours 165

the presence of necrosis is entirely dependent on thesampling site. A positive correlation was found be-tween differentiation and nuclear variation, whilstthere was no correlation between these parametersand MIB-1 index or the expression of immunohisto-chemical markers, with the exception of lower vWFexpression and higher VEGFC expression in poorlydifferentiated HSA.

Previous immunohistochemical studies of canineHSA have been restricted to the use of endothelialcell-specific markers such as vWF and CD31 to distin-guish poorly differentiated examples from other mes-enchymal tumours. In the present study we appliedan extended panel of antibodies in order to test theirdiagnostic utility and contribute to the understandingof the biological characteristics of these tumours.

Vimentin is an intermediate filament protein that ispart of the cytoskeleton of mesenchymal cells and waswidely expressed in the cytoplasm of both neoplasticand non-neoplastic ECs. Immunohistochemical la-belling for vimentin could therefore be of value inthe diagnosis of atypical variants of these tumourssuch as epithelioid HSA.

The strong expression of smooth muscle actin by theendothelial cells from all of the vascular lesions studiedhere reflects the contractile ability of these cells. Therewas, 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 forthe vascular marker vWF, whilst labelling was oftenfocal 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 poorlydifferentiated.

C-kitproto-oncogeneproduct (KIT,CD117) is a ty-rosine kinase growth factor receptor for stem cell fac-tor (SCF, mast cell growth factor) involved in thedevelopment and maintenance of haematopoieticstem 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 genehave been detected in mastocytoma, seminoma andgastrointestinal 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-immunoreactiveHSAs do not have mutations of c-kit, so such positivityis more likely related to an immature phenotype of theneoplastic cells (Miettinen et al., 2000). CD117 ex-pression has also been reported in 7 canine splenicHSAs (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 consistentlyCD117-negative, except for two HAs with mixed cap-illary-cavernous pattern. These observations may betaken to support the theory whereby canine HSAand human angiosarcoma originate from primitive,poorly differentiated endothelial cells identified bythe expression of CD117 and other surface proteins re-stricted to bone marrow precursor cells (EPC),whereas HAwould originate frommature, well differ-entiated endothelial cells. This may provide a theoret-ical explanation for multicentric involvement andrepresents not only a major diagnostic advancement,but also an appealing target for the development ofnovel therapy based on kinase-inhibitors (Fosmireet al., 2004; Lamerato-Kozicki et al., 2006). CD117was also expressed, albeit weakly, by endothelial cellsfrom all sections of granulation tissue, except for thesample with a low level of angiogenesis. This findingis consistent with the immature phenotype of angio-blasts, and precludes the use of KIT as a marker todistinguish HSA from GT.

The VEGFR family comprises a group of three ty-rosine kinase receptors for vascular endothelialgrowth factors. VEGFR-3 (the receptor for VEGFCand VEGFD) has been found in most endothelia dur-ing embryogenesis, whilst later in development ex-pression of this molecule is restricted to lymphaticendothelium 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 vascularneoplasms (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 proteinmay be promising candidates for use inmolecular-tar-geted therapies.

In the present study, themajority of canineHSAs ex-pressed VEGFR-3, suggesting that this marker wouldnot be suitable for the immunohistochemical discrimi-nation between lymphangiosarcoma and HSA (Neu-hauser et al., 2000). In contrast, expression of theVEGFR-3 ligand (VEGFC) was limited to foci withintwo HAs with capillary differentiation and sevenpoorly differentiated HSAs. The greater expression ofVEGFR-3 in HSA compared with HA suggests thatHSAmay bemore influenced byVEGFC. AsVEGFCwas not over-expressed in HSA, other growth promo-tion mechanisms rather than autocrine may be impor-tant in this tumour. No correlation was found betweenVEGFR-3 expression and cell proliferation activity.

CD44 is a ubiquitous multi-structural and multi-functional cell surface adhesion molecule involved incell-to-cell and cell-to-matrix interactions, cell traffic,

166 S. Sabattini and G. Bettini

lymph node homing, transmission of growth signalsand signals mediating haematopoiesis and apoptosis.Hyaluronic acid, an important component of the ex-tracellular matrix (ECM), is the principal ligand forCD44; others include collagen, fibronectin, lamininand chondroitin sulphate (Naor et al., 1997; Hidalgoet al., 2002). Many malignant cell types express highlevels of CD44 and it has been shown in animalmodelsthat injection of reagents interfering with CD44-li-gand interaction can inhibit local tumour growthand metastatic spread (Kajita et al., 2001). These ob-servations suggest that CD44may confer a growth ad-vantage on some neoplastic cells and, therefore, thatthis molecule may be used as a target for cancer ther-apy (Naor et al., 1997). The results of the present studyare consistent with these concepts as all HAs failed toexpress this marker, whilst more than half of HSAswere immunoreactive. CD44 was also expressed bythe proliferating ECs of GT, confirming their molecu-lar similarity with malignant ECs.

Proliferative activity, as determined by theMIB-1 in-dex, was markedly variable in HSA and not correlatedto differentiation and nuclear pleomorphism, suggestingthat tumourswith similarmorphologymayhaveadiffer-ent kinetic behaviour.TheMIB-1 indexofHSAsdidnotdiffer significantly from that of granulation tissue,whereas almost no cycling cells were identified in HAs.

During the course of these immunohistochemicalstudies, we often identified isolated VEGFC,VEGFR-3 and CD117-positive cells in the interstitialstroma of HAs.Morphologically, these were oblong toround cells with round nuclei that were consistentwith either angioblasts or infiltratingmast cells. In or-der to further identify these cells, toluidine blue stain-ing was performed on replicate sections of the tissuesexamined. This staining confirmed that these weremast 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 mediatorsmay 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 anddegrading the extracellular matrix (e.g. chymase,tryptase, matrix metalloproteinases, urokinase, inter-leukins-3, -4 and -8). It has also been postulated thatMCs may play both pro-angiogenic and anti-angio-genic roles in the proliferative and involuting phasesof 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 receptoris SCF, which acts to promote the proliferation, differ-

entiation, migration and secretory activity of thesecells. Endothelial cells in murine are known to secreteSCF, which may act to recruit MCs into the neoplas-tic microenvironment (Meininger et al., 1995). Yama-moto et al. (2000) detected a significantly increasednumber ofMCs in the lesions of human angiosarcomacompared with normal skin, additionally demonstrat-ing the presence of SCF-positive cells in these neoplas-tic tissues.

The above findings may be interpreted to suggestthat tumour cell-derived SCF might recruit infiltrat-ing MCs, which may further contribute to the prolif-eration and progression of tumour cells (Yamamotoet al., 2000). The fact that fewer MCs were detectedin canine HSA compared with HA is at odds withthis hypothesis, but could be explained if there werealternative proliferative mechanisms of an autocrinenature that meant that canine HSA cells were inde-pendent of external stimuli (Yonemaru et al., 2006).

In conclusion, the present study has shown that thepanel ofmarkers employed here is able to distinguish be-nign frommalignant vascular tumours, but not discrim-inate between neoplastic ECs and normal ECsproliferating in GT. This finding is consistent with theontogenetic hypothesis that states that HSA originatesfrom incompletely differentiated, bone marrow-derivedstemcells that arenear or at the stage of endothelial com-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 formolecular interventional therapy for this tumour. Fi-nally, the highnumber ofmast cells infiltratingHAs sug-gests that these cellsmighthavea role in thepathogenesisof benign vascular tumours.

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½ RA

eceived, August 8th, 2008ccepted, October 31st, 2008 �