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Tumour Endothelial Marker-1 is Expressed in Canine Haemangiopericytomas
J. Comp. Path. 2013, Vol. 149, 172e181
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NEOPLASTIC DISEASE
Tumour Endothelial Marker-1 is Expressed in Canine Haemangiopericytomas Y. Fujii*, T. Tsuchiya*, R. Morita*,x, M. Kimura*, K. Suzuki†, N. Machida‡, K. Mitsumori* and M. Shibutani* *Laboratory of Veterinary Pathology, † Laboratory of Veterinary Toxicology, ‡ Laboratory of Veterinary Clinical Oncology, Tokyo University of Agriculture and Technology, Tokyo and x Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan
Summary The aim of this study was to characterize immunohistochemically 18 cases of canine haemangiopericytoma (CHP) using two new candidate markers for pericytes, tumour endothelial marker (TEM)-1 and new glue (NG)-2, as well as the conventional mesenchymal cellular markers, vimentin, a-smooth muscle actin (a-SMA), desmin and von Willebrand factor (vWF). Because pericytes may have the same origin as endothelial or smooth muscle cells or the same differentiation potential as myofibroblasts, 17 cases of leiomyosarcoma (LMS), 20 cases of haemangiosarcoma (HS) and three cases of myofibroblastic sarcoma (MFS) were also examined. Expression of TEM-1 by >10% of the neoplastic population was observed in 94.4% (17/18) of haemangiopericytomas, 23.5% (4/17) of LMSs, 30.0% (6/20) of HSs and 66.7% (2/3) of MFSs. NG-2 expression by >10% of the neoplastic population was observed in 16.7% (3/18) of haemangiopericytomas, 52.9% (9/17) of LMSs, 0% (0/20) of HSs and 33.3% (1/3) of MFSs. Vimentin was expressed by all of tumours. In haemangiopericytoma, the incidence of positive immunoreactivity in >10% of the neoplastic population was 5.6% (1/18) for both a-SMA and desmin and 0% (0/18) for vWF. Considering the phenotypic features of cells expressing TEM-1, CHPs are thought to originate from immature vascular mural cells sharing their phenotype with myofibroblasts. NG-2 expression may be a phenotype of smooth muscle cells rather than pericytes in dogs. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; haemangiopericytoma; new glue-2; tumour endothelial marker-1
Introduction Human haemangiopericytomas are believed to originate from neoplastic pericytes (Battifora, 1973); however, according to the World Health Organization (WHO) classification, this hypothesis is still controversial (Guillou et al., 2002). The histogenesis of canine haemangiopericytomas (CHPs) is also not yet clear, because pericytic features have not been confirmed definitively in these tumours (Donald, 2002). It is also considered that haemangiopericytoma may be a non-specific term applied indiscriminately to several types of canine tumours that are actually of different histological origins Correspondence to: M. Shibutani (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.12.005
(Avallone et al., 2007). Haemangiopericytoma is uncommon in man (Yin et al., 2011), but common in dogs (Santos et al., 2009). Haemangiopericytomas of dogs and people are cutaneous tumours found in adults, and are most commonly found on the limbs. However, human haemangiopericytomas have also been found in non-cutaneous locations, while in dogs they are mostly found in skin and subcutaneous tissue (Pulley and Stannard, 1990). According to the WHO International Histological Classification of Tumours of Skin and Soft Tissues of Domestic Animals, the hallmark of CHP is the presence of perivascular whorls of fusiform cells (Hendrick et al., 1998). This feature may be present in other sarcomas, but it is usually dominant in haemangiopericytomas (Hendrick et al., 1998). Ó 2012 Elsevier Ltd. All rights reserved.
Immunohistochemical Characterization of Canine Haemangiopericytoma
The walls of microvessels are composed of two principal types of cells: endothelial cells, which form the inner lining of the vascular tube, and pericytes, which form the outer sheath (Ozerdem et al., 2002). Pericytes and endothelial cells are closely related and play an important role in neoangiogenesis (Beck and D’Amore, 1997). The mural cells of arteries, arterioles and veins, on the other hand, are smooth muscle cells and the mural cells of capillaries and venules are pericytes (Rhodin, 1968). Smooth muscle cells and pericytes are thought to develop from a common precursor cell (Hughes and Chan-Ling, 2004; Klein et al., 2011). However, the potential for mural precursor cells to differentiate into smooth muscle cells and pericytes has not definitively been confirmed by in-vitro analysis in human cells (Klein et al., 2011). Adiposederived mesenchymal stem cells have been shown to differentiate into vascular cell types including endothelial cells and pericytes (Natesan et al., 2011). The immunohistochemical features of CHPs have been reported (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). Neoplastic cells are positive for vimentin (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004), negative for cytokeratin (Perez et al., 1996; Mazzei et al., 2002), negative for von Willebrand factor (vWF; Perez et al., 1996; Mazzei et al., 2002) and negative for glial fibrillary acidic protein (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). A variable population of neoplastic cells is positive for a-smooth muscle actin (a-SMA), desmin, S100, muscle actin, CD34 and neuron-specific enolase (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). However, there is no definitive cellular marker for pericytes, making it difficult to differentiate this type of tumour from other mesenchymal tumours. Tumour endothelial marker (TEM)-1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumourigenesis, but not in normal mature vessels in man (Bagley et al., 2008). TEM-1 has been identified as a marker for endothelial cells within human tumours (Rettig et al., 1992). In contrast, recent studies have suggested that TEM-1 is expressed preferentially by vascular mural cells (both pericytes and smooth muscle cells; Tomkowicz et al., 2010). However, the functional significance of TEM-1 expression in pericytes and stromal cells, its up-regulation during cancer development and its role in angiogenesis remain largely unknown. New glue (NG)-2 is a transmembrane chondroitin sulphate proteoglycan that is widely expressed in newly formed blood vessels (Fukushi et al., 2004). In the rat retina, NG-2 is expressed both in pericytes and smooth muscle cells (Hughes and Chan-Ling, 2004). These cells share a common origin of precursor
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cells that express NG-2. NG-2 is also considered to exert a role in the facilitation of angiogenesis and NG-2 knock-out mice have been shown to have profoundly decreased proliferation of both pericytes and endothelial cells in the retina (Ozerdem and Stallcup, 2004). The aim of the present study was to elucidate the cellular characteristics of CHPs using two new candidate marker molecules for pericytes, TEM-1 and NG2, in addition to conventional mesenchymal cell markers. We also examined the immunohistochemical features of leiomyosarcomas (LMSs) and haemangiosarcomas (HSs), as they may share identical precursor cells, in order to determine the specificity of the candidate molecules for haemangiopericytomas. Because TEM-1 has also been reported to be expressed in tumour-associated myofibroblasts in man (Christian et al., 2008), we also examined the immunohistochemical features of myofibroblastic sarcomas (MFSs).
Materials and Methods The study included samples of 18 haemangiopericytomas, 17 LMSs, 20 HSs and three MFSs taken from the archives of two laboratories (the Laboratory of Veterinary Pathology and the Laboratory of Veterinary Clinical Oncology, Tokyo University of Agriculture and Technology). Tumours were diagnosed as haemangiopericytomas if they exhibited a typical cellular arrangement of perivascular whorled pattern with fusiform cells having short to long cytoplasmic processes and vesicular nuclei, with a lacy to moderately coarse chromatin pattern containing one or two round, indistinct to prominent nucleoli (Hendrick et al., 1998; Cho and Park, 2006). Cells may also be arranged in interlacing bundles and storiform or staghorn patterns, and sometimes have accompanying myxoid matrix (Hendrick et al., 1998). LMS was diagnosed if tumours were composed of interwoven fascicles of long, plump spindle to strap-like cells with abundant pink cytoplasm, distinct cell borders and elongated, blunt-ended nuclei (Hendrick et al., 1998). HS was diagnosed if tumours were composed of spindle-shaped, polygonal or oval neoplastic cells forming recognizable vascular clefts or channels somewhere in the tumour (Hendrick et al., 1998). MFS was diagnosed if tumours were composed of spindle-shaped neoplastic cells with illdefined cytoplasmic borders arranged in fascicles or bundles and storiform or whorled patterns intersected with variable amounts of collagenous tissue and a mucinous matrix (Mentzel and Fletcher, 2002; Hojo et al., 2012; Tsuchiya et al., 2012). Tumour tissues were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin
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Table 1 Clinical presentation of dogs investigated Case number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
Tumour type
Sex
Breed
Age (years)
Tumour location
CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS MFS MFS MFS
M F FN M FN M F M FN M M FN F M F MN M F F FN F FN F FN F F FN MN F MN MN FN FN FN F FN MN M MN MN FN MN M F M MN F F MN MN FN M MN MN F M F F
Crossbred Crossbred Crossbred Maltese Crossbred Crossbred Welsh corgi Crossbred Crossbred Labrador retriever Miniature schnauzer Shih Tzu Crossbred Crossbred Golden retriever Welsh corgi Crossbred Welsh corgi Miniature dachshund Shih Tzu Miniature dachshund Crossbred NR Cavalier King Charles spaniel Crossbred Labrador retriever Golden retriever Crossbred Maltese Welsh corgi Beagle Shiba Inu Cocker spaniel Shetland sheepdog Miniature dachshund Golden retriever Crossbred Chihuahua Spitz Beagle Golden retriever Golden retriever Flat-coated retriever Miniature dachshund Crossbred Welsh corgi Springer spaniel Crossbred Crossbred Welsh corgi Golden retriever Golden retriever Miniature dachshund Golden retriever Cocker spaniel Crossbred Polish Lowland sheepdog Crossbred
14 10 10 11 12 15 11 13 15 11 13 7 15 13 7 14 14 11 10 14 11 11 12 9 7 10 11 9 11 9 8 11 10 11 11 13 10 12 14 9 11 13 10 8 10 8 12 15 10 9 11 10 6 7 10 6 11 12
NR Skin of the forelimb Skin of the rump Skin of the forelimb Skin of the hindlimb Skin of the neck Skin of the upper perianal area Skin of the forelimb Skin of the forelimb Skin of the thorax Skin of the shoulder Skin of the shoulder Skin of the hindlimb Skin of the forelimb Skin of the hindlimb Skin of the dorsum Skin of the forelimb Skin of the hindlimb Spleen Small intestine Peritoneal region Rectum Uterus Caecum Vagina NR NR Jejunum Stomach Spleen Ileum Stomach Rump Bladder Spleen Spleen Spleen Skin of the thorax Skin of the forelimb and hindlimb Skin of the penis Liver Skin of the head and dorsum Spleen Spleen Spleen Spleen Spleen Skin of the neck Spleen Spleen Spleen Spleen Liver Spleen Ovary Skin of the abdomen Spleen Duodenum
M, male; F, female; FN, neutered female; MN, neutered male; NR, not recorded.
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Immunohistochemical Characterization of Canine Haemangiopericytoma
wax, sectioned and stained with haematoxylin and eosin (HE). For immunohistochemistry (IHC), the avidinebiotineperoxidase complex technique was employed, using a VECTASTAINÒ Elite ABC Kit (Vector Laboratories Inc., Burlingame, California, USA) with primary antibodies against vimentin, aSMA, desmin, vWF, TEM-1 and NG-2 (Table 2). The chromogen was 3, 30 -diaminobenzidine and the counterstain was haematoxylin. Dermal tissues were prepared from a dog as a positive control for vimentin, small intestine for a-SMA and desmin, and cerebrum for vWF. Non-immunized sera were substituted for the primary antibody as negative controls for IHC. The expression of each molecule was assessed visually by two independent readers in a blinded fashion. The percentage of positively labelled neoplastic cells was determined in five randomly selected fields at 100 magnification (+, 0e10%; ++, 10e50%; +++, 50e100%). The incidence of immunoreactive cases was compared statistically using Fisher’s exact probability test. The immunoreactivity analyzed by scoring was compared statistically using the ManneWhitney’s U-test. Comparisons were made between haemangiopericytomas and other types of tumour.
Results Table 1 presents the details of the dogs included in the study. The mean age of the dogs with haemangiopericytoma was 12.0 years (range 7e15 years). Of the 18 dogs with haemangiopericytoma, nine were male (one neutered) and nine were female (four neutered). Seventeen tumours had developed on the skin and the location of one was not specified. The mean age of the dogs with LMS was 10.3 years (range 7e14 years). Three of the 17 dogs with LMS were neutered males (MNs) and 14 were females (seven neutered). The mean age of the dogs with HS was 10.4 years (range 6e15 years). Thirteen of the 20 dogs with HS were male (nine neutered) and seven were female (three
neutered). The mean age of the dogs with MFS was 9.7 years (range 6e12). One of these three dogs was male and two were female. Table 3 and Table 4 show the results of the immunohistochemical labelling for cellular markers in the different types of tumours studied and Table 5 summarizes the incidence and grade of immunoreactivity of positive cases for each cellular marker. There was strong cytoplasmic immunoreactivity for vimentin in neoplastic cells in all cases of haemangiopericytoma, LMS and HS. In these cases, >50% of the neoplastic cells were positive for vimentin except for one LMS in which there was approximately 40% positivity. There were no significant differences in the incidence and grade of vimentin immunoreactivity between haemangiopericytomas and LMSs or HSs. There was strong cytoplasmic immunoreactivity for a-SMA in neoplastic cells in all cases of LMS. Among these, most cases had >50% positive cells (16 cases, 94.1% incidence) and one case had <10% positive cells (5.9%). There was cytoplasmic immunoreactivity for a-SMA in six cases of haemangiopericytoma (33.3%). Among these, five cases had <10% positive cells (27.8%), while one case had approximately 60% positive cells (5.6%). There was no immunoreactivity for a-SMA in neoplastic endothelial cells of HSs. However, positive immunoreactivity for desmin was observed in the stromal cell population among the neoplastic vascular structures. Both incidence and grade of a-SMA-immunoreactivity in haemangiopericytomas were significantly different from those in LMSs and HSs. There was strong cytoplasmic immunoreactivity for desmin in neoplastic cells in 15 out of 17 cases of LMS (88.2%). Among these, 13 cases had >50% positive cells (76.5%), one case had approximately 30% positive cells (5.9%) and one case had <10% positive cells (5.9%). Three cases of haemangiopericytoma (16.7%) had strong cytoplasmic immunoreactivity for desmin. Among these, two cases had <10% positive cells (11.1%) and one case had approximately 40% positive cells (5.6%). In HSs, immunoreactivity
Table 2 Immunohistochemical methodology Antibody
Antibody type
Dilution
Antigen retrieval
Vimentin a-SMA Desmin
Mouse monoclonal Mouse monoclonal Mouse monoclonal
1 in 100 1 in 100 1 in 50
Autoclaving (121 C, 10 min) Microwaving (90 C, 10 min) Microwaving (90 C, 10 min)
vWF TEM-1 NG-2 chondroitin sulphate proteoglycan
Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal
1 in 500 1 in 200 1 in 200
Microwaving (90 C, 10 min) None None
Source Dako, Glostrup, Denmark Dako Thermo Fisher Scientific, Waltham, Massachusetts, USA Dako Abcam, Cambridge, UK Millipore, Billerica, Massachusetts, USA
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Table 4 Immunohistochemical labelling intensity in MFS
Table 3 Immunohistochemical labelling intensity in haemangiopericytoma, HS and LMS Case Tumour number type 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS
Case number
Immunohistochemical reactivity Vimentin a-SMA Desmin
vWF
TEM-1
NG-2
+ + + +++ + + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + +++
+ +++ +++ + + + +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ ++ +++ ++ + ++
+++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + + +++ + + + + +++ ++ +++ ++ +++ ++
+ ++ + + + + + +++ +++ + + + + +++ ++ + ++ +++ ++ ++ ++ ++ +++ + +
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
+ + ++ ++ + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
, 0% positive cells; +, 1e10% positive cells; ++, 10e50% positive cells; +++, 50e100% positive cells.
56 57 58
Immunohistochemical reactivity Vimentin
a-SMA
Desmin
vWF
TEM-1
NG-2
+++ +++ +++
+++ + +
+
+
+++ +++
+++ +
, 0% positive cells; +, 1e10% positive cells; ++, 10e50% positive cells; +++, 50e100% positive cells.
for desmin was only observed in the non-neoplastic stromal cell population (Fig. 1). Both incidence and grade of desmin immunoreactivity in haemangiopericytomas were significantly lower than those in LMSs. There was cytoplasmic immunoreactivity for vWF in neoplastic cells of all cases of HS. Among these, 12 cases had >50% positive cells (60.0% incidence), four cases had 10e50% positive cells (20.0%) and four cases had <10% positive cells (20.0%). All cases of haemangiopericytoma were negative for vWF. All cases of LMS were negative for vWF except for one case showing <10% positive cells (5.9%). Both incidence and grade of vWF immunoreactivity in haemangiopericytomas were significantly lower than those in HSs. In normal tissues, TEM-1 was expressed by a small population of pericytes in capillaries and venules (Fig. 2A). There was no expression by vascular smooth muscle cells and vascular endothelial cells of most cases (Fig. 2B); however, a few endothelial cells in a few cases did show TEM-1 labelling. There was cytoplasmic immunoreactivity for TEM-1 in neoplastic cells in 17 out of 18 cases of haemangiopericytoma (94.4% incidence). Among these, 15 cases had >50% positive cells (83.3%; Fig. 3A) and two cases had 10e50% positive cells (11.1%). There was cytoplasmic immunoreactivity for TEM-1 in the neoplastic cells in eight out of 17 cases of LMS (47.1%). Among these, four cases had <10% positive cells (23.5%; Fig. 3B), three cases had 10e50% positive cells (17.6%) and one case had approximately 50% positive cells (5.9%). There was cytoplasmic immunoreactivity for TEM-1 in neoplastic cells in 10 out of 20 cases of HS (50.0%). Among these, four cases had <10% positive cells (20.0%; Fig. 3C), three cases had 10e50% positive cells (15.0%) and three cases had >50% positive cells (15.0%). The percentages of cases in which >10% of the neoplastic cells expressed TEM-1 were 94.4% in haemangiopericytomas, 23.5% in LMSs and 30.0% in HSs. Both incidence and grade of TEM-1-immunoreactivity in haemangiopericytomas were significantly higher than those in LMSs and HSs.
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Table 5 Incidence and grade of immunoreactivity of positively labelled tumours Tumour type
Number of tumours
Immunohistochemical reactivity Vimentin
CHP LMS HS MFS
18 17 20 3
*
†
18 (0, 0, 18) 17 (0, 1, 16) 20 (0, 0, 20) 3 (0, 0, 3)
a-SMA
Desmin
vWF
TEM-1
NG-2
6 (5, 0, 1) 17‡ (1, 0, 16){ 0‡ (0, 0, 0){ 3 (2, 0, 1)x
3 (2, 1, 0) 15‡ (1, 1, 13){ 0 (0, 0, 0) 1 (1, 0, 0)
0 (0, 0, 0) 1 (1, 0, 0) 20‡ (4, 4, 12){ 1 (1, 0, 0)x
17 (0, 2, 15) 8‡ (4, 3, 1){ 10‡ (4, 3, 3){ 2 (0, 0, 2)
13 (10, 1, 2) 11 (2, 6, 3) 1‡ (1, 0, 0){ 2 (1, 0, 1)
*
Incidence of positive cases. Number of animals with each grade of immunoreactivity in the order of +, ++ and +++. ‡ Significantly different from CHP by Fisher’s exact probability test (P <0.01). x Significantly different from CHP by ManneWhitney U-test (P <0.05). { Significantly different from CHP by ManneWhitney U-test (P <0.01). †
NG-2 was negative in pericytes of capillaries and venules, and negative in vascular endothelial cells in normal tissues (Fig. 4A). Diffuse immunoreactivity for NG-2 was observed in intestinal smooth muscle cells (Fig. 4B). However, vascular smooth muscle cells were mostly negative for NG-2. In the neoplastic tissues, there was cytoplasmic immunoreactivity for NG-2 in neoplastic cells in 13 out of 18 cases (72.2%) of haemangiopericytomas. Among these, 10 cases had <10% positive cells (55.6%; Fig. 5A), one case had approximately 20% positive cells (5.6%) and two cases had >50% positive cells (11.1%). There was cytoplasmic immunoreactivity for NG-2 in neoplastic cells in 11 out of 17 cases of LMS (64.7%). Among these, six cases had 10e50% positive cells (35.3%; Fig. 5B), three cases had >50% positive cells (17.6%) and two cases had <10% positive cells (11.8%). NG-2 immunoreactivity was found in only one case of HS (5.0%), with <10% of the neoplastic cells being positive. Nineteen cases of HS showed no
Fig. 1. Positive immunoreactivity for desmin in stromal cells in a HS (number 53). Neoplastic endothelial cells lack desmin expression. IHC. Bar, 50 mm.
expression of NG-2 (95.0%; Fig. 5C). Both incidence and grade of NG-2 immunoreactivity in haemangiopericytomas were significantly higher than those in HSs. All three cases of MFS showed immunoreactivity for vimentin in >50% of the neoplastic cells. In one case >50% of cells expressed a-SMA (33.3% incidence) and two cases had <10% a-SMA-positive cells (66.7%). One case had <10% of labelled cells (33.3%) and two cases were entirely negative for both desmin and vWF. Two cases had >50% of cells labelled for TEM-1 (66.7%) and one was negative. With regard to NG-2, one case had >50% positive cells (33.3%), one case had <10% positive cells (33.3%) and one was negative. Grade of immunoreactivity of a-SMA and vWF in haemangiopericytomas was significantly lower than that in MFSs.
Discussion In the present study, most cases of LMS expressed aSMA and desmin. However, only 33.3% of haemangiopericytomas showed positive immunoreactivity for a-SMA, while even fewer cases showed positive immunoreactivity for desmin. There was consistent expression of vWF by neoplastic endothelial cells in HSs. With regard to TEM-1, haemangiopericytomas showed expression by >50% of cells, but few LMSs and HSs displayed this level of expression. Both incidence and grade of TEM-1-immunoreactivity in haemangiopericytomas were significantly higher than those in LMSs and HSs, suggesting that TEM-1 expression is associated most closely with haemangiopericytomas. NG-2 expression was restricted to a small number of cells in haemangiopericytomas and LMSs. These results suggest that diagnostically, haemangiopericytomas can be distinguished from HSs by immunoreactivity for vWF in the latter tumour type. Moreover, haemangiopericytomas can be distinguished from LMSs using immunoreactivity
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Fig. 2. TEM-1 expression in vascular mural cells. (A) Some pericytes in a capillary show positive immunoreactivity for TEM-1. IHC. Bar, 50 mm. (B) Smooth muscle cells and endothelial cells in an arteriole do not express TEM-1. IHC. Bar, 50 mm.
for TEM-1 and desmin, respectively, having greater expression in these two tumour types. In contrast, NG-2 does not contribute to the differential diagnosis between LMS and haemangiopericytoma. TEM-1 was expressed by two of three cases of MFS, suggesting that TEM-1 could be used as a marker for neoplastic myofibroblasts.
TEM-1 was not expressed by the majority of vascular endothelial cells. TEM-1 expression was originally found in embryonic or tumour-associated endothelial cells (St Croix et al., 2000), suggesting that TEM-1 can be expressed by immature proliferating endothelial cells. However, we found TEM-1 expression in a small number of neoplastic cells in HSs. TEM-1 is reported
Fig. 3. TEM-1 expression in haemangiopericytomas, LMSs and HSs. (A) Diffuse distribution of neoplastic cells showing cytoplasmic immunoreactivity for TEM-1 in a haemangiopericytoma (number 5). IHC. Bar, 50 mm. (B) A few neoplastic cells show positive immunoreactivity for TEM-1 in a LMS (number 26). IHC. Bar, 50 mm. (C) A few neoplastic cells show positive immunoreactivity for TEM-1 in an HS (number 36). IHC. Bar, 50 mm.
Immunohistochemical Characterization of Canine Haemangiopericytoma
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Fig. 4. NG-2 expression in pericytes, endothelial cells and smooth muscle cells. (A) Pericytes and endothelial cells in a capillary do not express NG-2. IHC. Bar, 50 mm. (B) Smooth muscle cells in the muscle layer of the jejunum show positive immunoreactivity for NG-2. IHC. Bar, 200 mm.
to be a specific marker for tumour-associated myofibroblasts and neovessel mural cells (i.e. pericytes and smooth muscle cells) and is thought to control the migration and proliferation of these cells (Christian et al., 2008). This hypothesis is consistent with partial immunoreactivity for a-SMA in CHPs (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). In the present
study there was scattered TEM-1 expression in capillary pericytes. However, there was no TEM-1 expression by vascular smooth muscle cells. When blood vessels grow and mature, pericytes differentiate into vascular smooth muscle cells where TEM-1 expression is quickly down-regulated to below detection level (Tomkowicz et al., 2010).
Fig. 5. NG-2 expression in haemangiopericytomas, LMSs and HSs. (A) Neoplastic cells showing cytoplasmic immunoreactivity for NG-2 in a haemangiopericytoma (number 9). IHC. Bar, 50 mm. (B) Diffuse distribution of neoplastic cells showing cytoplasmic immunoreactivity for NG-2 in a LMS (number 28). IHC. Bar, 50 mm. (C) HS cells do not express NG-2 (number 37). IHC. Bar, 50 mm.
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Conflicting immunohistochemical data have suggested that CHPs are actually a heterogeneous tumour population with different histological origins (Avallone et al., 2007). In the present study a subpopulation of LMSs and haemangiopericytomas expressed TEM-1, with this expression being more common in haemangiopericytomas. Pericytes and vascular smooth muscle cells share common precursor cells (Hughes and Chan-Ling, 2004; Klein et al., 2011) and pericytes express TEM-1 strongly during angiogenesis (Tomkowicz et al., 2010). TEM-1 therefore represents the activated immature phenotype of mural cells, most likely pericytes. The finding of consistent expression of TEM-1 by CHPs suggests a single cellular origin of this tumour. TEM-1 was also expressed by the majority of neoplastic cells in two of three MFSs, which is in accordance with expression patterns in tumour-associated myofibroblasts (Christian et al., 2008). These results suggest that TEM-1 expression is found in populations of myofibroblasts as well as immature vascular mural cells, with preferential expression in pericytes. Histologically, there are some similarities between CHPs and MFSs. For example, cells in both tumour types may be arranged in fascicles and storiform, staghorn or whorled patterns (Donald, 2002; Tsuchiya et al., 2012). In contrast, several types of human sarcomas with different cellular origin have recently been shown to variably express TEM-1, and it has been suggested that this molecule plays a role in malignant behaviour of these tumours rather than representing a marker of a specific cell type (Rouleau et al., 2011). These findings contrast with the consistent expression of TEM-1 in CHPs, so further studies are necessary in order to determine the specificity of TEM-1 expression in canine sarcomas of different cellular origin. NG-2 expression by >10% of neoplastic cells was more frequent among LMSs than haemangiopericytomas. HSs did not express NG-2 except for one case with minor immunoreactivity. In the rat retina, NG-2 is expressed both in pericytes and smooth muscle cells (Hughes and Chan-Ling, 2004; Murfee et al., 2005), and NG-2 is considered to play a role in microvascular remodelling (Ozerdem and Stallcup, 2004; Murfee et al., 2006). However, NG-2 is not expressed in endothelial cells (Murfee et al., 2005). In the present study, NG-2 was not expressed by vascular cells (i.e. endothelial cells, pericytes and smooth muscle cells); however, intestinal smooth muscle cells all expressed NG-2. The reason for the lack of NG-2 expression in vascular smooth muscle cells is not clear, but the findings of the present study suggest that NG-2 is a marker of smooth muscle cells rather than pericytes in dogs.
It has been suggested that pericytes are involved in supporting the neoplastic vascular channels that characterize HSs (Kusewitt et al., 2000). However, in the present study, cells positive for a-SMA or desmin were negative for TEM-1 expression. TEM-1 is down-regulated in pericytes during development, with a significant decrease in expression in adult tissues (MacFadyen et al., 2007). These results suggest that HS cells positive for a-SMA or desmin, but negative for TEM-1 expression, were phenotypically mature mural cells. In conclusion, on the basis of TEM-1 expression, the neoplastic cells of CHPs are considered to have originated from immature vascular mural cells, sharing a phenotype with myofibroblasts. The specificity of TEM-1 expression should be examined further in canine sarcomas. An immature cellular phenotype may be the reason for partial expression of a-SMA in these tumours. NG-2 expression may be associated with smooth muscle cells rather than pericytes in dogs.
Acknowledgment The authors thank Mrs. S. Suzuki for technical assistance in preparing the histological specimens.
Conflict of Interest Statement The authors declare that there are no conflicts of interest.
References Avallone G, Helmbold P, Caniatti M, Stefanello D, Nayak RC et al. (2007) The spectrum of canine cutaneous perivascular wall tumors: morphologic, phenotypic and clinical characterization. Veterinary Pathology, 44, 607e620. Bagley RG, Honma N, Weber W, Boutin P, Rouleau C et al. (2008) Endosialin/TEM 1/CD248 is a pericyte marker of embryonic and tumor neovascularization. Microvascular Research, 76, 180e188. Battifora H (1973) Hemangiopericytoma: ultrastructural study of five cases. Cancer, 31, 1418e1432. Beck L Jr., D’Amore PA (1997) Vascular development: cellular and molecular regulation. FASEB Journal, 11, 365e373. Chijiwa K, Uchida K, Tateyama S (2004) Immunohistochemical evaluation of canine peripheral nerve sheath tumours and other soft tissue sarcomas. Veterinary Pathology, 41, 307e318. Cho HS, Park NY (2006) Primary haemangiopericytoma in the pelvic cavity of a dog. Journal of Veterinary Medicine A: Physiology, Pathology and Clinical Medicine, 53, 198e201. Christian S, Winkler R, Helfrich I, Boos AM, Besemfelder E (2008) Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-
Immunohistochemical Characterization of Canine Haemangiopericytoma
associated mural cells. American Journal of Pathology, 172, 486e494. Donald JM (2002) Tumors of the skin and soft tissues. In: Tumors in Domestic Animals, 4th Edit., MH Goldschmidt, MJ Hendrick, Eds., Iowa State Press, Ames, pp. 45e117. Fukushi J, Makagiansar IT, Stallcup WB (2004) NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and a3b1 integrin. Molecular Biology of the Cell, 15, 3580e3590. Guillou L, Fletcher JA, Fletcher CDM, Mandahl N (2002) Extrapleural solitary fibrous tumour and haemangiopericytoma. In: Pathology and Genetics of Tumours of Soft Tissue and Bone: World Health Organization Classification of Tumours, CDM Fletcher, KK Unni, F Mertens, Eds., IARC Press, Lyon, pp. 86e90. Hendrick MJ, Mahaffey EA, Moore FM, Vos JH, Walder EJ (1998) Unclassified tumors. In: Histological Classification of Mesenchymal Tumors of Skin and Soft Tissues of Domestic Animals, 2nd Series, Vol. 2, FY Schulman, Ed., Armed Forces Institute of Pathology, Washington DC, pp. 32e33. Hojo Y, Tsuchiya T, Shiraki A, Suzuki K, Shibutani M et al. (2012) High-grade myofibroblastic sarcoma of inguinal region in a dog. Journal of Veterinary Medical Science, 74, 625e628. Hughes S, Chan-Ling T (2004) Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo. Investigative Ophthalmology and Visual Science, 45, 2795e2806. Klein D, Weisshardt P, Kleff V, Jastrow H, Jakob HG et al. (2011) Vascular wall-resident CD44+ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to new vessel maturation. PloS One, 6, e20540. Kusewitt DF, Hubbard GB, Warbritton AR, McLeskey SW, Miska KB et al. (2000) Cellular origins of ultraviolet radiation-induced corneal tumours in the grey, short-tailed South American opossum (Monodelphis domestica). Journal of Comparative Pathology, 123, 88e95. MacFadyen J, Savage K, Wienke D, Isacke CM (2007) Endosialin is expressed on stromal fibroblasts and CNS pericytes in mouse embryos and is downregulated during development. Gene Expression Patterns, 7, 363e369. Mazzei M, Millanta F, Citi S, Lorenzi D, Poli A (2002) Haemangiopericytoma: histological spectrum, immunohistochemical characterization and prognosis. Veterinary Dermatology, 13, 15e21. Mentzel T, Fletcher JA (2002) Low grade myofibroblastic sarcoma. In: Pathology and Genetics of Tumours of Soft Tissue and Bone: World Health Organization Classification of Tumours, CDM Fletcher, KK Unni, F Mertens, Eds., IARC Press, Lyon, pp. 91e95. Murfee WL, Rehorn MR, Peirce SM, Skalak TC (2006) Perivascular cells along venules upregulate NG2 expression during microvascular remodeling. Microcirculation, 13, 261e273. Murfee WL, Skalak TC, Peirce SM (2005) Differential arterial/venous expression of NG2 proteoglycan in peri-
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vascular cells along microvessels: identifying a venulespecific phenotype. Microcirculation, 12, 151e160. Natesan S, Zhang G, Baer DG, Walters TJ, Christy RJ et al. (2011) A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation. Tissue Engineering Part A, 17, 941e953. Ozerdem U, Monosov E, Stallcup WB (2002) NG2 proteoglycan expression by pericytes in pathological microvasculature. Microvascular Research, 63, 129e134. Ozerdem U, Stallcup WB (2004) Pathological angiogenesis is reduced by targeting pericytes via the NG2 proteoglycan. Angiogenesis, 7, 269e276. on E, de Lara FC, Carrasco L Perez J, Bautista MJ, Roll et al. (1996) Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog. Veterinary Pathology, 33, 391e397. Pulley LT, Stannard AA (1990) Tumors of the skin and soft tissue. In: Tumors in Domestic Animals, 3rd Edit., JE Moulton, Ed., University of California Press, Berkeley, pp. 48e51. Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA et al. (1992) Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proceedings of the National Academy of Sciences of the USA, 89, 10832e10836. Rhodin JAG (1968) Ultrastructure of mammalian venous capillaries, venules, and small collecting veins. Journal of Ultrastructure Research, 25, 452e500. Rouleau C, Smale R, Fu YS, Hui G, Wang F et al. (2011) Endosialin is expressed in high grade and advanced sarcomas: evidence from clinical specimens and preclinical modeling. International Journal of Oncology, 39, 73e89. Santos SV, Torres LN, Da Silva TC, De S a LRM, Matera JM et al. (2009) Canine hemangiopericytomas: cell proliferation and apoptosis in the perivascular, storiform and epithelioid histological subtypes and their significance for prognosis. Brazilian Journal of Veterinary Pathology, 2, 23e28. St Croix B, Rago C, Velculescu V, Traverso G, Romans KE et al. (2000) Genes expressed in human tumor endothelium. Science, 289, 1197e1202. Tomkowicz B, Rybinski K, Sebeck D, Sass P, Nicolaides NC et al. (2010) Endosialin/TEM-1/CD248 regulates pericyte proliferation through PDGF receptor signaling. Cancer Biology and Therapy, 9, 908e915. Tsuchiya T, Suzuki K, Hojo Y, Shiraki A, Imaoka M et al. (2012) Low-grade myofibroblastic sarcoma of the maxillary region in a dog. Journal of Comparative Pathology, 147, 42e45. Yin B, Liu L, Li YD, Geng DY, Du ZG (2011) Retroperitoneal hemangiopericytoma: case report and literature review. Chinese Medical Journal, 124, 155e156.
August 5th, 2012 ½ Received, Accepted, December 17th, 2012
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Tumour Endothelial Marker-1 is Expressed in Canine Haemangiopericytomas Y. Fujii*, T. Tsuchiya*, R. Morita*,x, M. Kimura*, K. Suzuki†, N. Machida‡, K. Mitsumori* and M. Shibutani* *Laboratory of Veterinary Pathology, † Laboratory of Veterinary Toxicology, ‡ Laboratory of Veterinary Clinical Oncology, Tokyo University of Agriculture and Technology, Tokyo and x Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan
Summary The aim of this study was to characterize immunohistochemically 18 cases of canine haemangiopericytoma (CHP) using two new candidate markers for pericytes, tumour endothelial marker (TEM)-1 and new glue (NG)-2, as well as the conventional mesenchymal cellular markers, vimentin, a-smooth muscle actin (a-SMA), desmin and von Willebrand factor (vWF). Because pericytes may have the same origin as endothelial or smooth muscle cells or the same differentiation potential as myofibroblasts, 17 cases of leiomyosarcoma (LMS), 20 cases of haemangiosarcoma (HS) and three cases of myofibroblastic sarcoma (MFS) were also examined. Expression of TEM-1 by >10% of the neoplastic population was observed in 94.4% (17/18) of haemangiopericytomas, 23.5% (4/17) of LMSs, 30.0% (6/20) of HSs and 66.7% (2/3) of MFSs. NG-2 expression by >10% of the neoplastic population was observed in 16.7% (3/18) of haemangiopericytomas, 52.9% (9/17) of LMSs, 0% (0/20) of HSs and 33.3% (1/3) of MFSs. Vimentin was expressed by all of tumours. In haemangiopericytoma, the incidence of positive immunoreactivity in >10% of the neoplastic population was 5.6% (1/18) for both a-SMA and desmin and 0% (0/18) for vWF. Considering the phenotypic features of cells expressing TEM-1, CHPs are thought to originate from immature vascular mural cells sharing their phenotype with myofibroblasts. NG-2 expression may be a phenotype of smooth muscle cells rather than pericytes in dogs. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; haemangiopericytoma; new glue-2; tumour endothelial marker-1
Introduction Human haemangiopericytomas are believed to originate from neoplastic pericytes (Battifora, 1973); however, according to the World Health Organization (WHO) classification, this hypothesis is still controversial (Guillou et al., 2002). The histogenesis of canine haemangiopericytomas (CHPs) is also not yet clear, because pericytic features have not been confirmed definitively in these tumours (Donald, 2002). It is also considered that haemangiopericytoma may be a non-specific term applied indiscriminately to several types of canine tumours that are actually of different histological origins Correspondence to: M. Shibutani (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.12.005
(Avallone et al., 2007). Haemangiopericytoma is uncommon in man (Yin et al., 2011), but common in dogs (Santos et al., 2009). Haemangiopericytomas of dogs and people are cutaneous tumours found in adults, and are most commonly found on the limbs. However, human haemangiopericytomas have also been found in non-cutaneous locations, while in dogs they are mostly found in skin and subcutaneous tissue (Pulley and Stannard, 1990). According to the WHO International Histological Classification of Tumours of Skin and Soft Tissues of Domestic Animals, the hallmark of CHP is the presence of perivascular whorls of fusiform cells (Hendrick et al., 1998). This feature may be present in other sarcomas, but it is usually dominant in haemangiopericytomas (Hendrick et al., 1998). Ó 2012 Elsevier Ltd. All rights reserved.
Immunohistochemical Characterization of Canine Haemangiopericytoma
The walls of microvessels are composed of two principal types of cells: endothelial cells, which form the inner lining of the vascular tube, and pericytes, which form the outer sheath (Ozerdem et al., 2002). Pericytes and endothelial cells are closely related and play an important role in neoangiogenesis (Beck and D’Amore, 1997). The mural cells of arteries, arterioles and veins, on the other hand, are smooth muscle cells and the mural cells of capillaries and venules are pericytes (Rhodin, 1968). Smooth muscle cells and pericytes are thought to develop from a common precursor cell (Hughes and Chan-Ling, 2004; Klein et al., 2011). However, the potential for mural precursor cells to differentiate into smooth muscle cells and pericytes has not definitively been confirmed by in-vitro analysis in human cells (Klein et al., 2011). Adiposederived mesenchymal stem cells have been shown to differentiate into vascular cell types including endothelial cells and pericytes (Natesan et al., 2011). The immunohistochemical features of CHPs have been reported (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). Neoplastic cells are positive for vimentin (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004), negative for cytokeratin (Perez et al., 1996; Mazzei et al., 2002), negative for von Willebrand factor (vWF; Perez et al., 1996; Mazzei et al., 2002) and negative for glial fibrillary acidic protein (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). A variable population of neoplastic cells is positive for a-smooth muscle actin (a-SMA), desmin, S100, muscle actin, CD34 and neuron-specific enolase (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). However, there is no definitive cellular marker for pericytes, making it difficult to differentiate this type of tumour from other mesenchymal tumours. Tumour endothelial marker (TEM)-1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumourigenesis, but not in normal mature vessels in man (Bagley et al., 2008). TEM-1 has been identified as a marker for endothelial cells within human tumours (Rettig et al., 1992). In contrast, recent studies have suggested that TEM-1 is expressed preferentially by vascular mural cells (both pericytes and smooth muscle cells; Tomkowicz et al., 2010). However, the functional significance of TEM-1 expression in pericytes and stromal cells, its up-regulation during cancer development and its role in angiogenesis remain largely unknown. New glue (NG)-2 is a transmembrane chondroitin sulphate proteoglycan that is widely expressed in newly formed blood vessels (Fukushi et al., 2004). In the rat retina, NG-2 is expressed both in pericytes and smooth muscle cells (Hughes and Chan-Ling, 2004). These cells share a common origin of precursor
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cells that express NG-2. NG-2 is also considered to exert a role in the facilitation of angiogenesis and NG-2 knock-out mice have been shown to have profoundly decreased proliferation of both pericytes and endothelial cells in the retina (Ozerdem and Stallcup, 2004). The aim of the present study was to elucidate the cellular characteristics of CHPs using two new candidate marker molecules for pericytes, TEM-1 and NG2, in addition to conventional mesenchymal cell markers. We also examined the immunohistochemical features of leiomyosarcomas (LMSs) and haemangiosarcomas (HSs), as they may share identical precursor cells, in order to determine the specificity of the candidate molecules for haemangiopericytomas. Because TEM-1 has also been reported to be expressed in tumour-associated myofibroblasts in man (Christian et al., 2008), we also examined the immunohistochemical features of myofibroblastic sarcomas (MFSs).
Materials and Methods The study included samples of 18 haemangiopericytomas, 17 LMSs, 20 HSs and three MFSs taken from the archives of two laboratories (the Laboratory of Veterinary Pathology and the Laboratory of Veterinary Clinical Oncology, Tokyo University of Agriculture and Technology). Tumours were diagnosed as haemangiopericytomas if they exhibited a typical cellular arrangement of perivascular whorled pattern with fusiform cells having short to long cytoplasmic processes and vesicular nuclei, with a lacy to moderately coarse chromatin pattern containing one or two round, indistinct to prominent nucleoli (Hendrick et al., 1998; Cho and Park, 2006). Cells may also be arranged in interlacing bundles and storiform or staghorn patterns, and sometimes have accompanying myxoid matrix (Hendrick et al., 1998). LMS was diagnosed if tumours were composed of interwoven fascicles of long, plump spindle to strap-like cells with abundant pink cytoplasm, distinct cell borders and elongated, blunt-ended nuclei (Hendrick et al., 1998). HS was diagnosed if tumours were composed of spindle-shaped, polygonal or oval neoplastic cells forming recognizable vascular clefts or channels somewhere in the tumour (Hendrick et al., 1998). MFS was diagnosed if tumours were composed of spindle-shaped neoplastic cells with illdefined cytoplasmic borders arranged in fascicles or bundles and storiform or whorled patterns intersected with variable amounts of collagenous tissue and a mucinous matrix (Mentzel and Fletcher, 2002; Hojo et al., 2012; Tsuchiya et al., 2012). Tumour tissues were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin
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Table 1 Clinical presentation of dogs investigated Case number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
Tumour type
Sex
Breed
Age (years)
Tumour location
CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS MFS MFS MFS
M F FN M FN M F M FN M M FN F M F MN M F F FN F FN F FN F F FN MN F MN MN FN FN FN F FN MN M MN MN FN MN M F M MN F F MN MN FN M MN MN F M F F
Crossbred Crossbred Crossbred Maltese Crossbred Crossbred Welsh corgi Crossbred Crossbred Labrador retriever Miniature schnauzer Shih Tzu Crossbred Crossbred Golden retriever Welsh corgi Crossbred Welsh corgi Miniature dachshund Shih Tzu Miniature dachshund Crossbred NR Cavalier King Charles spaniel Crossbred Labrador retriever Golden retriever Crossbred Maltese Welsh corgi Beagle Shiba Inu Cocker spaniel Shetland sheepdog Miniature dachshund Golden retriever Crossbred Chihuahua Spitz Beagle Golden retriever Golden retriever Flat-coated retriever Miniature dachshund Crossbred Welsh corgi Springer spaniel Crossbred Crossbred Welsh corgi Golden retriever Golden retriever Miniature dachshund Golden retriever Cocker spaniel Crossbred Polish Lowland sheepdog Crossbred
14 10 10 11 12 15 11 13 15 11 13 7 15 13 7 14 14 11 10 14 11 11 12 9 7 10 11 9 11 9 8 11 10 11 11 13 10 12 14 9 11 13 10 8 10 8 12 15 10 9 11 10 6 7 10 6 11 12
NR Skin of the forelimb Skin of the rump Skin of the forelimb Skin of the hindlimb Skin of the neck Skin of the upper perianal area Skin of the forelimb Skin of the forelimb Skin of the thorax Skin of the shoulder Skin of the shoulder Skin of the hindlimb Skin of the forelimb Skin of the hindlimb Skin of the dorsum Skin of the forelimb Skin of the hindlimb Spleen Small intestine Peritoneal region Rectum Uterus Caecum Vagina NR NR Jejunum Stomach Spleen Ileum Stomach Rump Bladder Spleen Spleen Spleen Skin of the thorax Skin of the forelimb and hindlimb Skin of the penis Liver Skin of the head and dorsum Spleen Spleen Spleen Spleen Spleen Skin of the neck Spleen Spleen Spleen Spleen Liver Spleen Ovary Skin of the abdomen Spleen Duodenum
M, male; F, female; FN, neutered female; MN, neutered male; NR, not recorded.
175
Immunohistochemical Characterization of Canine Haemangiopericytoma
wax, sectioned and stained with haematoxylin and eosin (HE). For immunohistochemistry (IHC), the avidinebiotineperoxidase complex technique was employed, using a VECTASTAINÒ Elite ABC Kit (Vector Laboratories Inc., Burlingame, California, USA) with primary antibodies against vimentin, aSMA, desmin, vWF, TEM-1 and NG-2 (Table 2). The chromogen was 3, 30 -diaminobenzidine and the counterstain was haematoxylin. Dermal tissues were prepared from a dog as a positive control for vimentin, small intestine for a-SMA and desmin, and cerebrum for vWF. Non-immunized sera were substituted for the primary antibody as negative controls for IHC. The expression of each molecule was assessed visually by two independent readers in a blinded fashion. The percentage of positively labelled neoplastic cells was determined in five randomly selected fields at 100 magnification (+, 0e10%; ++, 10e50%; +++, 50e100%). The incidence of immunoreactive cases was compared statistically using Fisher’s exact probability test. The immunoreactivity analyzed by scoring was compared statistically using the ManneWhitney’s U-test. Comparisons were made between haemangiopericytomas and other types of tumour.
Results Table 1 presents the details of the dogs included in the study. The mean age of the dogs with haemangiopericytoma was 12.0 years (range 7e15 years). Of the 18 dogs with haemangiopericytoma, nine were male (one neutered) and nine were female (four neutered). Seventeen tumours had developed on the skin and the location of one was not specified. The mean age of the dogs with LMS was 10.3 years (range 7e14 years). Three of the 17 dogs with LMS were neutered males (MNs) and 14 were females (seven neutered). The mean age of the dogs with HS was 10.4 years (range 6e15 years). Thirteen of the 20 dogs with HS were male (nine neutered) and seven were female (three
neutered). The mean age of the dogs with MFS was 9.7 years (range 6e12). One of these three dogs was male and two were female. Table 3 and Table 4 show the results of the immunohistochemical labelling for cellular markers in the different types of tumours studied and Table 5 summarizes the incidence and grade of immunoreactivity of positive cases for each cellular marker. There was strong cytoplasmic immunoreactivity for vimentin in neoplastic cells in all cases of haemangiopericytoma, LMS and HS. In these cases, >50% of the neoplastic cells were positive for vimentin except for one LMS in which there was approximately 40% positivity. There were no significant differences in the incidence and grade of vimentin immunoreactivity between haemangiopericytomas and LMSs or HSs. There was strong cytoplasmic immunoreactivity for a-SMA in neoplastic cells in all cases of LMS. Among these, most cases had >50% positive cells (16 cases, 94.1% incidence) and one case had <10% positive cells (5.9%). There was cytoplasmic immunoreactivity for a-SMA in six cases of haemangiopericytoma (33.3%). Among these, five cases had <10% positive cells (27.8%), while one case had approximately 60% positive cells (5.6%). There was no immunoreactivity for a-SMA in neoplastic endothelial cells of HSs. However, positive immunoreactivity for desmin was observed in the stromal cell population among the neoplastic vascular structures. Both incidence and grade of a-SMA-immunoreactivity in haemangiopericytomas were significantly different from those in LMSs and HSs. There was strong cytoplasmic immunoreactivity for desmin in neoplastic cells in 15 out of 17 cases of LMS (88.2%). Among these, 13 cases had >50% positive cells (76.5%), one case had approximately 30% positive cells (5.9%) and one case had <10% positive cells (5.9%). Three cases of haemangiopericytoma (16.7%) had strong cytoplasmic immunoreactivity for desmin. Among these, two cases had <10% positive cells (11.1%) and one case had approximately 40% positive cells (5.6%). In HSs, immunoreactivity
Table 2 Immunohistochemical methodology Antibody
Antibody type
Dilution
Antigen retrieval
Vimentin a-SMA Desmin
Mouse monoclonal Mouse monoclonal Mouse monoclonal
1 in 100 1 in 100 1 in 50
Autoclaving (121 C, 10 min) Microwaving (90 C, 10 min) Microwaving (90 C, 10 min)
vWF TEM-1 NG-2 chondroitin sulphate proteoglycan
Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal
1 in 500 1 in 200 1 in 200
Microwaving (90 C, 10 min) None None
Source Dako, Glostrup, Denmark Dako Thermo Fisher Scientific, Waltham, Massachusetts, USA Dako Abcam, Cambridge, UK Millipore, Billerica, Massachusetts, USA
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Table 4 Immunohistochemical labelling intensity in MFS
Table 3 Immunohistochemical labelling intensity in haemangiopericytoma, HS and LMS Case Tumour number type 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP CHP LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS LMS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS
Case number
Immunohistochemical reactivity Vimentin a-SMA Desmin
vWF
TEM-1
NG-2
+ + + +++ + + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + +++
+ +++ +++ + + + +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ ++ +++ ++ + ++
+++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + + +++ + + + + +++ ++ +++ ++ +++ ++
+ ++ + + + + + +++ +++ + + + + +++ ++ + ++ +++ ++ ++ ++ ++ +++ + +
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
+ + ++ ++ + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
, 0% positive cells; +, 1e10% positive cells; ++, 10e50% positive cells; +++, 50e100% positive cells.
56 57 58
Immunohistochemical reactivity Vimentin
a-SMA
Desmin
vWF
TEM-1
NG-2
+++ +++ +++
+++ + +
+
+
+++ +++
+++ +
, 0% positive cells; +, 1e10% positive cells; ++, 10e50% positive cells; +++, 50e100% positive cells.
for desmin was only observed in the non-neoplastic stromal cell population (Fig. 1). Both incidence and grade of desmin immunoreactivity in haemangiopericytomas were significantly lower than those in LMSs. There was cytoplasmic immunoreactivity for vWF in neoplastic cells of all cases of HS. Among these, 12 cases had >50% positive cells (60.0% incidence), four cases had 10e50% positive cells (20.0%) and four cases had <10% positive cells (20.0%). All cases of haemangiopericytoma were negative for vWF. All cases of LMS were negative for vWF except for one case showing <10% positive cells (5.9%). Both incidence and grade of vWF immunoreactivity in haemangiopericytomas were significantly lower than those in HSs. In normal tissues, TEM-1 was expressed by a small population of pericytes in capillaries and venules (Fig. 2A). There was no expression by vascular smooth muscle cells and vascular endothelial cells of most cases (Fig. 2B); however, a few endothelial cells in a few cases did show TEM-1 labelling. There was cytoplasmic immunoreactivity for TEM-1 in neoplastic cells in 17 out of 18 cases of haemangiopericytoma (94.4% incidence). Among these, 15 cases had >50% positive cells (83.3%; Fig. 3A) and two cases had 10e50% positive cells (11.1%). There was cytoplasmic immunoreactivity for TEM-1 in the neoplastic cells in eight out of 17 cases of LMS (47.1%). Among these, four cases had <10% positive cells (23.5%; Fig. 3B), three cases had 10e50% positive cells (17.6%) and one case had approximately 50% positive cells (5.9%). There was cytoplasmic immunoreactivity for TEM-1 in neoplastic cells in 10 out of 20 cases of HS (50.0%). Among these, four cases had <10% positive cells (20.0%; Fig. 3C), three cases had 10e50% positive cells (15.0%) and three cases had >50% positive cells (15.0%). The percentages of cases in which >10% of the neoplastic cells expressed TEM-1 were 94.4% in haemangiopericytomas, 23.5% in LMSs and 30.0% in HSs. Both incidence and grade of TEM-1-immunoreactivity in haemangiopericytomas were significantly higher than those in LMSs and HSs.
Immunohistochemical Characterization of Canine Haemangiopericytoma
177
Table 5 Incidence and grade of immunoreactivity of positively labelled tumours Tumour type
Number of tumours
Immunohistochemical reactivity Vimentin
CHP LMS HS MFS
18 17 20 3
*
†
18 (0, 0, 18) 17 (0, 1, 16) 20 (0, 0, 20) 3 (0, 0, 3)
a-SMA
Desmin
vWF
TEM-1
NG-2
6 (5, 0, 1) 17‡ (1, 0, 16){ 0‡ (0, 0, 0){ 3 (2, 0, 1)x
3 (2, 1, 0) 15‡ (1, 1, 13){ 0 (0, 0, 0) 1 (1, 0, 0)
0 (0, 0, 0) 1 (1, 0, 0) 20‡ (4, 4, 12){ 1 (1, 0, 0)x
17 (0, 2, 15) 8‡ (4, 3, 1){ 10‡ (4, 3, 3){ 2 (0, 0, 2)
13 (10, 1, 2) 11 (2, 6, 3) 1‡ (1, 0, 0){ 2 (1, 0, 1)
*
Incidence of positive cases. Number of animals with each grade of immunoreactivity in the order of +, ++ and +++. ‡ Significantly different from CHP by Fisher’s exact probability test (P <0.01). x Significantly different from CHP by ManneWhitney U-test (P <0.05). { Significantly different from CHP by ManneWhitney U-test (P <0.01). †
NG-2 was negative in pericytes of capillaries and venules, and negative in vascular endothelial cells in normal tissues (Fig. 4A). Diffuse immunoreactivity for NG-2 was observed in intestinal smooth muscle cells (Fig. 4B). However, vascular smooth muscle cells were mostly negative for NG-2. In the neoplastic tissues, there was cytoplasmic immunoreactivity for NG-2 in neoplastic cells in 13 out of 18 cases (72.2%) of haemangiopericytomas. Among these, 10 cases had <10% positive cells (55.6%; Fig. 5A), one case had approximately 20% positive cells (5.6%) and two cases had >50% positive cells (11.1%). There was cytoplasmic immunoreactivity for NG-2 in neoplastic cells in 11 out of 17 cases of LMS (64.7%). Among these, six cases had 10e50% positive cells (35.3%; Fig. 5B), three cases had >50% positive cells (17.6%) and two cases had <10% positive cells (11.8%). NG-2 immunoreactivity was found in only one case of HS (5.0%), with <10% of the neoplastic cells being positive. Nineteen cases of HS showed no
Fig. 1. Positive immunoreactivity for desmin in stromal cells in a HS (number 53). Neoplastic endothelial cells lack desmin expression. IHC. Bar, 50 mm.
expression of NG-2 (95.0%; Fig. 5C). Both incidence and grade of NG-2 immunoreactivity in haemangiopericytomas were significantly higher than those in HSs. All three cases of MFS showed immunoreactivity for vimentin in >50% of the neoplastic cells. In one case >50% of cells expressed a-SMA (33.3% incidence) and two cases had <10% a-SMA-positive cells (66.7%). One case had <10% of labelled cells (33.3%) and two cases were entirely negative for both desmin and vWF. Two cases had >50% of cells labelled for TEM-1 (66.7%) and one was negative. With regard to NG-2, one case had >50% positive cells (33.3%), one case had <10% positive cells (33.3%) and one was negative. Grade of immunoreactivity of a-SMA and vWF in haemangiopericytomas was significantly lower than that in MFSs.
Discussion In the present study, most cases of LMS expressed aSMA and desmin. However, only 33.3% of haemangiopericytomas showed positive immunoreactivity for a-SMA, while even fewer cases showed positive immunoreactivity for desmin. There was consistent expression of vWF by neoplastic endothelial cells in HSs. With regard to TEM-1, haemangiopericytomas showed expression by >50% of cells, but few LMSs and HSs displayed this level of expression. Both incidence and grade of TEM-1-immunoreactivity in haemangiopericytomas were significantly higher than those in LMSs and HSs, suggesting that TEM-1 expression is associated most closely with haemangiopericytomas. NG-2 expression was restricted to a small number of cells in haemangiopericytomas and LMSs. These results suggest that diagnostically, haemangiopericytomas can be distinguished from HSs by immunoreactivity for vWF in the latter tumour type. Moreover, haemangiopericytomas can be distinguished from LMSs using immunoreactivity
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Fig. 2. TEM-1 expression in vascular mural cells. (A) Some pericytes in a capillary show positive immunoreactivity for TEM-1. IHC. Bar, 50 mm. (B) Smooth muscle cells and endothelial cells in an arteriole do not express TEM-1. IHC. Bar, 50 mm.
for TEM-1 and desmin, respectively, having greater expression in these two tumour types. In contrast, NG-2 does not contribute to the differential diagnosis between LMS and haemangiopericytoma. TEM-1 was expressed by two of three cases of MFS, suggesting that TEM-1 could be used as a marker for neoplastic myofibroblasts.
TEM-1 was not expressed by the majority of vascular endothelial cells. TEM-1 expression was originally found in embryonic or tumour-associated endothelial cells (St Croix et al., 2000), suggesting that TEM-1 can be expressed by immature proliferating endothelial cells. However, we found TEM-1 expression in a small number of neoplastic cells in HSs. TEM-1 is reported
Fig. 3. TEM-1 expression in haemangiopericytomas, LMSs and HSs. (A) Diffuse distribution of neoplastic cells showing cytoplasmic immunoreactivity for TEM-1 in a haemangiopericytoma (number 5). IHC. Bar, 50 mm. (B) A few neoplastic cells show positive immunoreactivity for TEM-1 in a LMS (number 26). IHC. Bar, 50 mm. (C) A few neoplastic cells show positive immunoreactivity for TEM-1 in an HS (number 36). IHC. Bar, 50 mm.
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Fig. 4. NG-2 expression in pericytes, endothelial cells and smooth muscle cells. (A) Pericytes and endothelial cells in a capillary do not express NG-2. IHC. Bar, 50 mm. (B) Smooth muscle cells in the muscle layer of the jejunum show positive immunoreactivity for NG-2. IHC. Bar, 200 mm.
to be a specific marker for tumour-associated myofibroblasts and neovessel mural cells (i.e. pericytes and smooth muscle cells) and is thought to control the migration and proliferation of these cells (Christian et al., 2008). This hypothesis is consistent with partial immunoreactivity for a-SMA in CHPs (Perez et al., 1996; Mazzei et al., 2002; Chijiwa et al., 2004). In the present
study there was scattered TEM-1 expression in capillary pericytes. However, there was no TEM-1 expression by vascular smooth muscle cells. When blood vessels grow and mature, pericytes differentiate into vascular smooth muscle cells where TEM-1 expression is quickly down-regulated to below detection level (Tomkowicz et al., 2010).
Fig. 5. NG-2 expression in haemangiopericytomas, LMSs and HSs. (A) Neoplastic cells showing cytoplasmic immunoreactivity for NG-2 in a haemangiopericytoma (number 9). IHC. Bar, 50 mm. (B) Diffuse distribution of neoplastic cells showing cytoplasmic immunoreactivity for NG-2 in a LMS (number 28). IHC. Bar, 50 mm. (C) HS cells do not express NG-2 (number 37). IHC. Bar, 50 mm.
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Conflicting immunohistochemical data have suggested that CHPs are actually a heterogeneous tumour population with different histological origins (Avallone et al., 2007). In the present study a subpopulation of LMSs and haemangiopericytomas expressed TEM-1, with this expression being more common in haemangiopericytomas. Pericytes and vascular smooth muscle cells share common precursor cells (Hughes and Chan-Ling, 2004; Klein et al., 2011) and pericytes express TEM-1 strongly during angiogenesis (Tomkowicz et al., 2010). TEM-1 therefore represents the activated immature phenotype of mural cells, most likely pericytes. The finding of consistent expression of TEM-1 by CHPs suggests a single cellular origin of this tumour. TEM-1 was also expressed by the majority of neoplastic cells in two of three MFSs, which is in accordance with expression patterns in tumour-associated myofibroblasts (Christian et al., 2008). These results suggest that TEM-1 expression is found in populations of myofibroblasts as well as immature vascular mural cells, with preferential expression in pericytes. Histologically, there are some similarities between CHPs and MFSs. For example, cells in both tumour types may be arranged in fascicles and storiform, staghorn or whorled patterns (Donald, 2002; Tsuchiya et al., 2012). In contrast, several types of human sarcomas with different cellular origin have recently been shown to variably express TEM-1, and it has been suggested that this molecule plays a role in malignant behaviour of these tumours rather than representing a marker of a specific cell type (Rouleau et al., 2011). These findings contrast with the consistent expression of TEM-1 in CHPs, so further studies are necessary in order to determine the specificity of TEM-1 expression in canine sarcomas of different cellular origin. NG-2 expression by >10% of neoplastic cells was more frequent among LMSs than haemangiopericytomas. HSs did not express NG-2 except for one case with minor immunoreactivity. In the rat retina, NG-2 is expressed both in pericytes and smooth muscle cells (Hughes and Chan-Ling, 2004; Murfee et al., 2005), and NG-2 is considered to play a role in microvascular remodelling (Ozerdem and Stallcup, 2004; Murfee et al., 2006). However, NG-2 is not expressed in endothelial cells (Murfee et al., 2005). In the present study, NG-2 was not expressed by vascular cells (i.e. endothelial cells, pericytes and smooth muscle cells); however, intestinal smooth muscle cells all expressed NG-2. The reason for the lack of NG-2 expression in vascular smooth muscle cells is not clear, but the findings of the present study suggest that NG-2 is a marker of smooth muscle cells rather than pericytes in dogs.
It has been suggested that pericytes are involved in supporting the neoplastic vascular channels that characterize HSs (Kusewitt et al., 2000). However, in the present study, cells positive for a-SMA or desmin were negative for TEM-1 expression. TEM-1 is down-regulated in pericytes during development, with a significant decrease in expression in adult tissues (MacFadyen et al., 2007). These results suggest that HS cells positive for a-SMA or desmin, but negative for TEM-1 expression, were phenotypically mature mural cells. In conclusion, on the basis of TEM-1 expression, the neoplastic cells of CHPs are considered to have originated from immature vascular mural cells, sharing a phenotype with myofibroblasts. The specificity of TEM-1 expression should be examined further in canine sarcomas. An immature cellular phenotype may be the reason for partial expression of a-SMA in these tumours. NG-2 expression may be associated with smooth muscle cells rather than pericytes in dogs.
Acknowledgment The authors thank Mrs. S. Suzuki for technical assistance in preparing the histological specimens.
Conflict of Interest Statement The authors declare that there are no conflicts of interest.
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August 5th, 2012 ½ Received, Accepted, December 17th, 2012