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Introduction

Osteopontin (OPN) is an acidic, secreted protein classified as a member of the SIBLING (Small Integrin-Binding, N-Linked Glycoprotein) family [10]. It is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer in human and has been shown to facilitate tumorigenesis in vivo [7]. OPN has long been implicated in the process of tumorigenesis. It was originally identified as a secreted marker for transformed cells, because the level of OPN mRNA is elevated in a wide variety of transformed cells in mice [24].

In recent years, substantial progress has been made in the detection and diagnosis of early stage of cancers in humans. However, there are still no molecular indicators that distinguish highly aggressive tumors from moderately aggressive and non-aggressive ones. But a few markers that predict invasiveness have been established [12, 14, 18, 31, 35]. New tumor markers and markers of tumor progression are needed for improved staging and for better assessment of treatments of many cancers [1, 2]. OPN may be one candidate marker for the progression of various malign tumors. In cancer, this molecule can support cell invasion and anchorage independence, thus enhancing tumor progression and metastasis formation [12, 14, 18, 19, 26, 30, 35].

Numerous studies are available about OPN and tumor interaction in human and experimental animals [12, 14,

18, 19, 26, 30, 35]. Contradictory results are available about OPN expression in human tumors especially in early reports. For example, Zhou et al. [36] reported that although OPN expression increased in metastatic melanomas compared to nonmetastatic melanoma, they found no correlation between OPN expression and tumor thickness, metastasis or patient survival. However, in contrast to this study, a recent study investigating OPN in primary melanoma found an association between increasing OPN staining and tumor thickness, a higher invasive level and mitotic index. They reported that higher OPN in tumors correlated with decreased recurrence-free and disease-specific survival [21]. Only one report is available on OPN expression in canine tumors. Klopfleisch et al. [16] reported that there is an increased OPN level in canine mammary carcinomas compared with adenomas by PCR, but levels were not found to be statistically significant.

The objective was to investigate the immunohistochemical expression of OPN and evaluate a possible relationship with tumor malignancy in canine and feline tumors.

Material and Methods

In this study, a total of 40 (26 malign and 14 benign) canine and feline tumors (32 dogs and 8 cats) collected from the archive of the department of pathology were used. They were composed of 15 malign mammary tumors, 5 benign mammary tumors, 9 benign soft tissue tumors, 5 malign soft tissue tumors, 2 malign skin tumors, 3 malign bone tumors

SUMMARY

Osteopontin (OPN) is a glycoprotein expressed by various tissues and cells. It is also implicated in tumor progression. The protein can mediate cell adhesion and is strongly associated with transformation and tumorigenesis. Overexpression of OPN influences invasion and metastasis of different human tumors, and OPN expression may be use as a possible prognostic marker. It has been detected in a growing number of human tumor types, by immunohistochemistry on tumor tissue sections. The objective of this study was to assess the immunohistochemical expression of OPN in different canine and feline tumors and to examine any possible relation with malignancy. To achieve these aim 40 different kinds of canine and feline tumors were evaluated. OPN was either not expressed or at low levels in benign tumors, but strongly expressed in malign tumors. This study showed that OPN may be associated with malignancy of cat and dog tumors.

Keywords: Osteopontin (OPN), immunohistochemistry, dog, cat, tumor

RÉSUMÉ

Expression de l’ostéopontine dans les tumeurs canines et félines

L’ostéopontine (OPN) est une glycoprotéine exprimée dans différents tissus et cellules. Elle est également impliquée dans la progression tumorale. La protéine intervient dans l’adhésion cellulaire et est fortement associée à la transformation et de la tumorigenèse. La surexpression de l’OPN modifie le pouvoir invasif et le risque de métastases de différentes tumeurs chez l’homme, et son expression peut être utilisée comme un marqueur pronostique.. L’objectif de cette étude était d’évaluer l’expression de l’OPN mesurée par immunohistochimie dans différentes tumeurs canines et félines et d’examiner une possible relation avec leur malignité. Pour cela 40 types différents de tumeurs canines et félines ont été évalués. L’OPN n’était pas exprimée ou exprimée à de faibles niveaux dans les tumeurs bénignes, mais fortement exprimé dans les tumeurs malignes.

Mots-clés : ostéopontine (OPN), immunohistochimie, chien, chat, tumeur

Immunohistochemical expression of osteopontin in canine and feline tumors

O. OZMEN1*, M. HALIGUR1, V. IPEK1

1University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Department of Pathology, Istiklal Yerleskesi , 15030, Burdur, Turkey

*Corresponding Author: ozlemozmen@mehmetakif.edu.tr

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and 1 hematopoietic tissue tumor. Diagnoses of the tumors and malignancy criterion were made according to the WHO [32] classification. Three different blind pathologist were evaluated the malignancy of the tumors. For gross findings, archive notes and pictures were used.

For histopathology, histochemistry and immuno-histochemistry, paraffin blocks were cut at a thickness of approximately 5 µm. All slides were stained routinely with hematoxylin and eosin (HE). For fat tissues, oil red O method was used to cryostat section. The Van Gieson method and immunohistochemistry were used for the evaluation of soft tissue tumors. Then tumor samples were stained with Ki67, proliferating cell nuclear antigen (PCNA), desmin, vimentin, pancytokeratin, smooth muscle actin (SMA), S100, glial fibrilary acidic protein (GFAP), carcinoembryonic protein (CEA) and alpha fetoprotein (AFP) for the identification of origin and determination of malignancy. After this evaluation, sections were stained immunohistochemically in order to demonstrate OPN activity. Commercial kits provided from, Abbiotech, San Diego, CA were used for immunohistochemical examination of OPN [Rabbit Polyclonal Osteopontin, 250801, 1:200 dilution], Ki67 [Rabbit Polyclonal Ki-67 Antibody, 250733, 1:100 dilution]; PCNA [Rabbit Polyclonal PCNA Antibody, 250812, 1:100 dilution); desmin [Mouse Monoclonal Desmin Antibody, 251743, 1:100 dilution]; Vimentin [Mouse Monoclonal Vimentin Antibody, 251809, 1:100 dilution]; Pancytokeratin [Mouse Monoclonal Pancytokeratin Antibody, 251788, 1:100 dilution], SMA [Mouse Monoclonal SMA, 251813, 1:200 dilution]; S100 [Mouse Monoclonal S-100 Antibody, 251795, 1:100 dilüution], GFAP [Rabbit Polyclonal GFAP Antibody, 250661, 1:200 dilution]; CEA [Rabbit Polyclonal CEA Antibody, 250598, 1:200 dilution]; and AFP [Mouse Monoclonal Alpha-Fetoprotein Antibody, 251700, 1:100 dilution]; NSE [Rabbit Polyclonal NSE Antibody 251399, 1:100 dilution], using a routine streptavidine-biotin peroxidase technique.

To evaluate the severity of the immunohistochemical reaction of tumor cells with markers, semiquantitative

analysis was performed using an arbitrary visual scale with a grading score ranging from (-) to (+++) as follows: (-) = negative, (+)= focal weak staining, (++) = diffuse weak staining, (+++)= diffuse strong staining. To evaluate the percentage of immunopositive cells, 100 cells were calculated in 10 different microscopic high-powered fields of each slide, and then were examined under the 40x objective of a trinocular microscope (Nikon E600) and microphotography apparatus.

Statistical analysis was done with SPSS (Statistical Package for Social Sciences) 13.0 software (SPSS Inc, Chicago, Ill, USA). Results are expressed as mean ± SD. In the statistical evaluations, 1-way analysis of variance test was used to observe any differences between immunopositive cells of malignant and benign tumors control group. Duncan multiple comparison method was used. In addition, logistic regression, and Pearson’s correlation tests were used for association between parameters. P values <0.05 were accepted as statistically significant.

Results

MACROSCOPICAL FINDINGS

In this study 20 mammary tumors, 14 soft tissue tumors, 3 bone tumors, 2 skin tumors and 1 hematopoietic tissue tumor were examined. Species and diagnosed tumors are shown in Tables I-II. Ages of the cats with tumors ranged from 5-12 years and for dogs 7-17 years. Most of the dog tumors were observed in small breeds. Grossly tumor masses were between 1x1.5 cm and 15x17 cm in diameter (Fig.1A-1D). Generally, malign tumors were bigger than the benign ones and they usually diagnosed in older animals. Mammary tumors were the most common tumors in both cats and dogs.

Breast tumors in cats were observed between 5-12 years of age, the age of the dogs ranged from 7-17. Most of the animals were tumor small breed dogs and owned cats. Macroscopically tumor masses were varied from 1x1.5 cm, up to 15x17 cm in diameter. Generally, big tumors

Case Diagnosis OPN Ki67 PCNA Des Vim PCK SMA NSE S100 GFAP CEA AFP1 Mammary AC +++ +++ +++ _ + ++ _ _ _ _ ++ _2 Lymphoma + + + _ _ _ _ _ _ _ _ _3 Vaccine

sarcoma+++ +++ ++ _ _ _ _ _ + _ _ _

4 Malignant melanoma

++ + ++ _ + _ _ _ + _ _ _

5 Mammary AC ++ ++ ++ _ + ++ _ _ + _ ++ _6 Fibrosarcoma ++ + ++ _ + _ _ _ _ + _ _7 Mammary AC +++ ++ +++ _ _ ++ _ _ _ _ ++ _8 Mammary

adenoma+ _ _ _ + + _ _ _ _ ++ +

AC: Adenocarcinoma, OPN: Osteopontin, PCNA: Proliferating cell nuclear antigen, Des: Desmin, Vim: Vimentin, PCK: Pancytokeratin, SMA: Smooth muscle actin, NSE: Neuron specific enolase, GFAP: Glial fibriler acidic protein, CAE: Carcinoembrionic antigen, AFP: Alpha fetoprotein.

Table I: Immunoreactions scores of the cat tumors with examined markers

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were diagnosed as malignant. Five of the canine mammary tumors consisted cartilage and bone formation. Breast adenocarcinomas were lobular structure and mostly whitish-yellow color at macroscopical examination. The majority of the malignant breast tumors had necrotic cavity in the middle part of the mass. These necrotic areas were usually reddish-brown, sometimes whitish - yellow colored liquid containing cystic formation were commonly observed. Bleeding was observed in malignant breast tumors.

Soft tissue tumors localized under the skin, vagina,

bladder, and ranging from 1x2 cm up to 4x6,4cm in diameter. The cut surface of the masses was generally soft and pink in color except malignant melanoma that was blackish. Fat, connective and muscular tissue tumors localized in the urogenital system, while histiocytoma, plasmacytoma and melanomas were located on the face and neck.

In the skin tumors, one squamous cell and 1 basal cell

carcinoma were present. Skin tumors localized at the face and ears and diameters ranging from 1x2 up to 2x5 cm. The masses were whitish- pink in color and slight hard, of the surface of the tumors were generally ulcerated. Metastases

were detected in some skin tumors to the regional lymph nodes. There were three cases of osteosarcoma (2 dogs and 1 cat) in our materials. In two tumors from dogs were observed lung and kidney metastasis.

HISTOPATHOLOGICAL FINDINGS

Histopathologically, the most prominent findings were severe hyperemia and which were seen in both malign and benign tumors. Hemorrhages were more common in surgically removed tumors. In mammary mixed tumors, cartilage and bone metaplasia were characteristic (Fig. 2A). Benign tumors kept the normal appearance of cells while marked pleomorphic features, numerous mitotic figures, papillary or cystic glands, necrosis and inflammatory reactions were seen in malignant tumors (Fig. 2B). Connective and myoepithelial tissue proliferations were also seen together with epithelial proliferation. In some malignant cases lymph node and lung metastases were diagnosed. Metastatic masses included numerous mitotic figures and anaplasia; more so than the than primary foci. No metastases were seen in benign cases.

No Diagnosis OPN Ki67 PCNA Des Vim PCK SMA NSE S100 GFAP CEA AFP1 Benign mix tumor + + + _ + + _ _ + _ _ _2 Leiomyoma _ + + ++ _ _ +++ _ _ _ _ _3 Fibroma _ + + _ + _ _ _ _ _ _ _4 Lipoma _ _ _ + _ _ _ _ _ _ _ _5 Mammary AC +++ ++ ++ _ _ ++ + _ + _ ++ +6 Osteosarcoma +++ ++ +++ _ + _ _ _ + _ _ _7 Fibroma _ _ + _ ++ _ _ _ _ _ _ _8 Benign mix tumor + + + _ + + _ _ _ _ _ _9 Mammary AC ++ ++ ++ _ _ +++ _ _ _ _ + _

10 Osteosarcoma ++ ++ ++ _ _ _ _ _ + _ _ _11 Histiocytoma + + + _ + _ _ _ _ + _ _12 Mammary AC +++ +++ +++ _ _ ++ _ _ _ _ ++ _13 Mammary AC ++ ++ ++ _ _ +++ + _ + + ++ _14 Osteoasrcoma +++ +++ ++ _ + _ + _ + _ _ _15 Malignant

histiocytoma+ + + _ _ _ _ _ _ _ _ _

16 Mammary AC ++ ++ +++ _ + +++ + _ _ _ ++ _17 Fibroma + + + _ ++ _ _ _ _ _ _ _18 Malign histiocytoma _ ++ ++ _ _ _ _ _ _ _ _ _19 Mammary AC ++ ++ ++ _ ++ ++ _ _ _ _ ++ _20 Malignant mix tumor ++ ++ +++ _ ++ + _ _ _ _ + _21 Benign mix tumor + + + _ + + + _ _ _ + _22 Mammary AC +++ +++ ++ _ ++ ++ + _ + _ ++ _23 Mammary AC ++ +++ ++ _ + ++ + _ + _ ++ _24 Mammary adenoma + + + _ _ +++ _ _ _ _ _ _25 Histiocytoma _ + + _ _ _ _ _ _ _ _ _26 Benign mix tumor + + + _ + + + _ _ _ _ _27 Fibrolipoma + + + _ _ _ + _ _ _ _ _28 Squamous cell carcino. ++ ++ ++ _ _ +++ + _ _ _ _ _29 Basal cell carcinoma + ++ ++ _ + +++ + _ + _ _ _30 Plasmacytoma + + + _ _ _ _ _ _ _ _ _31 Mammary AC ++ ++ ++ _ _ ++ _ _ _ _ ++ _32 Mammary AC ++ +++ ++ _ _ + _ _ _ _ + _

Table II: Immunoreactions scores of the dog tumors with examined markers

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Fourteen soft tissue tumors were diagnosed histopathologically, histochemically and immuno-histochemically according to cellular morphology (Figs. 2C-D). One feline vaccine sarcoma was examined in this study. Histopathologically, yellowish-brown pigments with numerous macrophage infiltrations were observed, with proliferated connective tissue and abundant mitotic figures.

Diagnosis of skin tumors was made according to characteristic histopathological appearance. For example, keratin pearls were typically observed in squamous cell carcinomas. An inflammatory reaction was seen in both squamous and basal cell carcinomas. Dermal involvement by epidermal cells was observed in both tumors. During the histopathological examination of osteosarcomas, high mitotic activity and disturbed bone tissue were commonly observed. There was less calcification or mineralization in metastatic masses than primary foci. Microscopically, hemorrhages and clusters of big lymphoblastic cells were common in a spleen with lymphoma. Generally metastases were the most prominent malignancy criterion. Mitotic activity, anaplasia or pleomorphism was also used for evaluation.

IMMUNOHISTOCHEMICAL FINDINGS

In this study, tumor tissues were immunostained with desmin, vimentin, pancytokeratin, SMA, GFAP, NSE, CAE and AFP in order to detect the origin of the cells. Ki67, PCNA and S100 were used to detect malignancy stages. Ki67 and PCNA reactions were especially used to evaluate the malignancy criteria. Marked expression was observed in malignant tumors in these markers. More than 25 malign and 15 benign tumors were immunostained with OPN antiserum and examined any differences in OPN expression in malign and benign ones were correlated the OPN expression of the tumor cells. The reaction between the tumors and markers is given in tables I and II. Statistical analysis for immunopositive cell numbers of dogs and cats bening and malignant tumors are shown in table III. This study shows that generally more than one cell type proliferated in mammary tumors. Especially in mixed tumors of dogs, numerous cells exhibited PCNA and Ki67 positive immunoreactions, and expressions were observed in epithelial, fibrous and even nervous cells.

OPN was strongly expressed in malignant tumors

and a correlation was observed with the Ki67 reaction,

Figure 1: A- Gross appearance of the malign mix mammary tumor from a dog, bone formations (arrows). B- Necrosis and cavities (arrows) at the cut surface of the mammary adenocarcinoma from a cat. C- Lung metastases of the mammary adenocarcinoma (arrows) in a dog, numerous metastatic foci can be seen. D- Osteosarcoma, tumoral mass at the caput humeri (arrow heads) in a dog.

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immunohistochemically. In contrast to malignant tumors, benign tumors showed minimal to no expression of OPN. Especially in malignant tumors, heterogenic appearance of OPN expression was seen in different areas and expressed in different severity of OPN in same tumor. While a strong reaction was observed in some areas; slight expression was seen in the other areas within the same tumor mass. Similar findings were also seen in the Ki67 reaction of tumors. In

mammary tumors, mitotically active tumor cells, both epithelial and mesenchymal origins were markedly expressed OPN.

In osteosarcomas, OPN activity was not influenced by decalcification. OPN expression was marked in proliferated cells. Interstitial areas tested negative for OPN in osteosarcomas. In skin tumors, squamous cell carcinomas

n OPN Ki67 PCNAMalign mammary tumors 14 50.73±9.18a 26.00±4.25a 44.00±5.39a

Benign mammary tumors 6 5.80±2.16b 5.20±2.49b 8.00±2.73b

Malign soft tissue tumors** 5 61.00±12.98ac 25.40±4.03a 46.00±6.24a

Benign soft tissue tumors*** 9 8.00±4.52b 7.11±3.05b 6.00±2.64b

Malign skin tumors **** 2 69.00±19.79bc 22.00±11.31a 40.00±4.24aMalign bone tumors***** 3 60.33±11.50ac 24.33±5.03a 41.66±2.51a

P value <0.001 <0.001 <0.001*:Differences statistically significant. The differences between the means of groups carrying different letters in the same column are statistically significant.**: Vaccine sarcoma. malignant melanoma. fibrosarcoma and malignant histiocytoma ***: Leiomyoma. lipoma. fibroma. histiocytoma and fibrolipoma. ****:Squamous cell carcinoma and basal cell carcinoma.*****: Ostesarcoma.

Table III: Statistical analysis results of the percentage of immunopositive cells of malign and benign dog and cat tumors.

Figure 2: A- Histopathological appearance of the malign mix tumor form a cat, cartilage and bone formation (arrows), HE, Bar=200µm. B- Proliferation of anaplastic and pleomorphic cells (arrows) in a mammary adenocarcinoma from a dog, HE, Bar=200µm. C- Micropscopical appearance of the malign histiocytoma, pleomophic cells (arrows), from a dog, HE, Bar=100µm. D- Histology of malign melanoma, numerous irregular melanocytes (arrows), dog, HE, Bar=100µm.

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more strongly expressed OPN than basal cell carcinomas (Figs. 3A-D).

Discussion

Although elevated levels of OPN expression have been observed in several types of human tumors, its expression in cat and dog tumors has not been reported in detail [2,8,12,14,18,23,26,35,36]. Only one report is available on OPN expression in canine mammary tumors it reported little increase of OPN level between adenomas and carcinomas by PCR [17]. In contrast, in this study, we observed a marked increased in OPN expression in different type of malign canine and feline tumors, including mammary tumors compared to benign tumors by immunohistochemical method. Marked OPN immunoreaction was seen in most cells in malignant tumor, while weak and a few immunopositive cells were seen in benign ones. To the best of our knowledge, this is the first study that comprehensively showed the

localization of OPN expression in canine and feline tumors by immunohistochemical method.

OPN was identified as a tumor-associated protein in transformed cells in cultures [24, 25] and has been shown to be present in some human tumor samples [4]. In human breast cancer, OPN has been shown to contribute functionally to the malign behavior of cells [29]. This study showed that a similar relation can be seen in canine and feline mammary tumors. In this study, we observed a heterogenic distribution at the OPN expression in different areas of same tumor. Immunohistochemical localization of the OPN was parallel to the expression of Ki-67 and PCNA. Microscopically marked OPN expression was observed in tumor areas composed of highly proliferated cells. Possible causes of the Klopfleisch et al. [17] results may be related to this heterogenic distribution of OPN expression in the canine mammary tumors.

Figure 3: A- OPN immunoreactions in an osteosarcoma,, marked OPN expression of tumoral cells (arrows) Streptoavidin biotin peroxidase method, with DAB, Harris hematoxylin counter-stain, Bar=100µm. B- Immunopositive OPN reaction in a, squamous cell carcinoma, from a dog, strong immunopositive reaction of squamous cells (arrows), Streptoavidin biotin peroxidase method, with DAB, Harris hematoxylin counter-stain, Bar=200µm. C- OPN immunoreactions of a mammary adenocarcinoma from a cat, marked OPN expression of tumoral celss (arrows), Streptoavidin biotin peroxidase method, with DAB, Harris hematoxylin counter-stain, Bar=200µm. D- OPN positive reaction in a mammary adenocarcinoma from a dog, marked intracytoplasmic OPN expression of tumoral celss (arrows) Streptoavidin biotin peroxidase method, with DAB, Harris hematoxylin counter-stain, Bar=50µm.

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Numerous studies reported that OPN overexpression is also related with breast cancer evolution and metastasis in humans; therefore, there is a potential use for OPN in monitoring the disease status of patients with breast cancer [11]. Most of our study material was composed of mammary tumors and OPN was more strongly expressed in malign mammary tumors than in benign ones. This result indicated that OPN may be used for a prognostic aim in canine and feline mammary tumors. But further studies are needed to explain the relation of OPN level and malignancy criteria in mammary tumors.

The use of cancer biomarkers to predict future patterns of disease has been an emerging issue, especially as cancer treatments have made such positive strides [3, 12-16, 18-20, 26, 35]. Breakthroughs in the development of reliable cancer biomarkers may be imminent due to advances in genomics and computer technology, which allows the analysis of vast quantities of data [9, 27, 34]. OPN has been studied in human and murine tumors and identified as a tumor-associated protein; this study showed that it can also be used for cat and dog tumors. Our study focuses on the role of OPN in cancer in dogs and cats, and its potential as a biomarker. OPN seems to be more than just a marker of malignancy because this protein may play a functional role in malign-gene expression and cancer cell behavior in cats and dogs. In addition, tumor diagnosis, anti-OPN treatment may be used for cancer in humans and animals.

Numerous in vitro and in vivo studies implicate OPN’s role in tumorigenesis, or more specifically, in tumor promotion [6, 12, 14, 18, 19, 22, 25, 26, 33, 35]. Authors reported that, the lack of induced OPN expression in OPN-null mice significantly suppresses benign squamous papilloma in vivo development, relative to wild-type mice, when subjected to the two-stage (initiation-promotion) mouse skin chemical carcinogenesis model [15]. OPN shows marked expression in squamous cell carcinomas, which have metastatic potential, but minimally expressed in solid basal cell carcinomas previously reported in humans [8]. Recently, marked suppression on papilloma development by lack of OPN expression in mice was reported [15]. Experimental evidence suggests that promoter-induced OPN expression plays a critical role in regulating the rate-limiting step of tumor promotion, possibly by providing the initiated cells a conducive environment in which to prolong their survival and, consequently, facilitate tumor development. In this study we also observed the marked expression of OPN in squamous cell carcinoma more than so basal cell carcinoma. But only two tumors were studied for this study. More comprehensive studies are needed for better evaluation of this issue.

High levels of OPN in several cancers are indicative of a poor prognosis. Overall and disease-free survivals are inversely related to OPN levels in several cancers in human [12, 14, 18, 19, 26,35]. There is strong correspondence between high OPN and lower mean survival time in tumors (82%) and plasma (100%) measurements, with large mean

differences in survival times, indicating a useful role for OPN in patient stratification. Patient survival is largely determined by tumor aggressiveness. Hence, it is not unexpected that OPN, a prognostic measure for survival, can also be a marker for grade, stage, and early progression [14, 31]. Although this study addressed the relation of OPN and malignancy of canine and feline tumors, studies are needed to determine for example, the blood or tissue OPN level and malignancy or survival time in these species. We have demonstrated that OPN may be using both tumor markers and progression markers in cats and dogs. The identification of OPN in tumor tissues can be used for diagnosis and clinical outcomes of tumors in these species. Furthermore, these observations have implications for the design of many experiments, on both cell lines and tissues of cats and dogs. Future research needs to assess whether the blood level of OPN, or possible combination with other markers, can further improve its diagnostic value in these species.

As a result, OPN was immunohistochemically identified as the leading candidate for tumor markers in cats and dogs tumors, from our initial results firstly. It is a secreted, integrin-binding protein that has already been reported as a marker of tumor progression in human tumors . The results presented here provide the first data to suggest that OPN may be a marker of cat and dog cancer progression.

Acknowledgement

This study was supported by scientific Projects Commission of University of Mehmet Akif Ersoy (Project number: 0116-NAP-10).

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