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Vet Pathol 44:276-284 (2007)
© 2007 American College of Veterinary Pathologists

Uveal Spindle Cell Tumor of Blue-Eyed Dogs: An Immunohistochemical Study

M. K. Zarfoss, G. Klauss, K. Newkirk, M. Kiupel, Y. Jones, C. M. H. Colitz and R. R. Dubielzig

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI (MZ, RD), the University of Minnesota Veterinary Medical Center, St. Paul, MN (GK), The Ohio State University Department of Veterinary Biosciences, Columbus OH (KN, CC), and the Michigan State University Diagnostic Center for Population and Animal Health, Lansing, MI (MK, YJ)


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Immunohistochemical techniques were used to investigate the origin of a spindle cell tumor in the anterior uveal tract of dogs and the influence of ultraviolet radiation on the development of this tumor. Thirteen tumors were identified from the 4,007 canine ocular samples examined at the Comparative Ocular Pathology Laboratory of Wisconsin between 1978 and 2005. Siberian Husky and Siberian Husky mix dogs were overrepresented (10/13 dogs, overall median age 10 years). Light microscopic evaluation (all dogs) and electron microscopy (2 dogs) were performed. Immunohistochemical staining included alpha-smooth muscle actin (SMA), vimentin, S-100, desmin, glial fibrillary acidic protein (GFAP), Melan A, microphthalmic transcription factor (MITF-1), protein gene product 9.5 (PGP 9.5), laminin, gadd45, p53, proliferating cell nuclear antigen (PCNA), anti-UVssDNA (antibody for detection of (6-4)-dipyrimidine photoproducts), and telomerase reverse transcriptase (TERT). All tumors occurred in the iris with or without ciliary body involvement and were composed of spindle cells arranged in fascicles and whorls (variable Antoni A and B behavior). All tumors were positive when immunostained for vimentin and S-100. Nine of 13 tumors exhibited GFAP immunopositivity. All tumors were negative for SMA, desmin, Melan A, and MITF-1. Tumors were variably positive for PGP 9.5, laminin, gadd45, p53, PCNA, anti-UVssDNA, and TERT. Electron microscopy revealed intermittent basal laminae between cells. These tumors are morphologically and immunohistochemically most consistent with schwannoma. The relationship between spindle cell tumors of the anterior uvea of dogs, altered neural crest, blue iris color, and ultraviolet radiation has not yet been fully elucidated.

Key words: Canine; eye; immunohistochemistry; iris; neural crest; schwannomas; spindle; tumor.

Intraocular spindle cell tumors of dogs are rare and include melanocytic tumors,11 peripheral nerve sheath tumors,43 and metastatic sarcomas. The most common metastatic spindle cell tumors of the canine eye at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) are hemangiosarcoma, osteosarcoma, metastatic malignant melanoma, and anaplastic mesenchymal tumors of undetermined origin. At the COPLOW, a morphologically distinct spindle cell tumor has been identified that is exclusively associated with partial or complete blue uveal color.28,29 The association between blue iridal color and tumors has been most extensively studied in humans in association with intraocular melanoma20,30 and patients with neurocristopathies who exhibit both pigment abnormalities and peripheral nerve sheath tumors.42 In blue eyes, there is virtually no histopathologically visible melanin within the iris stroma; it is present only in the posterior iridal epithelium. Melanin absorbs ultraviolet (UV) radiation;2,22 UV radiation has been implicated in the pathogenesis of human uveal melanoma2,22,44,46 and skin cancer.39

The purpose of this study was to describe a morphologically distinct uveal tumor of blue-eyed dogs and to perform immunohistochemical staining to investigate both the cell of origin and the cause of malignant transformation relative to (blue) uveal color. Immunohistochemical stains were used to rule out melanoma (Melan A, MITF-1), to rule in a tumor of peripheral nerve sheath origin (vimentin, S-100, glial fibrillary acidic protein [GFAP], protein gene product 9.5 [PGP 9.5], laminin), and to investigate the role of UV damage in the pathogenesis of this tumor (p53, anti-UVssDNA [antibody for detection of (6-4)-dipyrimidine photoproducts], proliferating cell nuclear antigen [PCNA], gadd45, and TERT).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Thirteen canine anterior uveal spindle cell tumors from the COPLOW collection were included in this study based solely on their morphologic features. All tumors in this study involved the anterior uvea (Fig. 1) of blue-eyed dogs. All tissues were formalin fixed and paraffin embedded. All stains performed on the 13 tumors included are reported. Immunohistochemistry for these dogs was performed based on tissue availability. Immunohistochemical staining results are reported in Table 1 as follows: negative = < 10% of neoplastic cells stained positively; + = 10–25% positive; ++ = 25–50% positive; and +++ = > 50% positive.


Figure 01
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  		Fig. 1. Subgross photo of an eye (anterior at top, optic nerve at bottom); dog No. 13. The image shows an asymmetric expansion of the anterior uvea by spindle-cell tumor. Retinal detachment and corneal defects are artifactual. HE stain.

 
The following stains were performed at the COPLOW: vimentin (Clone V9, No. M 0725, Dako Corp., Carpinteria, CA); GFAP (No. Z 0334, Dako); smooth muscle actin (Clone 1A4, No. M 0851, Dako); and desmin (Clone D33, No. M 0760, Dako). For these stains, 5-µm sections were mounted on slides and allowed to dry overnight at 56°C. Slides were placed in warm xylene, deparaffinized with 3 changes of xylene for 5 minutes each, and then hydrated in water. Samples were blocked for endogenous peroxidase with 3% H2O2 for 5 minutes. Samples were rinsed in distilled water before digestion with pronase for 5 minutes at room temperature or with 5% trypsin at 37°C for 8–20 minutes depending on antigen. Samples were cooled in distilled water followed by Tris-buffered saline (TBS, 0.05 M) for 2 minutes. Samples were drained and covered with 1–2 drops of Biogenex blocking serum for 30 minutes. Slides were drained and covered with 1–2 drops of primary antibody (rabbit or mouse origin) for 30 minutes or overnight. Sections were rinsed twice in 0.05 M TBS pH 7.4 for 2 minutes. Slides were drained and covered with secondary antibody for 15–30 minutes at room temperature. Slides were rinsed 3 times with 0.05 M TBS pH 7.4 for 2 minutes. Sections were developed in 3,3-diaminobenzidine solution (DAB), prepared by combining 2 ml of Tris, 50 µL of 3% H2O2, and 500 µl of stock DAB solution. Sections were developed for 30 seconds to 5 minutes, in parallel with positive controls. Sections were rinsed in TBS and distilled water. Slides were counterstained with Harris hematoxylin for 10–20 seconds, decolorized blue in L1CO3, and rinsed in tap water for 1 minute. Sections were dehydrated in 70%, 95%, 100% xylene and cover-slipped using Permount. Appropriate positive and negative controls were used in all cases. Immunohistochemical interpretation was carried out by the authors R. R. Dubielzig, G. Klauss, and M. K. Zarfoss.

The following immunohistochemical stains were performed at the Michigan State University Department of Pathobiology and Diagnostic Investigation: MITF-1, Melan A, S-100, PGP 9.5, laminin. Deparaffinization, antigen retrieval, and counterstaining were performed with an automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ). Paraffin-embedded tissue sections (5-µm thickness) were deparaffinized with EZ Prep (Ventana Medical Systems, Inc.), washed with Sodium Chloride Sodium Cirrate buffer (SSC) solution (Ventana Medical Systems, Inc.) and incubated with reaction buffer (Ventana Medical Systems, Inc.) at 37°C for 2 minutes. Slides were incubated at 37°C for 10 minutes with a mouse monoclonal anti-MITF-1 (Abcam, Cambridge, UK) and a mouse monoclonal anti-Melan A (DakoCytomation, Carpinteria, CA) at a concentration of 1 : 100 and 1 : 20, respectively. A biotinylated anti-mouse and anti-rabbit secondary antibody conjugated with alkaline phosphatase-streptavidin (enhanced alkaline phosphatase red detection kit, Ventana Medical Systems, Inc.) was applied to locate and visualize the bound primary antibody. After that, sections were incubated at 37°C for 32 minutes, counterstained with hematoxylin, followed with a bluing reagent to change the hue of hematoxylin to blue color (Ventana Medical Systems, Inc.). To remove the liquid cover slip, reagent sections were washed with a detergent and rinsed with warm tap water. Finally, sections were rinsed with 100% ethanol, followed by a 1 : 1 mixture of 50% ethanol and 50% xylene, then 100% xylene, and then cover-slipped. Immunohistochemical staining for PGP 9.5 (R&D Systems, Minneapolis, MN), laminin and S-100 (both Dako Corp.) was performed on a Dako autostainer as previously described21 with a labeled streptavidin-biotin-peroxidase complex system to visualize the immune reactions. Immunohistochemical positivity was observed and recorded by the authors M. Kiupel and Y. Jones.

The following antibodies were used in immunohistochemical staining at The Ohio State University College of Veterinary Medicine: p53 (Zymed 13-4100, 0.5 mg/ml, detects mutant p53, Zymed Laboratories, Inc., San Francisco, CA), PCNA (Clone PC10, No. M 0879, Dako Corp.), gadd45 (Santa Cruz; C-20, sc-792, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-UVssDNA (Trevigen Cat No. 4350-MC-100, Trevigen, Inc., Gaithersberg, MD), and TERT (Santa Cruz; L-20, sc7214, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protocol for anti-UVssDNA was as follows: 5 µm sections were deparaffinized on slides, incubated in 0.05 N HCl for 5 minutes on ice, and washed 3 times in 1x phosphate-buffered saline (PBS). Slides were incubated in 100 µg/ml RNase in 150 mM NaCl, 15 mM sodium citrate for 1 hour at 37°C, and then washed sequentially in 1x PBS, 50% ethanol, and 70% ethanol for 2 minutes each. DNA was denatured in situ with 0.15 N NaOH in 70% ethanol for 4 minutes at room temperature. Samples were washed sequentially in 70% ethanol, 50% ethanol, and 1x PBS for 2 minutes each, and then incubated in 5 µg/ml proteinase K in 20 mM Tris, 1 mM ethylenediamine tetraacetic acid (EDTA), pH 7.5 for 10 minutes at 37°C. Samples were then washed in 1x PBS. The anti-UVssDNA antibody (primary) was diluted to 6 µg/ml in 1x PBS, 0.01% Tween 20, on ice. Slides were incubated in diluted primary antibody for 1 hour at 37°C (or overnight at 4°C, or at room temperature for 3–4 hours), and then washed 3 times in 1x PBS for 5 minutes each. Slides were incubated in biotinylated anti-mouse antibodies (secondary) for 45 minutes at 37°C, and then washed 10 times in 1x PBS. Slides were incubated in peroxidase-conjugated streptavidin for 30 minutes at 37°C, and then washed 10 times in 1x PBS. Slides were then incubated in Vector NovaRED (chromogen, Vector Laboratories, Burlingame, CA) for 5 minutes at room temperature, and then washed 10 times in tap water. Slides were counterstained in Mayer's hematoxylin counterstain for 3 minutes at room temperature. Slides were then washed 10 times in tap water, dehydrated, mounted, and cover-slipped. Samples were visualized by light microscopy (Olympus BX-50, Olympus America Inc., Center Valley, PA) and digitally photographed (Olympus DP12 digital camera). Positivity was determined by CMHC and KN.

Protocol for PCNA: tissues were deparaffinized, hydrated, and then pretreated with Dako Target Retrieval Solution (catalog No. S1699, Dako Corp.). Retrieval solution was preheated to 90°C. Slides were incubated 20 minutes in steamer, and then cooled for 20 minutes. Slides were placed in Dako wash buffer (TBS/Tween 20, catalog No. S3006, Dako Corp.) for 5 minutes. Procedure on Dako autostainer was as follows: slides were rinsed once with water (rw) x 1, blocked for 5 minutes in peroxidase block in 3% H2O2, rw x 1, and rinsed once with wash buffer (rwb) x 1. Slides were then blocked with Dako serum-free protein block (catalog No. X0909, Dako Corp.) for 10 minutes. Samples were subsequently incubated for 30 minutes in primary anti-PCNA antibody, diluted 1 : 200 in Dako antibody diluent with background reducing components (catalog No. S3022, Dako Corp.), rwb x 2. Slides were incubated for 30 minutes in secondary antibody (Vector biotinylated horse anti-mouse, catalog No. BA-2000, diluted 1 : 200, Vector Labs.) in serum-free protein block, rwb x 2 and placed for 30 minutes in ABC (Vector R.T.U. Vectastain elite ABC, catalog No. PK-7100, Vector Labs.), rwb x 2, rw x 1, incubated for 5 minutes in Dako liquid DAB substrate (catalog No. K3466, Dako Corp.), rw x 1, rwb x 1. Slides were counterstained for 2 minutes in Richard-Allen hematoxylin 2 (Richard-Allen Scientific, Kalamazoo, MI) catalog No. 7231, rw x 2, dehydrated, and cover-slipped.

Protocol for TERT (1 : 350), p53, gadd45 (1 : 250) was as follows: samples were deparaffinized, rehydrated, and blocked with 3% peroxidase for 10 minutes at 37°C. Power Block (1x, catalog No. BS-1310-25, Biogenex, San Ramon, CA) was used for 10 minutes at 37°C. The sample was incubated with primary antibody for 1 hour at 37°C, and then washed. The sample was incubated in secondary antibody (super-sensitive label, peroxidase-conjugated streptavidin) for 30 minutes at 37°C, and then washed. Positivity was detected using Vector NovaRED for 5 minutes before washing. Samples were counterstained with Mayer's hematoxylin for 3 minutes at room temperature, washed, dehydrated, and cover-slipped. Positive controls for gadd45 and TERT were cataractous lens epithelial cells.8 Negative controls, where primary of secondary antibodies were omitted, were performed in all cases.

For electron microscopy, 1 mm3 of 2 formalin-fixed tumors were harvested and postfixed in gluteraldehyde. This tissue was postfixed a second time in osmium tetroxide and embedded in acrylic resin. Thin sections (approximately 90 nm) were cut, mounted on 200-mesh copper grids, stained with 5% methanolic uranyl acetate and Reynolds' lead citrate, and examined and photographed on a Philips 410 transmission electron microscope (Philips Medical Systems, Andover, MA).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
For signalment, follow-up, morphologic characteristics, and immunohistochemical results, see Table 1. While there was no sex predilection, Siberian Husky purebred or mix dogs were overrepresented (10/13 cases). The median age was 10 years. Histologically, all tumors asymmetrically expanded the iris and ciliary body and consisted entirely of spindle-shaped mesenchymal cells (Figs. 1–4). Tumor cells formed solid sheets of interwoven bundles, streams or whorls, often with palisading nuclei. Tumor morphology, nuclear features, necrosis, and mitotic index were highly variable. Antoni A and/or Antoni B behavior were exhibited by all tumors, consistent with human schwannomas (Figs. 3, 4).32 Tumors were graded as 1, 2, or 3 as previously described.34 All cases exhibited some degree of lymphoplasmacytic uveitis. Most cases (except for cases 2, 4, 11, and 13) exhibited either clinical or histopathologic evidence of glaucoma (historical ocular hypertension, loss of retinal ganglion cells, optic nerve cupping/gliosis). Other secondary findings within the globes included preiridal fibrovascular membranes, intraocular hemorrhage, peripheral corneal stromal neovascularization and inflammatory infiltrate, and cataract.


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  		Table 1. Signalment, morphology, immunopositivity, and follow-up for 13 uveal spindle-cell tumors of blue-eyed dogs.*

 

Figure 02
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  		Fig. 2. Iris; dog No. 3. Along the left, relatively normal iris stroma is bordered by posterior iridal epithelium. To the right, there is a round mass of mesenchymal cells lined anteriorly by hemorrhage. Within the center of the image, the neoplastic cells exhibit Antoni A behavior. HE stain.

 

Figure 03
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  		Fig. 3. Uveal spindle-cell tumor; dog No. 3. Antoni A behavior is shown. HE stain.
Fig. 4. Uveal spindle-cell tumor; dog No. 1. Antoni B behavior is shown. HE stain.
Fig. 5. Uveal spindle-cell tumor; dog No. 8. Intracytoplasmic immunopositivity is shown. S-100 immunohistochemistry.
Fig. 6. Uveal spindle-cell tumor; dog No. 13. Intracytoplasmic immunopositivity is shown. Glial fibrillary acidic protein (GFAP) immunohistochemistry.
Fig. 7. Uveal spindle-cell tumor; dog No. 7. Extracellular immunopositivity is shown. Laminin immunohistochemistry.
Fig. 8. Uveal spindle-cell tumor; dog No. 11. Nuclear and cytoplasmic immunopositivity is shown. Growth arrest and DNA damage45 (Gadd45) immunohistochemistry.
Fig. 9. Uveal spindle-cell tumor; dog No. 8. Nuclear immunopositivity is shown. UV single strand DNA (UVssDNA) immunohistochemistry.
Fig. 10. Uveal spindle-cell tumor; dog No. 6. Nuclear and cytoplasmic immunopositivity is shown. Telomerase reverse transcriptase (TERT) immunohistochemistry.

 
All dogs tested were negative for SMA (6/6), desmin (6/6), Melan A (7/7), and MITF-1 (7/7) and positive for vimentin (10/10) and S-100 (7/7). The tumors were positive for GFAP in 9/13 dogs, positive for PGP 9.5 in 7/9 dogs, and positive for laminin in 2/9 dogs (Figs. 5–7). Results for gadd45, p53, PCNA, anti-UVssDNA, and TERT were variable and are shown in Table 1 and Figs. 8–10.

Follow-up ranged from 0–28 months (see Table 1). While several patients died with clinical signs consistent with thoracic and abdominal metastasis, no necropsies were performed. Furthermore, dogs with possible metastases did not have any obvious morphologic or immunohistochemical similarities that might relate to prognosis.

Electron microscopic findings revealed that tumor cells had long interdigitating cytoplasmic processes and intermittent basal laminae at the plasma membrane (Fig. 11). The extracellular matrix consisted of collagen bundles.


Figure 04
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  		Fig. 11. Electron microscopic image of spindle-cell tumor; dog No. 10. The arrow is directed at the basal lamina.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Immunohistochemical stains were initially performed to rule out nonneural tumors. Vimentin immunoreactivity suggests that this tumor is of mesenchymal origin.13 Leiomyosarcoma or rhabdomyosarcoma are unlikely because the tumors did not stain for SMA and desmin.5 MITF-1 and Melan A immunonegativity suggest that this tumor is not of melanocyte origin.6,16 Immunohistochemical staining for c-kit was also performed on nine tumors (CD117, results not shown); all tumor cells were immunonegative. The GFAP immunopositivity makes a diagnosis of fibrosarcoma unlikely.38

Glial fibrillary acidic protein staining, in 9/13 dogs, suggests central nervous system/astrocyte origin; however, GFAP positivity has also been associated with both nonmyelinating Schwann cells52 and normal rodent uvea.4 Electron microscopy was also consistent with a tumor of peripheral nerve sheath origin due to the interrupted basal laminae between cells.32 Protein gene product 9.5 is a relatively neuron-specific protein similar to neuron-specific enolase.51 Laminin is found in basement membranes of endothelial cells, smooth muscle cells, and Schwann cells.5 Laminin staining was positive in only 2/9 cases. Formalin fixation may have interfered with stain uptake, or laminin expression may have been variable. This tumor is immunohistochemically and morphologically most consistent with a peripheral nerve sheath tumor, which arises from Schwann cells, neurofibroblasts, or other perineural cells.32 Such tumors in animals are generally classified as (benign) schwannomas or malignant peripheral nerve sheath tumors. The presence of Antoni A and B patterns in these tumors is also consistent with neural origin.26,32 While electron microscopy, light microscopy, and GFAP immunohistochemistry are most consistent with a schwannoma, these findings cannot reliably differentiate between a benign/malignant schwannoma, neurofibroma, neurofibrosarcoma, or other tumor of nerve origin in dogs.25,52

To the authors' knowledge, no intraocular peripheral nerve sheath tumors have been reported in the dog except for a recent case report by Sato et al.43 While morphologically similar to the tumors in this study, those authors clearly stated in a personal correspondence (Sato) that there were no identifiably blue areas in the affected (or unaffected) eye of their patient, a Shetland Sheepdog. This tumor immunohistochemically stained positively for S-100 and vimentin. At the COPLOW, no similar uveal tumors have been diagnosed in any other species or in brown-eyed dogs. Relative to brown-eyed dogs, blue-eyed dogs are at much higher risk for this tumor. Other studies have shown extraocular, benign, canine schwannomas to be vimentin positive (most consistent feature), desmin negative, variably GFAP positive, and often S-100 positive.32,38

Why do these tumors only occur in dogs with blue irides? To answer this question, the embryology and anatomy of the canine iris must be considered. While recent studies suggest that mammalian uveal stroma is formed from a mixture of neural crest and mesoderm, neural crest is believed to give rise to melanocytes within the iris.10,15,24 The color of the iris is normally dictated by heritable factors and determined by the pigment granules within the posterior iris epithelium, Rayleigh scattering of light by the stromal extracellular matrix, type of melanin, and the number and area of melanin granules within the iris stromal melanocytes.24,50 Aside from polygenic inheritance affecting melanin content of the iris, blue iridal color can result from a tyrosinase defect preventing melanin production in stromal melanocytes (heterochromic laboratory beagles,45 albinism, Siamese cats48), as well as abnormal neural crest migration into the uvea causing an absence of stromal melanocytes (white cats, associated with deafness3). At the COPLOW, two goniodysgenic canine irides (lacking tumor) were stained with GFAP. These irides showed increased linear GFAP positivity, consistent with nonmyelinated nerves, within the iris stroma compared with a similarly stained, bleached brown iris. Melan A immunohistochemistry on the same 2 goniodysgenic canine blue eyes found no convincingly positive melanocytes within the iridal stroma (results not shown). Lack of melanocytes within the uveal stroma would corroborate that this is not a primary intraocular melanocytic tumor; furthermore, it suggests a defect in neural crest. However, these findings cannot necessarily be extrapolated to all blue-eyed dogs; furthermore, the coat color of the patients with uveal spindle cell tumors was generally not recorded.

In addition to iridal melanocytes, neural crest is also responsible for the development of peripheral nerve tissue, including Schwann cells.35 In human neural crest–related diseases, genetic mutations in PAX 3 gene (Waardenburg syndrome) and NF1 gene (neurofibromatosis) are associated with hypopigmented irides or light brown iridal café au lait spots, respectively.23,42 While iris color is not necessarily blue in humans with neurofibromatosis, iridal lesions include pigmented hamartomas that are considered to be of neural crest origin (Lisch nodules).23 The NF1 gene is a tumor suppressor gene and neurofibromas and schwannomas are a feature of neurofibromatosis. Peripheral nerve sheath tumors related to human neurofibromatosis 1 are usually benign but can undergo malignant transformation.14,37 More research needs to be performed to determine whether or not a genetic defect in neural crest development contributes to oncogenesis in this tumor (particularly in Siberian Huskies).7

Ultraviolet radiation has been shown to contribute to oncogenesis in many tumors, including uveal melanoma.20,22,44,46 Schwannomas have been reported in humans in association with radiation therapy (but not UV damage).41 Positive immunohistochemical staining for p53, anti-UVssDNA, PCNA, gadd45, and TERT have all been associated with UV damage and oncogenesis. Specifically, research suggests that gadd45 and PCNA facilitate repair after DNA undergoes UV-related damage.1,47 Telomerase refers to a ribonucleoprotein complex that has 2 main components: an RNA subunit (TR) and a catalytic subunit (TERT). All cells express TR, but TERT is only expressed in cells that have telomerase activity. Telomerase activity facilitates repeated cell division and is therefore necessary for tumor proliferation.17,18 Up-regulation of TERT is strongly associated with exposure to the sun in normal human skin samples and exposure to UV radiation results in increased telomerase activity in lens epithelial cells, cultured splenocytes, and fibroblasts, suggesting a role in UV-induced oxidative stress.9,18,19,49 In one study, 100% of tumor samples with UV-specific p53 mutations also exhibited TERT activity; both telomerase activity and p53 mutations have been implicated in oncogenesis.49 The p53 gene is commonly mutated in human cancers and is integral in DNA repair after UV damage;47 it controls cell cycle arrest, apoptosis, and senescence, among other antitumor functions.12 Mutations in p53 cause increased p53 expression because the mutated form has a much longer half-life than the wild-type form.27 The UVssDNA antibody is specific for proteins that contain the (6-4)-dipyrimidine photoproduct, a product of UV damage.53 With this in mind, increased expression of gadd45, PCNA, TERT, p53, and anti-UVssDNA should all be up-regulated in tumors influenced by UV damage.

The results of this study were inconsistent for markers of UV-induced tissue damage and do not clearly support a role for UV damage in the development of this tumor. However, these results do not exclude UV damage as a factor because UV markers are in some cases only temporarily up-regulated after UV damage.36 There are other unmeasured effects of UV radiation36 and other UV-related antibodies that were not available for use in this study.31 Technically, inconsistent immunohistochemical results may be due to (delayed) fixation,13,52 anaplasia, or multiple cellular origins. Uveal spindle-cell tumors of blue-eyed dogs may arise as a result of a multifactorial process including a genetic neural crest defect and UV damage.40 Other melanin-related or genetic mechanisms may also be in play. Melanin has been shown to be associated with intraocular zinc storage (an antioxidant).33

We present 13 cases of a uveal spindle-cell tumor of blue-eyed dogs with morphologic and immunohistochemical features most consistent with a schwannoma. Morphologic features of malignancy were variable. While several of the dogs have died with clinical signs consistent with metastatic disease, no necropsies have been performed. The cell of origin, its relationship to neural crest, and the contribution of UV damage in the pathogenesis of uveal tumors in blue-eyed dogs have yet to be fully elucidated. Until that time, we will continue to refer to these tumors as uveal spindle-cell tumors of blue-eyed dogs. These tumors continue to accumulate at the COPLOW. We will continue to examine these cases and seek follow-up to better assess their behavioral malignancy. This tumor should be considered as a differential diagnosis in all intraocular masses arising in the blue canine uvea.


   Acknowledgements
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors would like to thank Drs. Steven Weisbrode and Donna Kusewitt for expert consultation. The authors would also like to thank the referring veterinarians and owners of these dogs for providing follow-up information.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Aboussekhra A, Wood RD. Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA. Exp Cell Res 221:326–332, 1995[CrossRef][ISI][Medline]
  2. Agar N, Young AR. Melanogenesis: a photoprotective response to DNA damage?. Mutat Res 571:121–132, 2005[ISI][Medline]
  3. Bergsma DR, Brown KS. White fur, blue eyes, and deafness in the domestic cat. J Hered 62:171–185, 1971[Free Full Text]
  4. Bjorklund H, Dahl D. Glial fibrillary acidic protein (GFAP)-like immunoreactivity in the rodent eye. J Neuroimmunol 8:331–345, 1985[CrossRef][ISI][Medline]
  5. Cerilli LA, Wick MR. Immunohistology of soft tissue and osseus neoplasms. In: Diagnostic Immunohistochemistry Dabbs DJ, ed. 59–112,Churchill Livingston, New York, NY. 2002
  6. Choi C, Kusewitt DF. Comparison of tyrosinase-related protein-2, S-100, and Melan A immunoreactivity in canine amelanotic melanomas. Vet Pathol 40:713–718, 2003[Abstract/Free Full Text]
  7. Clark LA, Wahl JM, Rees CA, Murphy KE. Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog. Proc Natl Acad Sci USA 103:1376–1381, 2006[Abstract/Free Full Text]
  8. Colitz CM, Davidson MG, McGahan MC. Telomerase activity in lens epithelial cells of normal and cataractous lenses. Exp Eye Res 69:641–649, 1999[CrossRef][ISI][Medline]
  9. Colitz CM, Whittington A, Carter R, Warren J. The effects of oxidative stress on telomerase activity and other stress-related proteins in lens epithelial cells. Exp Eye Res 78:235–242, 2004[CrossRef][ISI][Medline]
  10. Creuzet S, Vincent C, Couly G. Neural crest derivatives in ocular and periocular structures. Int J Dev Biol 49:161–171, 2005[CrossRef][ISI][Medline]
  11. Diters RW, Dubielzig RR, Aguirre GD, Acland GM. Primary ocular melanoma in dogs. Vet Pathol 20:379–395, 1983[Abstract]
  12. el-Deiry WS. Regulation of p53 downstream genes. Semin Cancer Biol 8:345–357, 1998[CrossRef][ISI][Medline]
  13. Elias JM. Immunohistopathology: A Practical Approach to Diagnosis ASCP Press, Chicago, IL. 1990
  14. Ferner RE, O'Doherty MJ. Neurofibroma and schwannoma. Curr Opin Neurol 15:679–684, 2002[CrossRef][ISI][Medline]
  15. Gage PJ, Rhoades W, Prucka SK, Hjalt T. Fate maps of neural crest and mesoderm in the mammalian eye. Invest Ophthalmol Vis Sci 46:4200–4208, 2005[Abstract/Free Full Text]
  16. Goding CR. Mitf from neural crest to melanoma: signal transduction and transcription in the melanocyte lineage. Genes Dev 14:1712–1728, 2000[Free Full Text]
  17. Greider CW. Telomerase activity, cell proliferation, and cancer. Proc Natl Acad Sci USA 95:90–92, 1998[Free Full Text]
  18. Hande MP, Balajee AS, Natarajan AT. Induction of telomerase activity by UV-irradiation in Chinese hamster cells. Oncogene 15:1747–1752, 1997[CrossRef][ISI][Medline]
  19. Hande MP, Lansdorp PM, Natarajan AT. Induction of telomerase activity by in vivo X-irradiation of mouse splenocytes and its possible role in chromosome healing. Mutat Res 404:205–214, 1998[ISI][Medline]
  20. Harbour JW, Brantley MA, Hollingsworth H, Gordon M. Association between choroidal pigmentation and posterior uveal melanoma in a white population. Br J Ophthalmol 88:39–43, 2004[Abstract/Free Full Text]
  21. Hoenerhoff MJ, Kiupel M, Rosenstein D, Pool RR. Multipotential osteosarcoma with various mesenchymal differentiations in a young dog. Vet Pathol 41:264–268, 2004[Abstract/Free Full Text]
  22. Hu DN. Photobiology of ocular melanocytes and melanoma. Photochem Photobiol 81:506–509, 2005[CrossRef][ISI][Medline]
  23. Huson S, Jones D, Beck L. Ophthalmic manifestations of neurofibromatosis. Br J Ophthalmol 71:235–238, 1987[Abstract/Free Full Text]
  24. Imesch PD, Bindley CD, Khademian Z, Ladd B, Gangnon R, Albert DM, Wallow IH. Melanocytes and iris color: electron microscopic findings. Arch Ophthalmol 114:443–447, 1996[Abstract]
  25. Kennedy PGE. Neural cell markers and their applications to neurology. J Neuroimmunol 2:35–53, 1982[CrossRef][ISI][Medline]
  26. Kim IT, Chang SD. Ciliary body schwannoma. Acta Ophthalmol Scand 77:462–466, 1999[CrossRef][ISI][Medline]
  27. Kindblom L-G, Ahlden M, Meis-Kindblom JM, Stenman G. Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell antigen and Ki67 in benign and malignant peripheral nerve sheath tumours. Virchows Arch 427:19–26, 1995[ISI][Medline]
  28. Klauss G, Dubielzig R. Characteristics of primary spindle cell neoplasms of the anterior uveal tract in eleven dogs. American College of Veterinary Pathologists Annual Conference. Vet Pathol 38:574, 2001
  29. Klauss G, Dubielzig R. Primary spindle cell neoplasms of the anterior uveal tract of fourteen dogs. American College of Veterinary Ophthalmologists Annual Conference 2001 p 55.
  30. Kliman GH, Augsburger JJ, Shields JA. Association between iris color and iris melanocytic lesions. Am J Ophthalmol 100:547–548, 1985[ISI][Medline]
  31. Kobayashi N, Katsumi S, Imoto K, Nakagawa A, Miyagawa S, Furumura M, Mori T. Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology. Pigment Cell Res 14:94–102, 2001[CrossRef][ISI][Medline]
  32. Koestner A, Higgins RJ. Tumors of the nervous system. In: Tumors in Domestic Animals Meuten DJ, ed. 697–738,Iowa State Press, Ames, IA. 2002
  33. Kokkinou D, Kasper HU, Bartz-Schmidt KU, Schraermeyer U. The pigmentation of human iris influences the uptake and storing of zinc. Pigment Cell Res 17:515–518, 2004[CrossRef][ISI][Medline]
  34. Kuntz CA, Dernell WS, Powers BE, Devitt C, Straw RC, Withrow SJ. Prognostic factors for surgical treatment of soft-tissue sarcomas in dogs: 75 cases (1986–1996). J Am Vet Med Assoc 211:1147–1151, 1997[ISI][Medline]
  35. Larsen WJ. Human Embryology. Churchill Livingstone, Philadelphia, PA. 2001
  36. Matsumura Y, Anathaswamy HN. Toxic effects of ultraviolet radiation on the skin. Toxicol Appl Pharmacol 195:298–308, 2004[CrossRef][ISI][Medline]
  37. Murovic JA, Kim DH, Kline DG. Neurofibromatosis-associated nerve sheath tumors: case report and review of the literature. Neurosurg Focus 20:E1, 2006[Medline]
  38. Perez J, Bautista MJ, Rollon E, de Lara FC, Carrasco L, Martin de las Mulas J. Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog. Vet Pathol 33:391–397, 1996[Abstract]
  39. Pfeifer GP, You YH, Besaratinia A. Mutations induced by ultraviolet light. Mutat Res 571:19–31, 2005[ISI][Medline]
  40. Robanus-Maandag E, Giovannini M, va der Valk M, Niwa-Kawakita M, Abramowski V, Antonescu C, Thomas G, Berns A. Synergy of Nf2 and p53 mutations in development of malignant tumours of neural crest origin. Oncogene 23:6541–6547, 2004[CrossRef][ISI][Medline]
  41. Rubinstein AB, Reichenthal E, Borohov H. Radiation-induced schwannomas. Neurosurgery 24:929–932, 1989[ISI][Medline]
  42. Sarnat HB, Flores-Sarnat L. Embryology of the neural crest: its inductive role in the neurocutaneous syndromes. J Child Neurol 20:637–643, 2005[Abstract/Free Full Text]
  43. Sato T, Yamamoto A, Shibuya H, Sudo H, Shirai W, Amemori T. Intraocular peripheral nerve sheath tumor in a dog. Vet Ophthalmol 8:283–286, 2005[CrossRef][ISI][Medline]
  44. Shah CP, Weis E, Lajous M, Shields JA, Shields CL. Intermittent and chronic ultraviolet light exposure and uveal melanoma: a meta-analysis. Ophthalmology 112:1599–1607, 2005[CrossRef][ISI][Medline]
  45. Shively JN, Phemister RD. Fine structure of the iris of dogs manifesting heterochromia iridis. Am J Ophthalmol 66:1152–1162, 1968[ISI][Medline]
  46. Singh AD, Rennie IG, Seregard S, Giblin M, McKenzie J. Sunlight exposure and pathogenesis of uveal melanoma. Surv Ophthalmol 49:419–428, 2004[CrossRef][ISI][Medline]
  47. Smith ML, Ford JM, Hollander MC, Bortnick RA, Amundson SA, Seo YR, Deng CX, Hanawalt PC, Fornace AJ. p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol Cell Biol 20:3705–3714, 2000[Abstract/Free Full Text]
  48. Thibos LN, Levick WR, Morstyn R. Ocular pigmentation in white and Siamese cats. Invest Ophthalmol Vis Sci 19:475–486, 1980[Abstract/Free Full Text]
  49. Ueda M, Ouhtit A, Bito T, Nakazawa K, Lubbe J, Ichihashi M, Yamasaki H, Nakazawa H. Evidence for UV-associated activation of telomerase in human skin. Cancer Res 57:370–374, 1997[Abstract/Free Full Text]
  50. Wilkerson CL, Syed NA, Fisher MR, Robinson NL, Wallow IH, Albert DM. Melanocytes and iris color: light microscopic findings. Arch Ophthalmol 114:437–442, 1996[Abstract]
  51. Wilkinson KD, Lee K, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246:670–673, 1989[Abstract/Free Full Text]
  52. Yen S-H, Fields KL. A protein related to glial filaments in Schwann cells. Ann N Y Acad Sci 455:538–551, 1985[CrossRef][ISI][Medline]
  53. Zavala AG, Lancaster T, Groopman JD, Strickland PT, Chandrasegaran S. Phage display of ScFv peptides recognizing the thymidine(6-4)thymidine photoproduct. Nucleic Acids Res 28:E24, 2000[CrossRef][Medline]
Request reprints from R. R. Dubielzig, University of Wisconsin-Madison, Department of Pathobiological Sciences, 2015 Linden Drive, Madison, WI 53706 (USA). E-mail: dubielzr{at}svm.vetmed.wisc.edu





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