Comparison of tyrosinase-related protein-2, S-100, and Melan A immunoreactivity in canine amelanotic melanomas

Tyrosinase-related protein-2 (TRP-2) is a highly conserved melanogenic enzyme expressed in both pigmented and unpigmented melanomas of the mouse. To determine whether TRP-2 would be a good diagnostic marker for amelanotic melanomas of the dog, we performed immunohistochemistry for TRP-2, S-100, and Melan A on 21 canine tumors identified as amelanotic melanomas based on routine histopathologic examination. Thirteen of the tumors were TRP-2 positive, 10 were Melan A positive, and 19 were S-100 positive. TRP-2 was expressed in the cytoplasm of tumor cells in both primary and metastatic melanomas. S-100 staining was positive in all of three schwannomas and two of three gastrointestinal stromal tumors (one fibrosarcoma and one leiomyosarcoma) tested. Neither Melan A nor TRP-2 antibodies reacted with these tumors. Our findings indicate that staining for TRP-2 is a sensitive and specific method for confirming the diagnosis of amelanotic melanoma in dogs.     

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.     

An osteoid variant of cutaneous melanoma in a dog detected by S100 and melan a markers

Osteoid malignant melanoma is a rare type of melanoma described in humans and dogs with some areas of bone differentiation. In this tumour, the origin of the bone matrix remains unclear. We report one case of this variant with, for the first time, a cutaneous origin in a dog. Malignant melanomas are aggressive tumours. Amelanotic tumours are sometimes difficult to recognize as they require immunohistochemical evaluation for an adequate diagnosis and we have used anti-vimentin, S100, and melan A antibodies for identification. Melan A is less sensitive but more specific than S100 in identifying amelanotic melanomas. This tumour was positive for vimentin, S100 and melan A, including the areas of osteoid. These results suggest osteoid differentiation of tumour cells rather than induced stromal metaplasia.     

Retrospective study of 338 canine oral melanomas with clinical, histologic, and immunohistochemical review of 129 cases

Diagnostic records from 338 canine oral melanomas in 338 dogs received at the Veterinary Medical Diagnostic Laboratory (1992-1999) were reviewed. Of these tumors, 122 plus an additional 7 metastatic melanomas of unknown origin were selected for clinical follow-up, histologic review, and immunohistochemistry. Chow Chow, Golden Retriever, and Pekingese/Poodle mix breeds were overrepresented, whereas Boxer and German Shepherd breeds were underrepresented. There was no gender predisposition and the average age at presentation was 11.4 years. Forty-nine dogs were euthanized due to recurrence or metastasis. The average postsurgical survival time was 173 days. The gingiva and the labial mucosa were the most common sites. Most tumors were composed of either polygonal cells (27 cases, 20.9%), spindle cells (44 cases, 34.1%), or a mixture of the two (polygonal and spindle) (54 cases, 41.9%). Clear cell (3 cases, 2.3%) and adenoid/papillary (1 case, 0.8%) patterns were uncommon. The metastases of 6/6 oral melanomas had morphologic and immunohistochemical features similar to those of the primary tumors. Immunohistochemically, Melan A was detected in 113/122 oral (92.6%) and 5/7 (71.9%) metastatic melanomas. Only 4/163 nonmelanocytic tumors were focally and weakly positive for Melan A. Antibodies against vimentin, S100 protein, and neuron-specific enolase stained 129 (100%), 98 (76%), and 115 (89.1%) of 129 melanomas, respectively. Antibodies against other melanocytic-associated antigens (tyrosinase, glycoprotein 100) did not yield adequate staining. We conclude that Melan A is a specific and sensitive marker for canine melanomas.     

Soft tissue sarcomas in the African hedgehog (Atelerix albiventris): microscopic and immunohistologic study of three cases.

Three soft tissue tumors from 2 female hedgehogs were examined microscopically and immunohistochemically. Two tumors involved haired skin and the third one was vaginal. Microscopically, the cutaneous tumors had features of malignant peripheral nerve sheath tumor (MPNST), whereas the vaginal tumor was classified only as a spindle cell sarcoma. Immunohistochemically, all 3 tumors were strongly positive for vimentin and strongly to moderately positive for CD10 and neuron-specific enolase but did not stain with antibody to S100 protein, an antigen typically present in human MPNST The cutaneous tumor from hedgehog no. 1 was examined ultrastructurally and the neoplastic cells resembled fibroblasts. Hedgehog no. 1 was euthanized at the time of the biopsy. The outcome of the other hedgehog was unknown.     

Immunoreactivity of A103, an antibody to Melan A, in canine steroid-producing tissues and their tumors.

The monoclonal antibody A103 to the melanocytic differentiation antigen Melan A stains human steroid-producing cells and their tumors. A total of 200 formalin-fixed, paraffin-embedded canine normal tissues and hyperplastic and neoplastic lesions of the adrenal gland, testis, and ovary were immunohistochemically tested for Melan A with antibody A103. Leydig cell tumors (23/23, 100%), Sertoli cell tumors (14/15, 93%), and adrenocortical adenomas (12/13, 92%) were consistently positive. Adrenocortical carcinomas (23/35, 65%) and granulosa cell tumors (10/17, 59%) were less frequently positive. All pheochromocytomas, seminomas, and dysgerminomas were negative. The pattern of staining was cytoplasmic, but nuclear staining was also frequently seen in normal Leydig cells and their tumors. As in human tumors, immunohistochemistry for Melan A stains many canine steroid-producing tumors and can be used to distinguish these tumors from those of nonstereidogenic cells.     

Immunohistochemical staining for S100 protein in the diagnosis of canine amelanotic melanoma

Formalin-fixed, paraffin-embedded tissue samples of canine amelanotic melanomas and normal canine tissues were studied immunohistochemically for the presence of S100 protein. Use of the avidin-biotin complex procedure demonstrated variable amounts of S100 protein in the tumor cell cytoplasm and nuclei in 26 of 31 tumors. S100 protein was not observed in some other common canine skin tumors stained by the avidin-biotin complex technique. These were a mast cell tumor, fibrosarcoma, mammary gland adenocarcinoma, histiocytoma, transmissible venereal tumor, and a thyroid gland adenocarcinoma. Among normal tissues the presence of S100 protein was demonstrated in chondrocytes in the trachea, myoepithelial cells in the breast, melanocytes in the skin, some sweat glands and ducts in the skin, stellate cells in the pituitary, and interdigitating reticulum cells in the lymph node and in Peyer's patches. These results indicate that the avidin-biotin complex procedure for demonstrating S100 protein is a useful diagnostic tool in the diagnosis of canine amelanotic melanoma.     

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.     

p53 expression and apoptosis in melanomas of dogs and cats

Twenty-seven melanocytic tumours from 20 dogs and four cats were examined for p53 expression and apoptosis. They included tumours that were histologically classified as benign (BM), primary malignant (PMM) and metastatic malignant melanomas (MMM). For all cases clinical follow-up was available. p53 expression was examined immunohistochemically using different monoclonal and polyclonal antibodies. Apoptosis was detected using the TUNEL technique. The tissue sections were analysed using a quantitative image analysing system. A p53 index (p53I) and an apoptotic index (AI) were determined. p53 over-expression was found infrequently in these canine and feline melanocytic tumours. Apoptosis was observed in some of the malignant tumours. In one feline case of malignant melanoma, p53 accumulation together with apoptosis was seen in three metastases but not in the primary tumour. p53I and AI were not significantly correlated with survival. These results are similar to those reported for human cutaneous melanomas.     

Feline melanoma: a comparative study of ocular, oral, and dermal neoplasms

Melanomas diagnosed in 29 cats over an 11 year period included 19 ocular (16 intraocular, three palpebral), five oral, and five dermal melanomas. Intraocular melanomas involved the ciliary body and iris in 12; the whole eye was involved in four. The average age of cats with intraocular melanomas was 11 years; the female : male ratio was 9 : 7. Histologically, eight intraocular tumors were mixed, six were epithelioid, and two were spindle cell. Ten of 16 cats (62.5%) with intraocular melanomas were killed because of the tumor at a mean of 156 days; four are living with no evidence of disease (average, 255 days). The mean time of death in cats with palpebral melanoma was 409 days. Metastasis occurred in 63% of cats with intraocular melanoma and all cats with palpebral melanoma. Four cats with oral melanoma were killed at a mean of 61 days; all had metastasis. Of five cats with cutaneous melanoma, one was killed with metastasis at 90 days; three cats were alive without evidence of recurrence or metastasis greater than 365 days after surgery. Results of this study indicate that in the cat, ocular melanomas are more common than oral and dermal melanomas, and ocular and oral melanomas are more malignant than dermal melanomas, with higher rates of mortality and metastasis.     

Aberrant p53 expression in feline vaccine-associated sarcomas and correlation with prognosis

Eighty spontaneously occurring feline vaccine-associated sarcomas (VAS) were evaluated to determine the immunohistochemical expression of the tumor suppressor gene p53. Sixty-five of 80 VAS (81%) exhibited positive immunoreactivity with Mab240, a murine monoclonal antibody that specifically recognizes mutated p53. Only 44 of 81 tumors (55%) were positive with rabbit polyclonal antibody CM-1. CM-1 often yielded nonspecific staining of nonneoplastic tissues. Nonspecific staining was greatly reduced or absent with Mab240. Cytoplasmic staining for p53 was a consistent pattern of VAS, occurring in 44% of tumors evaluated. Cats with tumors that exhibited cytoplasmic p53 had significantly shorter time to tumor recurrence compared to those cats with tumors that exhibited nuclear p53 staining (P = 0.0284), but no significant difference in survival outcome was observed. Immunohistochemical detection of p53 offers a prognostic tool for VAS, and, because abnormal p53 expression appears to be a common feature of feline VAS, molecular targeting of mutant p53, may offer a promising new therapeutic opportunity for this cancer.     

Anterior uveal spindle cell tumor in a cat

Purpose To describe a case of anterior uveal spindle cell tumor in a cat with features similar to spindle cell tumor of blue eyed dogs. Methods A 10‐year‐old female spayed domestic short‐haired cat was referred for an iris mass OS. The mass was solitary, nodular, nonpigmented, located medially, and causing dyscoria. A diagnosis of a benign epithelial tumor was suggested by a FNA of the mass. The cat was lost to follow‐up for 2 years, after which time she re‐presented with glaucoma, blindness and grossly evident iridal mass enlargement OS. Transconjunctival enucleation was performed and the globe submitted for histopathology. Results Histopathology of the enucleated globe revealed the superior iris to be infiltrated and effaced by a large population of neoplastic spindle cells. The cells were arranged in streams and bundles and exhibited Antoni‐A and Antoni‐B tissue patterns, which are characteristic of Schwann cell tumors. Mitotic figures were rare and cellular pleomorphism moderate. Immunohistochemical staining was positive for S‐100 protein and glial fibrillary acidic protein (GFAP), and negative for Melan‐A. Interestingly, there was no histological evidence of glaucoma. Conclusions Based on its histopathologic characteristics, this iris tumor was diagnosed as a Schwann cell variant of a peripheral nerve sheath tumor (PNST) closely resembling the spindle cell tumor of blue‐eyed dogs. Anterior uveal PNST has not been previously reported in cats to the authors’ knowledge. The presence of Antoni type A and type B tissue patterns along with immunohistochemical staining may facilitate a diagnosis of PNST and rule out malignant melanoma.     

Diagnostic immunohistochemistry of canine round cell tumors

Sixty-five canine skin neoplasms studied using immunocytochemistry, included 22 histiocytomas, 18 amelanotic melanomas, 14 cutaneous lymphosarcomas, six mast cell tumors, and five transmissible venereal tumors. Formalin-fixed, paraffin-embedded sections were stained using the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique for reactivity with S-100 protein, kappa and lambda immunoglobulin light chains, alpha-1-antitrypsin, alpha-1-antichymotrypsin, leukocyte common antigen (LCA), neuron-specific enolase, keratin, cytokeratin, muramidase, and vimentin. Detection of S-100, kappa and lambda light chains, neuron-specific enolase, and vimentin were most useful for screening these neoplasms. None of the markers examined was consistent in staining histiocytomas. While reactivity of S-100 (ten cases) and neuron-specific enolase (ten cases) was detected in some amelanotic melanomas, lambda light chain immunoglobulin (eight cases) was relatively consistent in cutaneous lymphomas. Mast cell neoplasms reacted with avidin and, therefore, were positive, even on negative control sections. Vimentin reacted strongly on all amelanotic melanomas and transmissible venereal tumors examined. These antibodies are helpful adjuncts in the differential diagnosis of canine skin tumors.     

Cutaneous melanoma in a ferret (Mustela putorius furo)

A 4-year-old spayed female ferret (Mustela putorius furo) was clinically evaluated for a slightly raised subcutaneous mass in the dorsal lumbar area. The mass was surgically excised and submitted for histopathologic evaluation. Histologically, the mass was composed of closely packeted large, atypical, polygonal to spindle-shaped cells arranged in sheets and short bundles. A few cells contained variable amounts of granular, brown to black intracytoplasmic pigment. Warthin-Starry and Fontana-Masson silver stains demonstrated variable numbers of fine black intracytoplasmic granules in most cells. The atypical cells stained positively for vimentin and S100 protein and negatively for cytokeratin and Melan A. Ultrastructurally, the neoplastic cells contained intracytoplasmic melanosomes in different stages of development. Compound melanosomes were not identified. To our knowledge, this report documents the first case of a spontaneous cutaneous melanoma in the ferret.     

Lack of detection of feline leukemia and feline sarcoma viruses in diffuse iris melanomas of cats by immunohistochemistry and polymerase chain reaction.

Diffuse iris melanoma was confirmed by light-microscopic examination in 10 formalin-fixed, paraffin-embedded globes from 10 cats. To determine if feline leukemia virus or a replication defective feline leukemia virus, feline sarcoma virus, was present in these anterior uveal melanomas, immunohistochemistry and polymerase chain reaction for feline leukemia virus were utilized. Immunohistochemical staining for feline leukemia virus glycoprotein 70 was performed on all 10 tumors using an avidin-biotin complex technique. The DNA was extracted from each specimen and a 166-base pair region of the feline leukemia virus long terminal repeat was targeted by polymerase chain reaction. Immunohistochemical staining for feline leukemia virus glycoprotein 70 and polymerase chain reaction amplification of a feline leukemia virus long terminal repeat region were negative in all cases. Feline leukemia virus/feline sarcoma virus was not detected in any neoplasms and therefore was unlikely to play a role in the tumorigenesis of these feline diffuse iris melanomas.     

Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection

Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifiable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids.     

Metastatic balloon cell melanoma in a dog

Background: Balloon cell melanoma is a rare variant of amelanotic melanoma that is difficult to differentiate from sebaceous cell carcinoma, liposarcoma, and other clear cell neoplasms without immunohistochemistry or ultrastructural evidence of melanin or melanosomes. Objective: The purpose of this report was to describe the clinical, cytologic, histologic, immunohistochemical, and ultrastructural findings in a dog with metastatic balloon cell melanoma. Methods: A 6‐year‐old female Golden Retriever was evaluated for a white, flocculent infiltrate in the anterior chamber of the left eye and an enlarged left prescapular lymph node. Cytologic evaluation of the eye and lymph node were performed following aqueocentesis and fine‐needle aspiration, respectively. The affected lymph node was examined histologically and stained for cytokeratin, vimentin, S‐100, and Melan A. Following euthanasia a necropsy was performed and samples of the affected lymph node were examined by electron microscopy. Results: Cytologic examination of the lymph node and aqueocentesis sample revealed round neoplastic cells that had abundant clear vacuolated cytoplasm. A tentative diagnosis of metastatic sebaceous cell carcinoma or clear cell neoplasm was made. Histologically, the affected lymph node had similar polygonal clear cells arranged in sheets and packets divided by delicate fibrovascular stroma. Immunohistochemical staining of the cells was negative for cytokeratin but positive for vimentin, weakly positive for S‐100, and strongly positive for Melan A. At necropsy, metastatic lesions were identified in the diaphragm, heart, lung, kidneys, left eye, prescapular and sublumbar lymph nodes, and multiple skin sites. Ultrastructural examination of neoplastic lymph nodes revealed many membrane‐bound vacuoles, myelinlike figures, and rare melanosomes. Conclusion: Immunohistochemical staining and ultrastructural features of the neoplastic cells supported a diagnosis of metastatic balloon cell melanoma.     

Cell proliferation and expression of connexins differ in melanotic and amelanotic canine oral melanomas

Melanoma is a malignant neoplasm occurring in several animal species, and is the most frequently found tumor in the oral cavity in dogs. Melanomas are classified into two types: melanotic and amelanotic. Prior research suggests that human amelanotic melanomas are more aggressive than their melanotic counterparts. This study evaluates the behavior of canine melanotic and amelanotic oral cavity melanomas and quantifies cell proliferation and the expression of connexins. Twenty-five melanomas (16 melanotic and 9 amelanotic) were collected from dogs during clinical procedures at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. After diagnosis, dogs were followed until death or euthanasia. Histopathology confirmed the gross melanotic or amelanotic characteristics and tumors were classified according to the WHO. HMB45 or Melan A immunostainings were performed to confirm the diagnosis of amelanotic melanomas. Cell proliferation was quantified both by counting mitotic figures and PCNA positive nuclei. Expressions of connexins 26 and 43 were evaluated by immunohistochemistry, qRT-PCR and Western blot. Dogs bearing amelanotic melanomas presented a shorter lifespan in comparison to those with melanotic melanomas. Cell proliferation was significantly higher in amelanotic melanomas. Expressions of Connexins 26 and 43 were significantly reduced in amelanotic melanomas. The results presented here suggest that oral cavity melanotic and amelanotic melanomas differ regarding their behavior, cell proliferation and connexin expression in dogs, indicating a higher aggressiveness of amelanotic variants.