A comparative review of melanocytic neoplasms

Melanoma is a devastating disease frequently encountered within both veterinary and human medicine. Molecular changes linked with neoplastic transformation of melanocytes include mutations in genes that encode proteins intrinsic to the regulatory pathways of two tumor suppressor proteins (retinoblastoma protein and p53), proto-oncogene mutation to oncogenes, altered expression of epithelial cadherin and CD44 adhesion molecules, and upregulation of angiogenic factors and other growth factors. Histologic evaluation of the primary mass is the most common means of diagnosis, with cytology used more frequently to document metastasis. Melanoma's highly variable histologic and cytologic patterns can make diagnosis by either method problematic. Adherent epithelioid morphology, including signet ring forms, and nonadherent round and spindle forms are recognized, with pigmentation an inconsistent finding. The site of the tumor, the thickness of the primary tumor or depth of invasion, and the number of mitotic figures per high-power field or per millimeter are used histologically to predict biologic behavior, whereas site and degree of pleomorphism are typically used for cytologic preparations. Diagnosis of amelanotic melanoma can be aided by ancillary diagnostic techniques. Tumor cells are usually positive for vimentin, S100, neuron-specific enolase, and Melan-A, and negative for cytokeratin. Melan-A as a positive marker is not as sensitive as the others are but is likely more specific. Monoclonal antibodies to human melanosome-specific antigens 1 and 5 cross-react with canine antigens for a combined sensitivity rate of 83%. Mouse monoclonal antibody IBF9 specifically recognizes canine melanoma antigen and also has good sensitivity. Serologic markers, including cytokines, cell adhesion molecules, and melanoma-inhibitory activity, are being investigated as potential sentinels of melanoma. Currently, there is no single diagnostic technique capable of differentiating benign from malignant melanocytic neoplasms or predicting survival time.     

A novel immunohistochemical marker of normal and neoplastic melanocytes in formalin-fixed, paraffin-embedded tissues of albino rats

In albino rats, spontaneous occurrence of melanocytic tumors is rare, with diagnosis difficult. This study evaluated immunoreactivity for PNL2 in normal and neoplastic melanocytes in formalin-fixed and paraffin-embedded tissues of albino rats. The samples consisted of 11 (1.57%) amelanotic melanomas in 700 rats (2 studies), 23 non-melanocytic tumors, and a wide variety of normal tissues. In normal albino rats, PNL2 stained the melanocytes in the iris and choroid of the eyeball and the hair bulb and basal cell layers of the epidermis of the whole body. In amelanotic melanoma, the tumor cells consisted of spindle cells with eosinophilic cytoplasm without melanin granules. PNL2 consistently stained cytoplasm in all amelanotic melanoma cells. In contrast, the nonmelanocytic tumor cells were not labeled. Electron microscopically, neoplastic, and normal melanocytes showed numerous cytoplasmic premelanosomes (stage II melanosome). In conclusion, PNL2 is direct against a fixative- and decalcific-resistant melanocyte-associated antigen, and has high specificity against normal and neoplastic melanocytes of albino rats.     

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.     

Pathology of canine oral malignant melanoma with cartilage and/or osteoid formation

Of 197 cases of canine oral malignant melanoma, 29 cases with myxoid, cartilage, and osteoid formation were studied pathologically and immunohistochemically. Tumor tissues were classified into spindle cell type (13 cases), epithelioid cell type (1 case), and mixed type (15 cases). Myxoid matrixes (29 tumors) were formed mainly in the tissues of spindle cell type and were positive for Alcian blue (pH 2.5). Cartilaginous matrixes (12 tumors) were formed in the myxoid tumor tissues. The morphology of atrophied neoplastic cells, which were embedded in the cartilage cavities, significantly differed from that of spindle cells proliferating in surroundings. There were reticular areas in the process of transitioning from myxoid to cartilaginous matrixes. Osteoid matrixes were not continuous with myxoid or cartilaginous matrixes, and arose as eosinophilic trabeculae in the dense collagenous connective tissues. A calcified bone trabecula was present among the osteoid trabeculae in a case. Melanin-producing melanocytes were proliferating in the collagenous matrixes, while amelanotic cells were in the osteoid matrixes. Immunohistochemistry demonstrated proliferating neoplastic cells as melanocytes. All cells in/out of these three matrixes were positive for Melan-A, S-100 protein, NSE, and vimentin. From these results, it is suggested that cartilage and osteoid matrixes are produced by dedifferentiated melanocytes.     

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.     

Canine oral primary melanoma cells exhibit shift to mesenchymal phenotype and phagocytic behaviour

Canine oral malignant melanoma (COMM) is a potentially lethal cancer disease. We established primary cell lines from mostly amelanotic primary COMM and metastases and assessed lesions and derived cells for Melan A, PNL2 and CD146 expression. Then, migration and invasion of CD146‐enriched vs ‐depleted COMM cells were analysed. Epithelial‐to‐mesenchymal transition (EMT) was addressed by Vimentin‐staining and MMP2/MMP9 zymography. Phagocytic behaviour was analysed by histopathological examination and phagocytosis assay. While Melan A‐ and PNL2‐staining yielded inconsistent data, 100% of COMM sections and primary cells showed CD146 expression, suggesting that this protein may serve as a prognostic marker. An overall correlation between CD146‐expression and migration/invasion was not observed. All primary cell lines consistently expressed Vimentin and secreted biologically active MMP2, indicating that they had undergone EMT. Importantly, COMM sections exhibited cell‐in‐cell structures, and all primary cell lines exhibited phagocytic activity, supporting the concept that cell cannibalism may have a role in COMM progression.     

Cyclooxygenase-2 expression is related with localization, proliferation, and overall survival in canine melanocytic neoplasms

A direct relationship has been firmly established between cyclooxygenase-2 (COX-2) expression and malignant behavior in human melanoma. This report examines the relationship between COX-2 expression and tumor location, mitotic and proliferative indices, degree of T CD3(+) lymphocyte infiltration, overall survival, and frequency of recurrence and metastasis of 57 melanocytic tumors (25 oral and 32 cutaneous). COX-2 was highly or moderately expressed in 88% of oral neoplasms (22 of 25), whereas for their cutaneous counterparts, COX-2 expression was low or insignificant in 75% of cases (24 of 32). High and moderate COX-2 expression levels were observed in 73% of melanocytic tumors with a mitotic index ≥ 3 per 10 high-power fields (26 of 36), whereas in 81% of tumors with a mitotic index < 3 (17 of 21), expression was mild or absent. There were 41 cases with known clinical outcomes; of those showing high, moderate, and mild COX-2 expression, 83.3% (10 of 12), 37.5% (3 of 8), and 25% (2 of 8) died, respectively, whereas 100% of animals showing no COX-2 expression (13 of 13) were still alive at the last follow-up. COX-2 expression was statistically correlated with tumor location, mitotic and percentage Ki-67 proliferative indices, and overall survival, frequency of neoplastic recurrence and metastasis. Regression analysis also showed disease-specific predictive value for COX-2 expression for subjects with melanocytic neoplasms. Additionally, only high COX-2 expression showed significant differences in overall survival, in comparison with moderate, mild, or absent expression. These results suggest that high COX-2 expression may be considered a prognostic biomarker and potentially as a target for therapeutic and preventive strategies in canine melanocytic neoplasms.     

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.     

C-kit expression in canine mucosal melanomas

The c-kit receptor is responsible for transmission of promigration signals to melanocytes; its downregulation may be involved in malignant progression of human melanocytic neoplasms. Expression of this receptor has not been examined in normal or neoplastic melanocytes from dogs. In this study, 14 benign dermal and 61 malignant mucosal melanocytic tumors were examined for c-kit (KIT) expression. Sites of the mucosal melanomas were gingiva (not further specified; n = 30), buccal gingiva (n = 6), soft palate (n = 4), hard palate (n = 5), tongue (n = 7), lip (n = 6), and conjunctiva (n = 3). Melan A was expressed in all 14 dermal melanocytomas and in 59 of 61 (96.7%) tumors from oral or conjunctival mucosa, confirming melanocytic origin. C-kit receptor expression was strong and diffuse throughout the cytoplasm in all 14 dermal melanocytomas and was identified in basilar mucosal melanocytes over submucosal neoplasms (27 of 61, 44.3%), junctional (neoplastic) melanocytes (17 of 61, 27.9%), and, less commonly, neoplastic melanocytes of the subepithelial tumors (6 of 61, 9.8%). KIT expression anywhere within the resected melanomas correlated with significantly longer survival. These results suggest that c-kit receptor expression may be altered in canine melanomas and may have potential as a prognostic indicator for mucosal melanomas.     

Use of immunocytochemical techniques in canine melanoma

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.     

Use of alkaline phosphatase staining to differentiate canine osteosarcoma from other vimentin-positive tumors

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowsky's stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multi-lobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.     

Melanocytic matricoma in a dog

We report a tumor that developed at the root of the tail in a male, 2-year-old Shih Tzu dog. Histologically, the tumor consisted of basaloid epithelial cells, with shadow cells and neoplastic melanocyte proliferation. Immunohistochemically, epithelial components were positive for keratin/cytokeratin but basaloid cells were negative. Mononuclear or multinuclear melanocytes were positive for vimentin, S-100 protein, and HMB-45. In conclusion, this dog was diagnosed with canine melanocytic matricoma on the basis of the similarity of the histologic and immunohistochemical features in humans, and it may be the first case in an animal.     

Soft tissue sarcomas and mast cell tumours in dogs; clinical behaviour and response to surgery

OBJECTIVE: To characterise the types of canine soft tissue sarcoma and mast cell tumour treated surgically at the University Veterinary Centre, Sydney. To evaluate the success of surgical treatment of these tumours and identify variables predictive of local recurrence and survival. To establish whether conclusions drawn from previous international studies are applicable to the University Veterinary Centre, Sydney, dog population and vice versa. DESIGN: Clinical presentation and results of surgical excision of 54 soft tissue sarcomas and 70 mast cell tumours affecting the trunk and limbs of dogs at the University Veterinary Centre, Sydney, between 1989 and 2001 were reviewed retrospectively. RESULTS: Cross-bred dogs and Rhodesian Ridgebacks were at significantly greater risk of developing soft tissue sarcomas, and Boxers, Australian Cattle Dogs and Staffordshire Bull Terriers were at significantly greater risk of developing mast cell tumours than other breeds. Fine needle aspiration biopsy yielded a correct diagnosis in 62.5% of soft tissue sarcomas and 96% of mast cell tumours. Local recurrence was encountered after surgical excision in 7.4% of soft tissue sarcomas and 7.3% of mast cell tumours. Metastasis occurred in 6% of soft tissue sarcomas and 12% of mast cell tumours. The most significant risk factors for local recurrence were contaminated surgical margins (soft tissue sarcomas) and histological grade (mast cell tumours). Due to the low number of animals experiencing metastasis, no conclusions could be drawn about significant risk factors. CONCLUSIONS: Aggressive surgical management of soft tissue sarcomas and mast cell tumours is associated with a low incidence of local recurrence. The type, location and behaviour of mast cell tumours and soft tissue sarcomas in the population of dogs presented to the University Veterinary Centre, Sydney are similar to those reported by others.     

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.     

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.     

An immunohistochemical study of cyclooxygenase-2 expression in various feline neoplasms

Cyclooxygenase (COX) enzymes catalyze the synthesis of prostaglandins and exist as two isoforms, COX-1 and COX-2. COX-2 is a potent inducible mediator of inflammation. COX-2 is also upregulated in several human tumors and in canine squamous cell, renal cell, and transitional cell carcinomas, prostatic adenocarcinoma, and intestinal neoplasia. The purpose of this study was to determine whether COX-2 is expressed in various feline tumors. Results of this study may help determine whether COX-2 is a potential target for therapeutic and preventive strategies in cats. Immunohistochemical studies were performed on paraffin-embedded tissues using the amplified streptavidin-biotin-horseradish peroxidase system. COX-2 was found in 7 of 19 (37%) feline transitional cell carcinomas and in 2 of 21 (9%) feline oral squamous cell carcinomas. No COX-2 immunoreactivity was detected in cutaneous squamous cell carcinomas (6), adenocarcinomas (nine mammary, eight pulmonary, seven intestinal), lymphomas (six nasal, six intestinal), or 10 vaccine-associated sarcomas. The widespread absence of COX-2 expression in most feline neoplasms might suggest that COX-2 inhibitors would have a low potential as anticancer agents.     

Amelanotic melanocytic tumors of the pinna in six F344 rats.

Spontaneous amelanotic melanocytic tumors of the pinna were found in six females of 960 male and 960 female albino (F344/DuCrj) rats which had been used in three different 24-month chronic toxicity studies. The age when the pinnal tumors were detected ranged from 37 to 59 weeks. The tumors were located unilaterally in the pinna and observed as subcutaneous spherical to irregular, solid white masses measuring 7 to 25 mm in diameter. The pinnal tumors were histologically classified into spindle cell and pleomorphic cell types. The spindle cell type was observed in four rats and composed of fusiform cells arranged in interlacing bundles. The pleomorphic cell type was observed in the remaining two rats and composed of pleomorphic large cells arranged in sheets. One tumor of the latter type metastasized to the submaxillary lymph node and lung. Melanin pigments were not demonstrated in any of the tumors. In immunohistochemistry, nuclei and cytoplasm of tumor cells in all the tumors were slightly positive for S-100 protein. Ultrastructurally, tumor cells contained a considerable number of premelanosomes in the cytoplasm. Desmosomes were occasionally observed between the cell membranes of the adjacent tumor cells. No distinct basal lamina was seen around tumor cells.