Malignant amelanotic melanoma behind the left eye in a female Crj:CD(SD)IGS rat: a case report

A tumor behind the left eye in a female Crj:CD(SD)IGS rat was investigated histopathologically, immunohistopathologically, and electron microscopically. The tumor invaded and destroyed orbital tissues and bones. It consisted of various tumor cells; namely, spindle-shaped, epithelioid, anaplastic melanoma cells, and had prominent eosinophilic cytoplasm and nuclei with a greater variation in size. Immunohistochemically, almost all of the tumor cells were positive for antimelanoma, PNL2 antibody. Ultrastructurally, the tumor cells were rich in small vesicles containing fine granules and filamentous structures. This is the first report describing an amelanotic melanoma in the head of an albino rat.     

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.     

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.     

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.     

Bovine undifferentiated alveolar rhabdomyosarcoma and its differentiation in xenotransplanted tumors.

At necropsy, a 7-year-old Holstein cow, clinically diagnosed via rectal palpation as having malignant abdominal neoplasia was found to have many metastatic tumors of various sizes in the abdominal and thoracic cavities and in the liver, lung, kidney, adrenal, uterus, and lymph nodes. These tumors were examined by histologic and immunohistochemical methods using anti-human myoglobin, anti-bovine myoglobin, and anti-desmin sera. Tumors were diagnosed as undifferentiated alveolar rhabdomyosarcomas. The tumors in the abdominal and thoracic cavities differed histologically from the metastases in organs. The former tumors consisted of only undifferentiated cells, most of which did not stain positively for desmin or myoglobin. The metastatic tumors contained a few rhabdomyoblastic cells that stained positively for desmin and myoglobin. Tumors tissues from the cow were transplanted and propagated through six passages in athymic nude mice. After one passage, the transplanted tumor histologically resembled those from the abdominal serosa and consisted of cells with scanty cytoplasm with an alveolar arrangement. Cells from this tumor stained positively for desmin and negatively for myoglobin. After two passages through nude mice, tumor cells were of two distinct histologic types: those cells with scanty cytoplasm and elongated cells with plentiful eosinophilic cytoplasm. Both of these cells stained positively for both desmin and myoglobin. After six passages, cross striations were detected in neoplastic cells by electron microscopy. These findings illustrate that a highly undifferentiated bovine rhabdomyosarcoma, in which most of the cells in the original tumor lacked desmin and myoglobin, became better differentiated and stained positively for desmin and myoglobin after serial transplantation in nude mice.     

Malignant melanoma of the eyelid in a red squirrel (Sciurus vulgaris orientis)

A 6-year-old male red squirrel (Sciurus vulgaris orientis) developed bilateral tumors of upper and lower eyelids. The tumors in the left lid recurred despite surgical removal. Necropsy revealed metastasis to the lung. The neoplastic cells were epithelioid and highly pleomorphic, and only a few cells contained melanin granules. Occasionally melanoma cells were immunoreactive for S100, neuron-specific enolase and vimentin, and a small number of cells for cytokeratin. Ultrastructurally, the presence of premelanosomes was confirmed in the cytoplasm. Possible presence of cytokeratin-positive neoplastic melanocytes should be taken into account when differentiating a nonpigmented epithelioid melanoma from other tumors such as anaplastic carcinomas.     

Tail-base mass from a "horse of a different color"

A 14-year-old bay Thoroughbred gelding was presented for evaluation of a mass at the base of the tail. The mass had been present for 1 year, and recently had begun to increase in size. Additional masses were found around the eye and shoulder. A fine-needle aspirate of the tail-base mass revealed highly anaplastic round to polyhedral cells containing dark green to black cytoplasmic granules interpreted to be melanin. Histologically, the mass was composed of pleomorphic, poorly pigmented, round to polyhedral cells interpreted to be neoplastic melanocytes. With immunohistochemistry, the cells were positive for vimentin and S-100, but negative for pancytokeratin and Melan-A. The cytologic and histopathologic diagnoses were amelanotic melanoma. The horse was treated with cimetidine, but the tumor continued to progress. In this report, we describe the cytopathologic features of an aggressive amelanotic melanoma in a non-grey horse and emphasize the unique correlation between cytologic and histologic findings.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

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.     

Spontaneous amelanotic melanomas of the uveal tract in F344 rats.

Five intraocular amelanotic melanomas were identified in the National Toxicology Program's database consisting of records from more than 60,000 female and 60,000 male F344 rats, which were used as control and treated animals in 2-year carcinogenicity studies. The five spontaneous melanomas were grossly observed as white or yellow, unilateral nodules, which originated in the region of the iris and ciliary body, often also involving the choroid. These amelanotic melanomas were composed predominantly of spindle cells arranged in a whorled pattern often with perivascular orientation. Mitotic figures were common in five tumors. The spindle cells had a positive immunoreactivity for S-100 protein but were negative for desmin. Electron microscopic studies provided clear evidence that these tumors originated from the uveal melanocytes. Ultrastructurally, the spindle cells contained numerous cytoplasmic premelanosomes (stage II melanosomes) that were not associated with melanin. Special histochemical studies showed that the spindle cells had a negative reaction for melanin. Although electron microscopic features are critical in the diagnosis of amelanotic melanomas of the uveal tract, the whorled pattern of spindle cells is a useful histologic criterion in differential diagnosis of this tumor in F344 rats.     

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.     

Immunohistochemical evaluation of a malignant intestinal carcinoid in a dog

An intestinal carcinoid with multiple metastases was identified in a 5-year-old male Shih Tzu with a clinical history of anemia, fatigue, anorexia, vomiting, intermittent diarrhea, intestinal bleeding, and progressive emaciation. There was a yellowish-white mass 15 mm in diameter in the anterior jejunum and white nodules consistent with metastases in many organs. Histopathologically, the mass consisted of neoplastic cells arranged in lobules, trabeculae, or closely interdigitating islands of cells. Neoplastic cells were generally polygonal with round hyperchromatic nuclei, modest amounts of eosinophilic cytoplasm, and eosinophilic cytoplasmic granules. Mitoses were common. Rosette formations of tumor cells were apparent in metastatic tumors. Immunohistochemically, tumor cells stained positive for cytokeratin 13, synaptophysin, protein gene product 9.5, neuron-specific enolase, chromogranin A, calcitonin gene-related peptide, serotonin (5-HT), and Leu-7. Serum 5-HT concentrations for this dog were increased 10-fold compared with those of normal dogs. All findings were consistent with a diagnosis of a malignant intestinal carcinoid.     

A Case of Olfactory Neuroblastoma Induced in A Rat by N-Nitrosobis(2-hydroxypropyl)amine

N-nitrosobis(2-hydroxypropyl)amine (BHP) is a well-known carcinogen and induces tumors in various tissues. In the present paper, a case of olfactory neuroblastoma (ONB) induced in a rat by BHP is described. The tumor was observed in one out of 25 male rats that received 2000 ppm of BHP in drinking water from 6 to 18 weeks of age and were sacrificed at 26 weeks of age. Histologically, the tumor arose in the posterior nasal cavity and consisted of small round cells and elongate cells with scant basophilic cytoplasm. The neoplastic cells proliferated with compartmentalization into the lobules by fibrovascular septa. True rosettes, pseudorosettes and an intercellular fibrillar matrix were occasionally observed. Immunohistochemically, the tumor cells were positive for NF120/200 and β III-tubulin. These results indicate that the present tumor is the first case of ONB induced in a rat by BHP treatment.     

Granular cell variants in a rat schwannoma. Evidence of neurogenic origin of granular cell tumor (myoblastoma).

A large, intraabdominal rat schwannoma had numerous granule-containing cells cytologically identical to cells of granular cell tumor (myoblastoma). The small eosinophilic granules stained positively by the periodic acid-Schiff (PAS) reaction, with intensity not reduced by diastase pretreatment. Granules stained positively with the Tibor Pap silver impregnation for reticulin and by electron microscopy were identical to myoblastoma cell granules. The nuclei of granular cells were morphologically identical to those of the neoplastic Schwann cells. The granular cells were in numerous foci within the tumor, frequently were seen in mitosis, and possessed an extremely variable volume of cytoplasm. They seemed to evolve from neoplastic Schwann cells. Cells with only a narrow perinuclear rim of granular cytoplasm were of the same size and general configuration as adjacent neoplastic Schwann cells, while cells with increasing volumes of granular cytoplasm were increasingly swollen and round. One area of the tumor was composed almost entirely of such large cells and was histologically identical to classic granular cell tumor.     

A case of adenocarcinoma of the endometrium extending into the leiomyoma of the uterus in a rabbit

In a pet rabbit, 2 tumor masses one on each horn were macroscopically seen in the wall of the uterus. On light microscopic examination, the right horn mass consisted of an admixture of neoplastic epithelial and mesenchymal element. The epithelial element was composed of neoplastic epithelial cells with numerous mitotic figures and formed varied sizes of acini, glandular, and solid structures. The tumor was diagnosed as an adenocarcinoma of the endometrium. The mesenchymal element was composed of well-differentiated smooth muscle cells and was diagnosed as a leiomyoma. While adenocarcinoma cells formed a protrusive mass in the uterine lumen, they also showed an extension into the leiomyoma of the myometrium. By immunohistochemistry, adenocarcinoma stained positive for cytokeratin (MNF116) and leiomyoma stained positive for smooth muscle actin, showing a substantial difference in the cytological nature of these tumor cells. The results may give a further evidence supporting the narrative of the tumor development that an adenocarcinoma of the endometrium extended into leiomyoma of the uterus. To the author's knowledge, this is the first report describing this type of combination of two independent tumors in a pet rabbit.     

Myoepitheliomas in inbred laboratory mice.

Myoepitheliomas are subcutaneous tumors that arise from myoepithelial cells of various exocrine glands. In a retrospective study of 142 tumors observed over a period of 3 years, myoepitheliomas occurred spontaneously in A/HeJ, A/J, BALB/cJ, BALB/cByJ, LLC.A/Ckc, and NOD/Lt inbred strains of mice. Tumors presented primarily in the subcutaneous tissues of the ventral neck (74% of the myoepitheliomas evaluated) but were observed in several other subcutaneous locations, including the head, perineum, and ventral abdomen. These areas were adjacent to salivary, mammary, clitoral, preputial, and Harderian glands. Forty myoepitheliomas were tested by the avidin-biotin complex technique with a panel of antisera specific for mouse keratins, intermediate filaments, and other cytoskeletal proteins to determine the cell type from which this neoplasm originated. Antibodies directed against the specific mouse keratins K5, K6, and K14, and a broadly cross-reactive cytokeratin antibody stained acinar and ductal myoepithelial cells in normal mammary, salivary, and Harderian glands, and neoplastic cells in all cases. Antisera directed against a smooth muscle actin (anti-alpha-sm-1) stained acinar myoepithelial cells of the glands and vascular smooth muscle but neither ductular myoepithelial cells nor tumor cells. This supports the notion that these tumors originate from extraglandular ductular myoepithelial cells. Southern blots, prepared from DNA extracted from nine myoepitheliomas, did not show restriction fragment length polymorphisms when mouse mammary tumor virus (MMTV) cDNA or Int-1 genomic DNA probes were used; this implies that a retrovirus is not the etiologic agent.     

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.     

Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.