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.     

Identification of matrix metalloproteinases in canine cutaneous mast cell tumors

Presence of matrix metalloproteinases has been associated with tumor invasion and metastasis in human neoplasia. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 was determined in canine mast cell tumor tissue and normal stromal tissue from 24 dogs with spontaneously occurring cutaneous mast cell tumors. Seventeen of the mast cell tumors were of histologic grade 2, and 7 were of histologic grade 3. Gelatin zymography and computer assisted densitometry image analysis were used to quantify matrix metalloproteinase concentration. Bands from canine tissues migrated in the same location as human proenzyme and active enzyme matrix metalloproteinase 2 and matrix metalloproteinase 9 standards. A semiquantitative value for each patient sample was obtained by comparing the optical assessment density of each unknown band to the optical density of the human standard. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 in histologic grade 2 mast cell tumors and histologic grade 3 mast cell tumors was compared, as was presence of matrix metalloproteinases in tumor and stromal tissue. There was dramatically more proenzyme matrix metalloproteinase 9 activity in histologic grade 3 mast cell tumors when compared to grade 2 tumors (P = .03). There was also dramatically more active enzyme matrix metalloproteinase 2 and active enzyme matrix metalloproteinase 9 activity in tumor tissue compared to stromal tissue (P = .02, P < .0001). This study demonstrates that the proenzyme and active enzyme forms of matrix metalloproteinase 2 and matrix metalloproteinase 9 are present in canine mast cell tumors. This appears to be related to the degree of histologic malignancy, although histologic grade 1 tumors were not evaluated.     

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.     

Transmission of transmissible venereal tumor of the dog to the coyote.

Three coyotes experimentally implanted with cells from a transmissible venereal tumor of a dog developed neoplasms. Histopathologic appearances of the tumors in the coyotes were similar to those described in the dog. Attempts to transmit the tumor to suckling mice, rats, hamsters, kittens, and opossums were not successful.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Use of a murine xenograft model for canine transmissible venereal tumor

OBJECTIVE: To develop a murine model for canine transmissible venereal tumor (CTVT). ANIMALS: Thirty-three 6-week-old NOD/LtSz-scid (NOD/SCID) mice and seven 6-week-old C57BL/6J mice. PROCEDURE: Samples of CTVT were excised from a 3-year-old dog and inoculated SC into ten 6-week-old NOD/SCID mice to induce growth of xenograft transmissible venereal tumor (XTVT). To establish mouse-to-mouse transmission, samples of XTVT were removed and inoculated SC into 4 groups of 6-week-old NOD/SCID mice and into a control group. Samples of CTVT were also inoculated into immunocompetent C57BL/6J mice for a mouse antibody production (MAP) test. The canine and xenografted tumors were evaluated cytologically and histologically, and polymerase chain reaction was performed for detection of the rearranged LINE/c-MYC junction. RESULTS: 8 of 10 NOD/SCID mice that were inoculated with CTVT developed tumors 3 to 10 weeks after inoculation. In the second-generation xenograft, all mice developed tumors by postinoculation day 47; 1 X 10(6) of XTVT cells were enough to create a xenograft. Metastases developed in 4 of 20 mice. Xenografted and metastatic tumors retained cytologic, histologic, and molecular characteristics of CTVT. Results of the MAP test were negative for all pathogens. CONCLUSION: We established an NOD/SCID murine model for XTVT and metastasis of CTVT. This model should facilitate study of tumor transplantation, progression, and metastasis and should decrease or eliminate the need for maintaining allogenic transfer in dogs.     

Mitosis and apoptosis in canine cutaneous histiocytoma and transmissible venereal tumour

Cell proliferation and apoptosis in canine cutaneous histiocytomas and transmissible venereal tumours were examined in twenty cases. The Ki-67 immunohistochemistry and Tunel methods were used to detect mitotic activity and apoptosis, respectively. The number of Ki-67 immunoreactive cells was 11.65 (+/- 1.1706) in canine cutaneous histiocytomas and 17 (+/- 2.1751) in transmissible venereal tumours. The mean values of apoptotic cells for canine cutaneous histiocytomas and transmissible venereal tumours were 13.25 (+/- 1.8758) and 8.52 (+/- 1.1007), respectively. It was considered that mitotic activity and apoptotic indices were useful in differentiation of canine cutaneous histiocytomas and transmissible venereal tumours. The correlation values for canine cutaneous histiocytomas and transmissible venereal tumours were 0.359 (+/- 0.330) and -0.232 (+/- 0.344), respectively. No significant (P > 0.05) correlation was found between mitosis and apoptosis in these two tumour types.     

Variation among pathologists in the histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference.

Ten veterinary pathologists independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors using the Patnaik classifications. The degree of agreement in grading among the pathologists was compared with the degree of agreement among the same pathologists in a previous study, in which each pathologist used the reference for grading that he/she uses routinely. Mean agreement improved significantly from 50.3% to 62.1% with uniform use of the Patnaik classifications (P = 0.00001), suggesting that there is value in uniform application of a single grading scheme for canine cutaneous mast cell tumors. Agreement among pathologists was still not 100%, suggesting that a more objective grading scheme should be developed and that other histologic indicators of prognosis should be investigated.     

Immunohistochemical detection of proliferating cell nuclear antigen and Ki-67 in mast cell tumors from dogs

OBJECTIVE: To determine whether immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and Ki-67 correlated with prognosis for dogs with cutaneous mast cell tumors (MCT). DESIGN: Case series. ANIMALS: 120 dogs with solitary cutaneous MCT that were excised. PROCEDURE: Information on signalment, history, and outcome was obtained by sending a questionnaire to referring veterinarians. Tumors were graded histologically, and immunohistochemical staining for Ki-67 and PCNA was performed. RESULTS: Survival rates 12, 18, and 24 months after surgery were significantly different among groups when dogs were grouped on the basis of histologic grade. Although mean number of PCNA-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, there was great overlap in values. Mean number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, without any overlap in values between groups, and number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly different among groups when tumors were grouped on the basis of histologic grades. For dogs with grade-II tumors, number of Ki-67-positive nuclei/1,000 tumor nuclei (< 93 vs > or = 93) was significantly associated with outcome (survived vs died). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for dogs with solitary cutaneous MCT, determining number of Ki-67-positive nuclei may be useful in predicting prognosis, particularly for dogs with grade-II tumors.     

Cutaneous neoplasia in 340 cats.

A total of 340 cases of cutaneous neoplasia were diagnosed in 340 of 3,564 cats that were examined by biopsy or necropsy during a 41-month period from January 1, 1986 through May 31, 1989. Eighteen types of tumor occurred, but four types comprised 77% of the cases. These were basal cell tumor, 89 cases (26%, mean age 10.3); mast cell tumor, 72 cases (21%, mean age 8.6); squamous cell carcinoma, 52 cases (15%, mean age 11.6); and fibrosarcoma, 50 cases (15%, mean age 10.2). For each of these four types of tumors, peak number of cases occurred in cats older than 10 years. Mast cell tumor was the only tumor diagnosed in cats younger than 1 year. The head was the most common site for basal cell tumors, mast cell tumors, and squamous cell carcinomas. The legs were the most common location of fibrosarcomas. Siamese cats had approximately three times as many mast cell tumors as statistically expected, but only one-fourth as many squamous cell carcinomas. Breed predilection for other skin tumors was not apparent. Sex predilection was not detected for any skin tumor.     

Variation among pathologists in histologic grading of canine cutaneous mast cell tumors.

Ten veterinary pathologists at 1 veterinary institution independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors (MCTs). There was significant variation among pathologists in grading the MCTs (P < 0.001). The probability of assigning a low grade was significantly higher for the pathologists in this study who use a published reference for histologic grading of canine cutaneous MCTs that allows subcutaneous MCTs or MCTs with mitotic figures to be included in the low-grade category (P < 0.0001 and P < 0.0001, respectively).     

The effect of prednisone on histologic and gross characteristics in canine mast cell tumors

The objective of the study was to determine whether neoadjuvant prednisone therapy affects histological features of cutaneous and subcutaneous mast cell tumors. Twenty-eight dogs with a treatment naïve > 1-cm diameter mast cell tumor (MCT) were randomly assigned (Random number generator; Random.org, Dublin, Ireland) in a blinded fashion to receive either prednisone or placebo (Quality Food Center Pharmacy, Kirkland, Washington, USA). Volumes of mast cell tumors were calculated before incisional and excisional biopsies. Following incisional biopsy, patients received either prednisone (1 mg/kg body weight) daily or a placebo for 7 to 14 days leading up to excisional biopsy. Tumor grade for cutaneous MCT, and mitotic count and atypia for all tumors were reported. Perioperative treatment with prednisone had no significant effect on tumor grade, atypia, or mitotic count. Tumor volume was significantly decreased with prednisone treatment. The use of neoadjuvant prednisone to decrease MCT volume in order to facilitate tumor excision, can be considered without significant concern for change of tumor histologic features in the common population of low- to intermediate-grade MCT.     

Radiation therapy for incompletely excised grade II canine mast cell tumors

Forty-five dogs with incompletely excised grade II mast cell tumors were treated with radiation using a cobalt 60 teletherapy unit (15 fractions of 3.2 Gy for a total of 48 Gy). Twenty-four of the dogs underwent prophylactic regional lymph node irradiation. Three (6.7%) dogs had tumor recurrence, two (4.4%) dogs developed metastasis, and 14 (31%) dogs developed a second cutaneous mast cell tumor. No difference in overall survival rate was observed between the dogs receiving and not receiving prophylactic irradiation of the regional lymph node.     

Immunohistochemical expression of p27 and p21 in canine cutaneous mast cell tumors and histiocytomas

The objective of this study was to evaluate by immunohistochemical means the nuclear expression of p27 and p21 proteins in cutaneous mast cell tumors and histiocytomas of dogs. In mast cell tumors, nine of the 13 grade I tumors, 13 of the 19 grade II tumors, and 10 of the 15 grade III tumors showed no detectable or mild p27 immunoreactivity. In contrast, one of the 13 grade I tumors, 12 of the 19 grade II tumors, and 11 of the 15 grade III tumors showed moderate or marked p21 immunoreactivity. Nineteen of the 28 histiocytomas showed no detectable or mild p27 immunoreactivity, and 24 cases showed moderate or marked p21 immunoreactivity. These findings indicate that a loss or absence of p27 expression is an early pathogenic event in mast cell and histiocyte tumorigenesis and that p21 expression may be a marker of mast cell tumor progression and histiocytoma cell proliferation.     

Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors.

By using flow cytometry, a retrospective analysis of the DNA content of 40 primary canine mast cell tumors and seven lymph nodes that contained metastatic mast cell tumor from 44 dogs of various breed, sex, and age was performed on formalin-fixed, paraffin-embedded samples of the tumors and nodes. These samples were chosen according to the following criteria: samples contained sufficient well-preserved tumor tissue in the paraffin block for processing, sufficient patient history data were available, clean and homogeneous cell suspensions were obtained after processing, and interpretable DNA histograms were produced on analysis. The ploidy data obtained were compared with the histopathologic grade, the anatomical site of occurrence, the clinical stage of the tumors, and the survival of the dogs. Over 70% (29/40) of the mast cell tumors were diploid. Three metastatic mast cell tumors in lymph nodes had the same ploidy status as their corresponding primary tumors. In five dogs, mast cell tumors from multiple sites in each dog displayed similar ploidy status. Of 26 dogs evaluated for survival times, 69% (18/26) had diploid tumors and 31% (8/26) had aneuploid tumors. When numbers of diploid versus aneuploid tumors were compared, no significant difference was found between any two grades, clinical stages, or anatomic sites. A significant difference (P = 0.02) was found, however, between aneuploid and diploid tumors when comparing Stage I and non-Stage I disease. The Kaplan-Meier survival plot indicated a tendency towards an increased survival within the first year in dogs with diploid versus aneuploid tumors (P = 0.06).     

Recurrence rates and sites for grade II canine cutaneous mast cell tumors following complete surgical excision.

A retrospective study was performed on 31 dogs with completely excised, grade II, cutaneous mast cell tumors in order to determine recurrence rates and sites. Distant tumor recurrence developed in 22% of dogs, and local tumor recurrence developed in 11% of dogs; however, the vast majority of these animals were incompletely staged initially. Complete surgical excision of grade II mast cell tumors was associated with effective local control in 89% of these dogs. Therefore, adjuvant radiation therapy might not be indicated in the majority of dogs with complete surgical excision.     

Canine and feline mast cell tumors: biologic behavior, diagnosis, and therapy

Our understanding of the etiology, behavior, and most effective form of mast cell tumor treatment is rudimentary. I have tried to indicate specific areas that need further study in order to resolve some of the present controversies. Clinicians should recognize that many of the published recommendations for treatment of mast cell tumors are based on opinion and should be viewed with skepticism. Because of the infrequence of this tumor in man, limited help can be expected from human oncologists, and thus the burden of responsibility for progress in predicting behavior and developing effective treatment for canine mast cell tumors falls on the shoulders of veterinarians.     

Expression of the KIT protein (CD117) in primary cutaneous mast cell tumors of the dog.

Thirty-one canine cutaneous masses, diagnosed as mast cell tumors (MCT) by histopathologic analysis, were used to evaluate the immunohistochemical pattern of expression of KIT protein (CD117), a type III tyrosine kinase protein involved in mast cell growth and differentiation. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Immunohistochemical KIT expression was compared with histologic grade and some histomorphologic features (cell differentiation and nuclear grade) evaluated separately. A possible predictive role of biologic behavior in MCTs for KIT expression was also investigated. Immunohistochemical analysis revealed three different patterns of KIT expression: a cytoplasmic diffuse pattern, a membranous pattern with immunostaining located on the cell surface, and a cytoplasmic perinuclear pattern, where KIT expression was detected in the cytoplasm of the neoplastic mast cells, close to the nucleus. Statistical analysis showed a close relationship between different KIT immunohistochemical patterns and histologic grade (P < 0.00000), cell differentiation (P < 0.00000), and nuclear grade (P < 0.0024). According to Kaplan-Meier-estimated survival curves compared by survival analysis, KIT expression was significantly associated with survival time (P = 0.037) but not cancer-free interval (P = 0.50). Similar to other well-known histomorphological features, KIT expression is a useful parameter of malignancy in cutaneous MCTs. KIT expression also predicted the biological behavior of the tumors in this study.     

Immunohistochemical characterization of intraocular metastasis of a canine transmissible venereal tumor

Little has been published on intraocular metastasis of transmissible venereal tumors (TVT) in dogs. This report presents a 4-year-old male Labrador Retriever with a previous history of subcutaneous TVT which underwent total remission after treatment with vincristine. The dog presented with clinical signs of uveitis and increased intraocular pressure (IOP) in both eyes. After enucleation of the left eye, a diagnosis of TVT was made based on morphology, histology and immunohistochemistry (IHC). IHC staining for vimentin, S-100 protein, cytokeratin and HMB45 was performed to differentiate this lesion from TVT, lymphoma, melanoma, carcinomas, neurogenic tumors and fibrosarcoma. The IHC findings supported the diagnosis of TVT for this round cell tumor.