Metallothionein Expression in Dogs With Chronic Hepatitis and Its Correlation With Hepatic Fibrosis, Inflammation, and Ki-67 Expression.

The chronic form of primary hepatitis occurs commonly in dogs, and the etiology is rarely found. Metallothionein (MT) is a heavy metal-binding protein found in many organs, including the liver. MT was recently shown to enhance liver regeneration and decrease hepatic fibrosis in human beings. This study examined the expression of MT in 24 cases of chronic hepatitis in dogs using immunohistochemistry. To understand the role of MT as a determinant of hepatic inflammation, fibrosis, bile duct proliferation, and regeneration, we correlated its expression with histologic lesions of chronic hepatitis, such as hepatic inflammation, fibrosis, and bile duct proliferation, as well as hepatocellular growth fraction as measured by Ki67 immunolabeling. Hepatocellular growth fraction was used as a measure of hepatic regeneration. Regression analysis revealed a significant positive correlation between MT labeling intensity and growth fraction (r(2) = 0.29, P < .05). The percentage of MT-positive cells and the overall MT expression were both positively correlated with growth fraction (r(2) = 0.25 and 0.26, respectively; P < .05). A negative correlation was found between the overall MT labeling and fibrosis (r(2) = 0.18, P < .05). A similar trend of negative correlation was also found between the percentage of MT-positive cells and fibrosis, but the P value was not statistically significant (r(2) = 0.14, P = .0684). These findings suggest a protective role of MT in dogs affected by chronic hepatitis, similar to its role in human beings. These dogs may respond to treatment modules focusing on enhancing the expression of MT.

Prognostic value of histologic grading for feline mammary carcinoma: a retrospective survival analysis.

Feline mammary carcinoma is highly malignant and generally associated with a poor prognosis, although studies suggest the range of survival times in affected cats is broad. Histologic grading of these tumors is achieved using the Elston and Ellis system, originally developed for human breast cancer. In cats, however, classification using this method has variable prognostic value. Therefore, objectives of this study were (1) to evaluate the Elston and Ellis grading system for feline mammary carcinoma in a predominantly spayed population and (2) to determine whether modification of this system or development of a novel system improved the prognostic value of histologic grading. Survey data and histologic features for 108 carcinomas from 97 cats were analyzed with respect to overall survival. Elston and Ellis grading failed to correlate significantly with overall survival. Using multivariable analysis, lymphovascular invasion, nuclear form, and mitotic count each demonstrated independent prognostic significance (P = .008, <.001, and .004, respectively). Modifications of the Elston and Ellis system and a novel grading system were proposed based on these results; all showed significant correlation with overall survival (P < .001). Median survival times were 27, 29, or 31 months for grade I; 14, 12, or 14 months for grade II; and 13, 5, or 8 months for grade III carcinomas using the mitotic-modified Elston and Ellis, the revised Elston and Ellis, or the novel grading system, respectively. Based on this retrospective study, adoption of the species-specific systems as proposed here may improve the prognostic value of histologic grading for feline mammary carcinoma.

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor in canine cutaneous fibrosarcomas.

The expression of five markers associated with tumour angiogenesis, proliferation and apoptosis was studied in 24 canine cutaneous fibrosarcomas. Tumours were assigned histological grades and were immunohistochemically evaluated for the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2). Additionally, intra-tumour microvessel density (iMVD) was assessed by immunohistochemical labelling for expression of von Willebrand factor (vWf) and tumour proliferation index (PI) was measured following labelling of Ki-67 antigen. Finally, tumour apoptotic index (AI) was determined by application of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP end-labelling method (TUNEL). VEGF and VEGFR-2 expression were detected in 22/24 (92%) and 24/24 (100%) of fibrosarcomas, respectively. There was correlation between VEGF and VEGFR-2 expression (r = 0.51) and between histological grade and PI (r = 0.82). A significant difference in PI between tumours of different histological grade was found (P < 0.05). The median PI in grade 2 and 3 tumours (30.6 and 54.7, respectively) was significantly higher than in grade 1 tumours (6.4). Therefore, only PI correlates significantly with the histological grade of canine cutaneous fibrosarcomas. The potential for autocrine activity for VEGF exists in canine cutaneous fibrosarcomas, as VEGF and VEGFR-2 expression was found in most tumours.

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor associated with tumor cell proliferation in canine cutaneous squamous cell carcinomas and trichoepitheliomas.

The expression of 5 markers associated with angiogenesis was studied in canine squamous cell carcinomas (SCCs) (n = 19) and canine trichoepitheliomas (TCPs) (n = 24). SCCs were assigned histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand's factor, Ki-67 antigen, and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate end-labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19 (100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial correlation between histologic grade and PI (r = 0.51); and moderate correlation between VEGF and histologic grade (r = 0.43), VEGFR-2 and histologic grade (r = 0.47), VEGF and PI (r = 0.47), and VEGFR-2 and PI (r = 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial correlation between VEGF and PI (r = 0.51) and a moderate correlation between VEGFR-2 and iMVD (r = 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs (9.05) (P value < .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with evidence for tumor cell proliferation.

Diagnoses and clinical outcomes associated with surgically amputated canine digits submitted to multiple veterinary diagnostic laboratories.

Amputation is commonly performed to both treat and diagnose conditions affecting the digits of dogs. Although histopathologic evaluation of these digits is routinely done, data on the prevalence and prognosis of neoplasms of the digit are scarce. The records of multiple veterinary diagnostic laboratories were searched to identify submissions of amputated digits from dogs. Four hundred twenty-eight separate submissions were reviewed for diagnosis, age, sex, limb of origin, and digits affected, and the original submitting clinics were surveyed to determine clinical outcome of the animal. No diagnosis could be agreed upon in 24 animals, and these were excluded from the study. Kaplan-Meier product-limit method was used to determine the disease-free interval and survival time. Neoplastic disease was identified in 296 of 404 submissions, with exclusively inflammatory lesions composing 108 cases. A total of 30 different neoplastic processes were identified. In 233 (77.7%) of the neoplastic cases, a malignant tumor was identified. Squamous cell carcinoma was the most commonly identified tumor (n = 109, 36.3%), and 11 of 42 dogs for which clinical follow-up information was available developed metastatic disease. Squamous cell carcinoma of the digit appears to have a greater metastatic potential than that occurring elsewhere in the body. Other common diagnoses included melanoma (n = 52, 17.3%), soft-tissue sarcoma (n = 29, 9.7%), and mast cell tumor (n = 20, 6.7%). Melanomas were associated with poor prognoses, with a median survival time of 365 days.

Diagnoses and clinical outcomes associated with surgically amputated feline digits submitted to multiple veterinary diagnostic laboratories.

Amputation is commonly performed in an attempt to both treat and diagnose conditions affecting the digits of cats. The records of multiple veterinary diagnostic laboratories were searched to identify submissions of amputated digits from cats. Eighty-five separate submissions were reviewed for diagnosis, age, sex, limb of origin, and digits affected; and the original submitting clinics were surveyed to determine clinical outcome. The Kaplan-Meier product-limit method was used to determine the disease-free interval and survival time. Neoplastic disease was identified in 63 of 85 submissions, with exclusively inflammatory lesions composing the other 22 cases. In 60 (95.2%) of the neoplastic cases, a malignant tumor was identified. Squamous cell carcinoma was the most commonly identified malignant tumor (n = 15; 23.8%) and was associated with a median survival time of 73 days. Other diagnoses included fibrosarcoma (n = 14; 22.2%); adenocarcinoma, likely metastases of a primary pulmonary neoplasm (n = 13; 20.6%); osteosarcoma (n = 5; 7.9%); mast cell tumor (n = 4; 6.3%); hemangiosarcoma (n = 5; 7.9%); malignant fibrous histiocytoma (n = 2; 3.2%); giant cell tumor of bone (n = 2; 3.2%); and hemangioma (n = 2; 3.2%). Giant cell tumor of bone has not been previously described in the digits of cats. Various neoplasms can occur in the digits of cats, and submission of the amputated digit for histopathologic diagnosis is essential to determine the histogenesis and predict the clinical outcome.

Kernicterus in an adult dog.

A 7-year-old, spayed female, Wheaton terrier dog was icteric, lethargic, and anorexic with increased activity of hepatocellular and cholestatic liver enzymes and an extreme hyperbilirubinemia level of 609 micromol/L (reference interval: 1.0-4.0 micromol/L). Necropsy findings included profound icterus and red and yellow mottling of the liver. Yellow discoloration of the thalamic and subthalamic nuclei was detected on subgross examination of the formalin-fixed brain. Histologic examination of the brain revealed neuronal necrosis within the discolored nuclei, necrosis of Purkinje cells, and Alzheimer type II astrocytes in the cerebrocortical gray matter and in the nuclei, with gross discoloration. Histologic examination of the liver revealed extensive necrosis in a periacinar-to-bridging pattern and often extending to portal triads. A case of naturally occurring kernicterus in an adult dog secondary to extreme hyperbilirubinemia resulting from fulminant hepatic failure is reported. The few reports of this disease in domestic species involved neonates, namely 1 foal and 1 kitten.

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.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for polyomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain polyomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for polyomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-murine polyomavirus polyclonal antiserum as the primary antibody. The DNA was extracted from sections of each tissue block, and a polymerase chain reaction assay was performed, using primers designed to amplify regions of the bovine polyomavirus genome and consensus polyomavirus primers designed to detect unknown polyomaviruses. RESULTS: Polyomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that polyomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Immunohistochemical detection of tumor suppressor gene p53 protein in feline injection site-associated sarcomas.

Sarcomas associated with injection sites are a rare but important problem in cats. Immunohistochemical detection of p53 protein may correlate to mutation of the p53 tumor suppressor gene, a gene known to be important in oncogenesis. The expression of nuclear p53 protein in 40 feline injection site-assocated sarcomas was examined by immunohistochemical staining. In 42.5% (17/40), tumor cell nuclei were stained darkly; in 20% (8/40), tumor cell nuclei were stained palely; and in 37.5% (15/40), tumor cell nuclei were unstained. Immunohistochemical detection of p53 protein in a proportion of injection site-associated sarcomas suggests that mutation of the p53 gene may play a role in the pathogenesis of these tumors.

Comparison of endogenous feline leukemia virus RNA content in feline vaccine and nonvaccine site-associated sarcomas.

OBJECTIVE: To determine whether feline vaccine site-associated sarcomas (VSS) contain a higher amount of endogenous FeLV (enFeLV) RNA, compared with feline nonvaccine site-associated sarcomas (non-VSS). SAMPLE POPULATION: Formalin-fixed paraffin-embedded (FFPE) tissues from 50 VSS and 50 cutaneous non-VSS. PROCEDURE: RNA was extracted from FFPE sections of each tumor, and regions of the long terminal repeat (LTR) and envelope (env) gene of enFeLV were amplified by use of reverse transcriptase-polymerase chain reaction (RT-PCR). The density of each RT-PCR product band for enFeLV was compared with that of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An integrated density value (IDV) was determined by use of densitometry, and the IDV ratio for enFeLV to GAPDH was calculated for each enFeLV primer set. RESULTS: The median (interquartile range) of the IDV ratio for the enFeLV LTR primer set was 0.52 (0.26 to 1.17) for the VSS group and 0.84 (0.21 to 1.53) for the non-VSS group. The median (interquartile range) of the IDV ratio for the enFeLV env primer set was 0.60 (0.37 to 0.91) for the VSS group and 0.59 (0.36 to 1.09) for the non-VSS group. CONCLUSIONS: Because the amount of enFeLV RNA within the LTR and env gene was not significantly different between the VSS and non-VSS groups, enFeLV replication or expression is unlikely to be involved in VSS development.

Mutational analysis of tumor suppressor gene p53 in feline vaccine site-associated sarcomas.

OBJECTIVES: To investigate the role of tumor suppressor gene p53 mutation in feline vaccine site-associated sarcoma (VSS) development and to evaluate the relationship between p53 nucleotide sequence and protein expression. SAMPLE POPULATION: Formalin-fixed paraffin-embedded tissues of 8 feline VSS with dark p53 immunostaining (high p53 expression) and 13 feline VSS with faint or no staining (normal p53 expression). PROCEDURE: DNA was extracted from neoplastic and normal tissue from each paraffin block. The following 3 regions of the p53 gene were amplified by polymerase chain reaction: 379 base pair (bp) region of exon 5, intron 5, and exon 6, 108 bp region of exon 7, and 140 bp region of exon 8. Amplified p53 products were sequenced and compared with published feline p53. The p53 mutations identified were correlated with p53 mutations predicted by immunostaining. RESULTS: Neoplastic cells of 5 of 8 (62.5%) VSS that had high p53 expression harbored single missense mutations within the p53 gene regions examined. The p53 gene mutations were not detected in the 13 tumors with normal p53 immunostaining. Nonneoplastic tissues adjacent to all 21 VSS lacked mutations of these p53 gene regions. CONCLUSIONS: The p53 gene mutations were restricted to neoplastic tissue and, therefore, were unlikely to predispose to VSS. However, p53 mutations may have contributed to cancer progression in 5 of the 21 VSS. There was very good (kappa quotient = 0.67 with a confidence limit of 0.3 to 1.0), although not complete, agreement between prediction of mutation by p53 immunostaining and identification of mutations by sequencing of key p53 gene regions.

Evaluation of formalin-fixed paraffin-embedded tissues obtained from vaccine site-associated sarcomas of cats for DNA of feline immunodeficiency virus.

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) method for detection of feline immunodeficiency virus (FIV) DNA, using formalin-fixed paraffin-embedded (FFPE) tissues, and to use this method to evaluate tissues obtained from vaccine site-associated sarcomas (VSS) of cats for FIV DNA. SAMPLE POPULATION: 50 FFPE tissue blocks from VSS of cats and 50 FFPE tissue blocks from cutaneous non-vaccine site-associated fibrosarcomas (non-VSS) of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor and regions of the gag gene of FIV were amplified by a PCR, using 3 sets of primers. Sensitivity of the method was compared between frozen and FFPE tissues, using splenic tissue obtained from a cat that had been experimentally infected with FIV. RESULTS: We did not detect FIV DNA in VSS or non-VSS tissues. Sensitivity of the PCR method was identical for frozen or FFPE tissues. CONCLUSIONS AND CLINICAL RELEVANCE: It is possible to detect FIV DNA in FFPE tissues by use of a PCR. We did not find evidence to support direct FIV involvement in the pathogenesis of VSS in cats.

Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog.

Affinity electrophoresis, using wheat germ lectin, was used to separate the alkaline phosphatase isoenzymes in the sera of 150 dogs with alkaline phosphatase values greater than or equal to 150 IU/L. The method provided clearer separation of the liver, bone and steroid-induced alkaline phosphatase isoenzymes commonly observed in canine serum, compared to conventional cellulose acetate electrophoresis. The dogs were divided into four patient groups determined by previous corticosteroid treatment, evidence of elevated endogenous corticosteroid levels, age and alanine aminotransferase values. The isoenzyme pattern of each patient was qualitatively assessed. The isoenzyme pattern most frequently observed was greater than 50% steroid induced alkaline phosphatase, which was present in 76 of 150 dogs. This pattern was observed in 18 of 22 dogs receiving corticosteroid therapy, two of three dogs with hyperadrenocorticism, and in dogs with a variety of other diagnoses. The majority of immature dogs (12 of 20) had an isoenzyme pattern consisting of greater than 50% bone. The majority of dogs with active hepatocellular injury (16 of 27) had greater than 50% liver isoenzyme. The isoenzyme pattern was not specific for certain diseases, therefore the diagnostic usefulness is limited. However the isoenzyme result is useful in some cases to determine which further diagnostic tests are indicated, and to determine the source of alkaline phosphatase elevation.