Growth Plate Lesions of Fattening Bulls.

Lameness related to growth plate lesions is an important problem in the beef industry. This article describes the macroscopic and microscopic lesions in the distal metatarsal physis of bulls from an association of farmers in northeastern Italy. The metatarsal bones of 62 bulls (12 with severe lameness and 50 without lameness), average age 16.44 ± 1.72 months, were examined at the abattoir. The animals came from the same geographic area and shared intensive husbandry practices and a diet based on maize starch. A total of 124 metatarsal bones were sectioned, and the distal metaphyseal growth plate was grossly examined. Twenty-three cases, including 12 lame and 9 nonlame animals with visible lesions on macroscopic examination, and 2 controls (a total of 46 physes) were examined microscopically. Eight of 12 bulls with severe lameness had a chronic purulent physitis in at least 1 limb. Segmental thickening of the hypertrophic zone, consistent with osteochondrosis (OC), was present contralaterally ( n = 3 cases) and bilaterally ( n = 3 cases) in 6 of these animals. In the group of nonlame bulls, 19 of 50 (38%) had similar segmental thickening of the physis consistent with OC. In the remaining bulls, minor findings included partial closure of the physis and a variable degree of metaphyseal hyperemia. A high incidence of OC was found in both lame and nonlame fattening bulls. It is likely that lame animals were clinically more severe due to secondary hematogenous implantation of bacteria, resulting in a purulent physitis and severe lameness that required emergency slaughter in some cases.

Ellis-van Creveld Syndrome in Grey Alpine Cattle: Morphologic, Immunophenotypic, and Molecular Characterization.

Ellis-van Creveld (EvC) syndrome is a human autosomal recessive disorder caused by a mutation in either the EVC or EVC2 gene, and presents with short limbs, polydactyly, and ectodermal and heart defects. The aim of this study was to understand the pathologic basis by which deletions in the EVC2 gene lead to chondrodysplastic dwarfism and to describe the morphologic, immunohistochemical, and molecular hallmarks of EvC syndrome in cattle. Five Grey Alpine calves, with a known mutation in the EVC2 gene, were autopsied. Immunohistochemistry was performed on bone using antibodies to collagen II, collagen X, sonic hedgehog, fibroblast growth factor 2, and Ki67. Reverse transcription polymerase chain reaction was performed to analyze EVC1 and EVC2 gene expression. Autopsy revealed long bones that were severely reduced in length, as well as genital and heart defects. Collagen II was detected in control calves in the resting, proliferative, and hypertrophic zones and in the primary and secondary spongiosa, with a loss of labeling in the resting zone of 2 dwarfs. Collagen X was expressed in hypertrophic zone in the controls but was absent in the EvC cases. In affected calves and controls, sonic hedgehog labeled hypertrophic chondrocytes and primary and secondary spongiosa similarly. FGF2 was expressed in chondrocytes of all growth plate zones in the control calves but was lost in most EvC cases. The Ki67 index was lower in cases compared with controls. EVC and EVC2 transcripts were detected. Our data suggest that EvC syndrome of Grey Alpine cattle is a disorder of chondrocyte differentiation, with accelerated differentiation and premature hypertrophy of chondrocytes, and could be a spontaneous model for the equivalent human disease.

Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

Angiogenesis in spontaneous tumors and implications for comparative tumor biology.

Blood supply is essential for development and growth of tumors and angiogenesis is the fundamental process of new blood vessel formation from preexisting ones. Angiogenesis is a prognostic indicator for a variety of tumors, and it coincides with increased shedding of neoplastic cells into the circulation and metastasis. Several molecules such as cell surface receptors, growth factors, and enzymes are involved in this process. While antiangiogenic therapy for cancer has been proposed over 20 years ago, it has garnered much controversy in recent years within the scientific community. The complex relationships between the angiogenic signaling cascade and antiangiogenic substances have indicated the angiogenic pathway as a valid target for anticancer drug development and VEGF has become the primary antiangiogenic drug target. This review discusses the basic and clinical perspectives of angiogenesis highlighting the importance of comparative biology in understanding tumor angiogenesis and the integration of these model systems for future drug development.

Providing high-quality research training for veterinary pathologists in Europe.

Despite their key role in a wide range of fields relating to animal and public health, there is currently a lack of veterinary pathologists in Europe. In 1999, to help address the problem, the European College of Veterinary Pathologists (ECVP) and the European Society of Veterinary Pathology (ESVP) established a joint Education Committee. In this Special Article, Professor Anja Kipar and colleagues, all members of the committee, describe the ECVP/ESVP Summer Schools in Veterinary Pathology programme, which aims to provide high-quality research training for veterinary pathologists from all over Europe and beyond.

Immunohistochemical Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Hyperplasia, Neoplasia and Supporting Stroma.

The ability of a tumour to become simultaneously resistant to different drugs is known as multidrug resistance and is often due to the expression of ATP-dependent binding cassette transporters (ABC-transporters) such as P-glycoprotein (PGP) and breast cancer resistance protein (BCRP). In this study, the expression of PGP and BCRP was determined in the components of hyperplastic and neoplastic canine mammary glands, including the supporting stroma. The variation of expression of these molecules in carcinomas was evaluated between lesions of different histological stage and grade of malignancy. Samples included 47 hyperplastic tissues and 10 benign and 46 malignant neoplasms. Tumours were classified into histological subtype, histological stage and grade. Immunohistochemical evaluation of PGP and BCRP expression showed that both markers are potentially expressed by epithelial cells, myoepithelial cells in complex tumours and mesenchymal cells in mixed tumours, but expression of both proteins was significantly higher in malignant epithelial cells versus hyperplastic epithelium or the epithelium of benign tumours. BCRP showed significantly higher expression in epithelial cells of simple carcinomas versus those of complex and mixed carcinomas. Grade II and III carcinomas had higher epithelial PGP expression than grade I tumours. The positivity of stromal fibroblasts was higher in histological stage II versus I carcinomas, and in histological grade II versus I carcinomas. Malignant and invasive tumours were more likely to express PGP and/or BCRP in luminal and stromal components and evaluation of these markers could provide valuable information for the identification of tumours characterized by an aggressive and chemoresistant phenotype.

Immunohistochemical expression of heat shock proteins, p63 and androgen receptor in benign prostatic hyperplasia and prostatic carcinoma in the dog.

This study compared heat shock proteins Hsp60, Hsp72 and Hsp73, along with p63 and androgen receptor (AR) immunoexpression between 16 cases of benign prostatic hyperplasia (BPH) and 11 prostatic carcinomas (PCa) in dogs. The proportion of Hsp60-positive cells was higher in PCa compared with BPH (P = 0.033), whereas the frequency and intensity of Hsp73 immunostaining did not differ significantly between the two groups. Hsp72-immunostained nuclei formed a discontinuous layer along the basement membrane in BPH, whereas cells in this layer in PCa were negative or weakly positive. Hsp72 nuclear score showed significant positive associations with both p63 (P = 0.016) and AR (P = 0.009) scores. Double immunofluorescence revealed Hsp72-p63 and Hsp72-AR co-expressions in basal cell nuclei. Aberrant cytoplasmic p63 immunolabelling was observed in 3 of 11 PCa cases. These results suggest a role of the combined expression of Hsp72, p63 and AR in basal epithelial cells in canine BPH and PCa.

Oral Squamomelanocytic Tumour in a Dog: a Unique Biphasic Cancer.

In human medicine, squamomelanocytic tumour is a malignant cutaneous neoplasm composed of closely intermingled neoplastic squamous cells and melanocytes. A multinodular gingival tumour in a 16-year-old, mixed breed neutered female dog was examined microscopically. Two populations of neoplastic cells, melanocytic and squamous epithelial cells were intermingled. The melanocytic cells were melan-A positive and cytokeratin AE1-AE3 negative and the squamous component was cytokeratin AE1-AE3 positive and melan-A negative. Bovine papillomavirus was not identified by immunohistochemistry or polymerase chain reaction. A diagnosis of squamomelanocytic tumour was made.

Validation of tissue microarray for molecular profiling of canine and feline mammary tumours.

Tissue microarray (TMA) is a high-throughput method adopted for simultaneous molecular profiling of tissue samples from large patient cohorts. The aim of this study was to validate the TMA method for the molecular classification of canine and feline mammary tumours. Twelve samples, five feline and five canine mammary tumours and two canine haemangiosarcomas, were collected. TMA construction was based on Kononen's method of extracting a cylindrical core of paraffin wax-embedded 'donor' tissue and inserting it into a 'recipient' wax block. Seven consecutive sections from each tissue array block were subjected to immunohistochemistry (IHC) using primary antibodies specific for oestrogen receptor (OR), progesterone receptor (PR), c-erbB-2, cytokeratin (CK) 5/6, CK14, CK19 and p63. The same panel of antibodies was applied to the full sections from all cases. Comparison between full sections and TMA scores revealed different results depending on the antibodies. Labelling for OR, PR, CK19 and p63 showed total concordance, c-erbB2 (score +2, +3) was concordant in nine out of ten cases, CK5/6 and CK14 in eight out of ten cases. The TMA platform preserves the molecular profile of canine and feline mammary tumour markers, representing a useful tool for rapid and cost-effective analysis for the first phenotypic screening using OR, PR and c-erbB2 antibodies. Basal cytokeratin, used for triple negative identification, shows a multifocal 'niche' expression pattern, for which IHC of the full section or multiple core array is recommended.

Giant cells in anaplastic mammary carcinoma of the dog and cat.

Four uncommon anaplastic mammary carcinomas containing numerous giant cells are described in three dogs and one cat. The giant cells of all cases were studied by means of monoclonal and polyclonal antibodies to detect epithelial (carcinoembryonic antigen and keratin) and mesenchymal (vimentin, lysozyme and S-100 protein) differentiation. Most of them proved to have an epithelial immunophenotype. Ultrastructurally, scattered bundles of tonofilaments but no lysosome-like bodies could be detected. One tumour had an additional, different type of giant cell, which had a benign multinucleated osteoclast-like appearance, gave positive staining for acid phosphatase, had a histiocytic-stromal immunohistochemical pattern, and was, ultrastructurally, multinucleate with irregular folds and no evidence of tonofilaments. In one case some giant cells had an epithelial immunophenotype and others a stromal immunophenotype, even though their histological and ultrastructural features were the same. In the least histologically differentiated tumour the giant cells presented a coexpression of intermediate filaments. This supported the theory that there might be a stem cell origin for most canine mammary tumours.

Basement membrane components in mammary tumours of the dog and cat.

The distribution of the basement membrane (BM) components, laminin, type IV collagen and type VII collagen were studied immunohistochemically in benign and malignant growths of the mammary epithelium of the dog and cat. Intact BMs were found in benign growths, but in well-differentiated malignant tumours they were generally discontinuous, and missing in poorly differentiated carcinomas. An increase in the histological grade of atypia was accompanied by a more marked disruption or fading of BM. Monoclonal antibody (LH 7.2) proved useful in demonstrating type VII collagen in tumours in which massive proliferation of blood vessels made the evaluation of BM features with antibodies to laminin and type IV collagen difficult. Type VII collagen is present in BM of the mammary epithelium but not under the endothelium of blood vessels; it may therefore enhance the value of BM markers as aids in the study of neoplastic progression.

Proliferative activity assessed by anti-PCNA and Ki67 monoclonal antibodies in canine testicular tumours.

The recent availability of monoclonal antibodies raised against cell cycle nuclear antigens makes possible, by means of immunohistochemical techniques, an easy and quick method of evaluating tumour kinetic activity, in addition to older methods such as measurement of the mitotic index. Some of these antibodies can be used on formalin-fixed paraffin wax-embedded samples, thus allowing the use of archival material. In the present study the proliferative activity of testicular tumours of the dog (seminomas and Sertoli and Leydig cell tumours) was investigated with two monoclonal antibodies to proliferating cell nuclear antigen (PCNA) clone PC10, and Ki67 clone MIB1. The former recognizes a formalin-resistant epitope of PCNA, and MIB1 the same antigen as Ki67 in formalin-fixed, paraffin wax-embedded sections after incubation in a microwave oven. Three parameters of proliferative activity were considered: PCNA and Ki67 indices (percentage of nuclear area positive to PCNA and to Ki67), and mitotic index (number of mitoses per 1000 cells). The PCNA index and Ki67 index revealed a good correlation in linear regression analysis (P < 0.001) as did the mitotic index (P < 0.01). None of the parameters considered revealed a significant difference in proliferative activity of the three types of tumour (P > 0.05-Spearman test), but in both seminomas and Sertoli cell tumours the progression from tubular to diffuse pattern paralleled an increase in growth fraction. It is interesting that some seminomas of the diffuse type, often considered on histological grounds to be the most malignant, showed the highest values of the above-mentioned parameters.

Histochemical and immunohistochemical characterization of canine mammary mucinous carcinoma.

Mucinous carcinoma is a rare mammary tumour, characterized by intracellular and extracellular mucin. It is still uncertain whether the origin of the mucin is epithelial, myoepithelial or fibroblastic. Eleven canine cases originally classified as mucinous carcinomas were reassessed and compared with myoepithelial nests of mixed tumours. All samples were examined (1) histochemically by the periodic acid-Schiff (PAS) and PAS-diastase methods, and with alcian blue (pH 2.5 and pH 1.0), mucicarmine and Grimelius silver stain, and (2) immunohistochemically for cytokeratin 19, vimentin, alpha-actin and chromogranin A. This examination revealed that only five of the 11 tumours were genuine mucinous carcinomas. In these five tumours the mucus-secreting cells showed cytoplasmic cytokeratin 19 positivity; the mucus showed PAS-diastase and mucicarmine positivity, and alcianophilia which was stronger at pH 2.5 than at 1.0. The remaining six cases were re-classified as mixed tumours because both mucus and mucus-producing cells shared the following similarities with myoepithelial nests of mixed tumours: vimentin and alpha-actin cytoplasmic positivity, PAS negativity, alcianophilia both at pH 2.5 and 1.0, and mucicarmine positivity.

Laminin expression in testicular tumours of the dog.

The expression of laminin was studied to determine the distribution pattern of basement membranes (BMs) in normal testes and in a series of 40 canine testicular tumours (seminomas, Leydig and Sertoli cell tumours). BM was always present around seminiferous tubules and blood vessels in normal testes and in seminomas and Sertoli cell tumours of the intratubular type without invasion. BM changes (fragmentation or loss, or both) were usually found in invasive neoplasms which retained their tubular structure; disruption or absence was observed in tumours, with a diffuse pattern. The BM was never expressed in Leydig cell tumours, except around vessels, irrespective of their histological growth pattern (cystic-vascular, pseudoadenomatous, diffuse). An attempt was made to relate the degree of BM modification to proliferative monoclonal antibodies and mitotic index. In parallel with the progressive loss of BM an increase in proliferative activity occurred, indicating that BM changes are additional useful prognostic indicators in testicular tumours of the dog.

Detection of proliferating cell nuclear antigen (PCNA) in canine and feline mammary tumours.

Quantitation of immunohistochemical staining of the proliferating cell nuclear antigen (PCNA, clone PC10) by image analysis was performed on benign and malignant mammary tumours of dogs and cats. Scoring of the slides was carried out by image analysis to assess the percentage of labelled nuclei (expressed as a ratio of areas). Either the strongly labelled nuclei (SP-PCNA index), or all of the stained nuclei (TP-PCNA index) were counted as positive to determine the growth fraction and its correlation with the histopathological classification and nuclear grade (degree of nuclear differentiation, considered a morphological correlate of tumour aggressiveness). A significant difference in the values of PCNA indices was seen between benign and malignant growths (P < 0.0001, dog; P < 0.05, cat). Neither of the PCNA indices showed correlation with nuclear grade in dogs (P = 0.14 for SP-PCNA index and P = 0.31 for TP-PCNA index) or cats (P = 0.09 for SP-PCNA index and P = 0.07 for TP-PCNA index). A significant difference in the number of mitoses, expressed as mitotic index, was seen between benign and malignant growths in the dog (P < 0.01) but not in the cat (P = 0.078). Good correlation of mitotic index with nuclear grade was revealed in canine malignant growths (P < 0.05), but in feline malignant tumours such correlation (P < 0.05) was shown only when the values of intermediate plus typical forms were compared with the data for atypical forms. It is concluded that quantitation of PCNA-positive nuclear area by image analysis provides an objective method for assessing proliferative activity in benign and malignant mammary tumours of dogs and cats.

Evaluating mitotic activity in canine and feline solid tumors: standardizing the parameter.

Three different methods for evaluating mitotic activity (mitotic count, mitoses/area, mitotic index) were applied to different types of canine and feline solid tumors to determine the method that is most objective and correlates best with other parameters of cell proliferation. Mitotic activity was evaluated on toluidine blue stained histological sections. Slides stained with histochemical (AgNOR proteins) and immunohistochemical (MIB1, PCNA) markers of cell proliferation were available for each case. Quantitation of mitotic activity and cell proliferation parameters was performed with an image analyzer. Mitotic activity assessment was compared with cell proliferation indices and its ability to discriminate tumors grouped on histologically based criteria including the histological type, malignant or benign characteristics, and grade. A significant correlation by linear regression analysis with other parameters assessing cell proliferation revealed that mitotic index correlated 1000% and mitoses/area and mitotic count correlated 40% of the time. In discriminating the proliferative activity of tumors grouped by histological criteria, mitotic index and mitotic count revealed 1000% concordance with the other parameters of cell proliferation, while mitoses/areas showed 80% concordance.

Multiparametric survival analysis of histological stage and proliferative activity in feline mammary carcinomas.

Mammary tumours are among the most frequent malignant neoplasms in the cat and determination of prognosis on histological grounds alone can be unsatisfactory because it does not always correspond to the clinical behaviour of the neoplastic disease. The aim of this two-year post-mastectomy survival study is to relate the histological stage or invasiveness (the most commonly used histological parameter to grade malignancy) to several parameters assessing the proliferative activity-mitotic index, MIB1 index, and AgNOR index. Invasiveness was graded as local and vascular invasion whilst values of the parameters expressing proliferative activity, all quantified by image analysis, have been classified into low and high proliferative activity groups according to their median values, (0.719 for mitotic index, 12.11 for MIB1 index, and 3.19 for AgNOR index). For each group, mean survival (months+/-SD) was calculated. Histological stage (local invasion 21.83+/-7.83 months, blood vessels and/or lymphatics invasion 13.38+/-8.99,P<0.01), mitotic index (low 22.43+/-88.78, high 12.37+/-7.49,P<0.001), and AgNOR index (low 21.86+/-10.68, high 13.82+/-7.11,P<0.05) revealed a significant association with survival in univariate analysis and had an independent prognostic value in multiparametric survival test (P<0.001).

Clinical and pathological features of viral haemorrhagic disease of rabbits and the European brown hare syndrome.

The authors review the clinical, macro- and microscopical features, and pathogenesis of viral haemorrhagic disease (VHD) of rabbits and the European brown hare syndrome (EBHS). The two diseases share similar clinical and pathological manifestations involving an acute syndrome, sometimes accompanied by nervous and respiratory symptoms and epistaxis, and in all cases by severe hepatic damage and multifocal haemorrhages leading to fatal shock. The hepatic lesions (necrosis and inflammation) result from direct cytolytic and indirect microthrombotic effects of the causal agent. Endothelial lesions and a primary or secondary defect of coagulation factors are possible causes of the haemorrhagic syndrome. Typical lesions consist of necrotic hepatitis and congestion, haemorrhaging and oedema of the lungs and trachea. The histological and ultrastructural alterations of the liver are similar to those found in certain cases of acute fatal hepatitis in man. The high correlation between histologically typical hepatic findings and immunohistochemistry and immunoelectron microscopy is of diagnostic value. Both microscopic lesions and pathogenesis favour the unifying definition of "infectious necrotic hepatitis of Leporids" for the two disease entities.

Heat shock protein 90 is associated with hyperplasia and neoplastic transformation of canine prostatic epithelial cells.

Heat shock protein 90 (HSP90) is a molecular chaperone that regulates critical signalling proteins of cancer development and progression. Abnormal levels of HSP90 have been observed in human prostatic carcinoma (PC), with prognostic and therapeutic implications. Since spontaneously arising canine PC is a valuable model for the human disease, the aim of this study was to evaluate the immunohistochemical expression of HSP90 in two normal canine prostates, 17 canine prostates with benign prostatic hyperplasia (BPH) and five canine prostates with PC. HSP90 was expressed in the cytoplasm of epithelial cells in all samples, with a significant increase in labelled cells in PCs. Nuclear labelling was observed occasionally in normal tissue, but was increased in BPH and PC. HSP90 immunoreactivity in preneoplastic lesions (proliferative inflammatory atrophy and prostatic intraepithelial neoplasia) was similar to that in PCs. Increased HSP90 expression in canine PCs suggests the involvement of this molecule in carcinogenesis and tumour progression, supporting HSP90 as a potential target for therapeutic intervention.

CD117 expression influences proliferation but not survival in canine mammary tumours.

CD117 is a transmembrane tyrosine kinase receptor encoded by the c-Kit proto-oncogene. The immunohistochemical expression of CD117 was examined in 49 specimens of canine mammary glands (eight normal/hyperplastic, 11 benign tumours and 30 malignant tumours). Expression was assessed as: (1) presence or absence of CD117; (2) membrane, cytoplasmic, or both, distributions; and (3) percentage of CD117-labelled cells. None of these three immunohistochemical parameters was correlated with the type of mammary tissue (i.e. normal, benign or malignant), histotypes or histological stage of malignant tumours, or survival. An association was observed between Ki67 index and all three CD117 labelling parameters only for malignant tumours, with a significant increase in proliferative activity in tumours expressing CD117, mainly with both cytoplasmic and membrane expression.