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The aim of this work was to document the value of morphometric analysis in quantifying neuronal loss in canine cerebellar degenerative disease. Measurements of the cerebellar cell populations in a dog affected by severe cortico-cerebellar degeneration (CCD) were compared with those of an age-matched control dog. Histological stainings (hematoxylin-eosin, thionin, Bielschowsky) were performed; the granule cell density and Purkinje cell linear density were quantified. GFAP immunohistochemistry was run to quantify the stained area. Morphometric analysis in the pathologic dog showed a significant decrease of Purkinje cell linear density (4.50/mm), granule cells density (81.34/10000 µm2) and an increase of GFAP stained area (40.28%) in comparison to the age-matched dog (Purkinje cell linear density=12.74/mm, granule cell neuron density=95.53/10000 µm2 and GFAP stained area=34.68%). The use of morphometric techniques is suggested in order to enable comparison between results obtained from different laboratories of pathology.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical–pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical–pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

It is commonly accepted that some form of skin barrier dysfunction is present in canine atopic dermatitis (AD), one of the most common cutaneous pruritic inflammatory diseases of dogs. The impaired skin barrier function facilitates the penetration of allergens and subsequently stronger sensitization responses. The role of the endocannabinoid system (ECS) in the physiology and pathology of the skin is becoming increasingly established. It has been demonstrated that cannabinoid receptors are expressed in healthy and diseased skin and, based on current knowledge, it could be stated that cannabinoids are important mediators in the skin. The present study has been designed to immunohistochemically investigate the expression of the cannabinoid receptors type 1 (CB1R) and 2 (CB2R) and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors alpha (PPARα), and serotoninergic receptor 1a (5-HT1aR) in keratinocytes of healthy dogs and of dogs with AD. Samples of skin tissues were collected from 7 healthy controls (CTRL-dogs) and from 8 dogs with AD (AD-dogs). The tissue samples were processed using an immunofluorescence assay with commercially available antibodies, and the immunolabelling of the receptors studied was quantitatively evaluated. The keratinocytes of the CTRL- and the AD-dogs showed immunoreactivity for all the receptors investigated with a significant upregulation of CB2R, TRPA1, and 5-HT1aR in the epidermis of the AD-dogs. The presence of cannabinoid and cannabinoid-related receptors in healthy keratinocytes suggested the possible role of the ECS in canine epidermal homeostasis while their overexpression in the inflamed tissues of the AD-dogs suggested the involvement of the ECS in the pathogenesis of this disease, having a possible role in the related skin inflammation and itching. Based on the present findings, the ECS could be considered a potential therapeutic target for dogs with AD.

BackgroundAtopic dermatitis (AD) is one of the most common cutaneous inflammatory and pruritic diseases in dogs. Considering its multifactorial nature, AD can be a challenging disease to manage, and the therapeutic strategy must often be multimodal. In recent years, research has been moving toward the use of natural products which have beneficial effects on inflammation and itching, and no side effects. Cannabinoid receptors have been demonstrated to be expressed in healthy and diseased skin; therefore, one of the potential alternative therapeutic targets for investigating AD is the endocannabinoid system (ECS).ObjectiveTo immunohistochemically investigate the expression of the cannabinoid receptor type 2 (CB2R), and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in mast cells (MCs), macrophages, dendritic cells (DCs), T cells, and neutrophils of the skin of dogs with AD.AnimalsSamples of skin tissues were collected from eight dogs with AD (AD-dogs).Materials and methodsThe immunofluorescent stained cryosections of the skins of 8 dogs with AD having antibodies against CB2R, GPR55, TRPV1, TRPA1 were semiquantitatively evaluated. The inflammatory cells were identified using antibodies against tryptase (mast cells), ionized calcium binding adaptor molecule 1 (IBA1) (macrophages/DCs), CD3 (T cells), and calprotectin (neutrophils). The proportions of MCs, macrophages/DCs,Tcells, and neutrophils expressing CB2R, GPR55, TRPV1 and TRPA1 were evaluated.ResultsThe cells of the inflammatory infiltrate showed immunoreactivity (IR) for all or for some of the cannabinoid and cannabinoid-related receptors studied. In particular, MCs and macrophages/DCs showed CB2R-, GPR55-, TRPA1-, and TRPV1-IR; T cells showed CB2R-, GPR55- and TRPA1-IR, and neutrophils expressed GPR55-IR. Co-localization studies indicated that CB2R-IR was co-expressed with TRPV1-, TRPA1-, and GPR55-IR in different cellular elements of the dermis of the AD-dogs.Conclusions and clinical importanceCannabinoid receptor 2, and cannabinoid-related receptors GPR55, TRPV1 and TRPA1 were widely expressed in the inflammatory infiltrate of the AD-dogs. Based on the present findings, the ECS could be considered to be a potential therapeutic target for dogs with AD, and may mitigate itch and inflammation.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

Simple Summary Multidrug resistance of neoplastic cells to chemotherapeutic drugs is a phenomenon mediated by several molecular mechanisms. Among these, P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) counteract the intracellular load of multiple drugs, preventing their efficacy. The basal (intrinsic) cellular expression can be further stimulated by drug exposure. P-gp and BCRP are a subject of intense investigation both in human and veterinary oncology since a better understanding of how their expression is distributed across different tumors allows planning alternative therapeutic strategies. In canine mammary carcinomas, a phenotypic classification similar to the one widely adopted for breast cancer is currently employed. For Basal- and Normal-like phenotypes, chemotherapy is still the main option. In this study, we observed that canine mammary carcinomas bear a high intrinsic expression of both P-gp and BCRP, regardless of their molecular phenotype, and their presence does not influence the outcome. Abstract P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are major actors in multidrug resistance (MDR) phenomenon in both human and canine mammary carcinomas (CMCs). The aim of this study was to investigate an association between the intrinsic expression of P-gp and BCRP compared to the immunophenotypes and outcome in CMCs. Fifty CMCs were evaluated at immunohistochemistry (IHC) for P-gp, BCRP, Estrogen receptor alpha (ER), Progesterone receptors (PR), Human Epidermal Growth Factor Receptor type 2 (HER2), basal cytokeratins 5/6 (CK5/6), Epidermal Growth Factor Receptor 1 (EGFR), and Ki67 proliferation index. P-gp and BCRP positive cases were, respectively, 52% and 74.5%, with a significantly higher expression of BCRP than P-gp. Five immunophenotypes were defined in 37 out of 50 CMCs: 9 (24.3%) Luminal A, 5 (13.5%) Luminal B, 9 (24.3%) HER2 overexpressing, 9 (24.3%) Triple-negative basal-like, and 5 (13.5%) Triple-negative non-basal-like. In all CMCs at least one marker was expressed. Follow-up data were available for 25 animals. The average cancer-specific survival was 739 ± 444 days. A number of CMCs bear a high expression of P-gp and BCRP but no significant association was found between their expression and the immunophenotypes, Ki67 index, the histological grade, and tumor-related death.

Lipid-rich carcinoma is a rare histotype of canine mammary tumors with cytoplasmic vacuolation. In humans, glycogen-rich carcinoma, secretory carcinoma, and myoepithelial neoplasms are included in the differential diagnosis for lipid-rich carcinoma. The aim of the study was to investigate the existence of histotypes other than lipid-rich in canine mammary carcinomas with vacuolated cytoplasm using a diagnostic algorithm based on histopathology, histochemistry, immunohistochemistry, and ultrastructure and to evaluate the molecular phenotype of these neoplasms. Ten mammary carcinomas were collected, histologically reviewed, and subjected to histochemistry (PAS, PAS with diastase, Alcian blue, Sudan III [1 case], and Congo red [1 case]); immunohistochemistry for CK19, CK5/6, CK14, p63, calponin, vimentin, ER, PR, and HER2; and transmission electron microscopy (TEM). Cytokeratin immunolabeling demonstrated the epithelial origin of all tumors. Sudan III and TEM confirmed the diagnosis of lipid-rich carcinoma in 8 tumors (one amyloid-producing). One tumor was reclassified as a glycogen-rich carcinoma based on PAS reactivity that was diastase-labile, and a second tumor was reclassified as a carcinoma-and-malignant myoepithelioma based on the differentiation markers. Lipid-rich carcinomas were basal-like (5/8), null-type (2/8), and luminal A phenotype (1/8). The glycogen-rich carcinoma was basal-like, while the carcinoma-and-malignant myoepithelioma was luminal A. Vacuolated morphology of neoplastic cells in canine mammary carcinoma can indicate either a neoplasm of luminal epithelial origin with cytoplasmic lipid or glycogen, or vacuolated neoplastic suprabasal myoepithelial cells. Glycogen-rich carcinoma is a novel histological type that should be considered in the differential diagnosis for canine mammary carcinomas with vacuolated cytoplasm.

Simple Summary Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and feline mammary carcinomas (FMCs). We previously reported the identification of MMTV-like sequences and viral protein in six of 78 FMCs collected in Tuscany, Italy. To corroborate this finding, FMCs samples collected from a different geographic area were investigated. MMTV-like sequences and p14 protein were identified in three of 24 FMCs collected at the University of Bologna, one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All the examined FMCs from Pisa and Bologna were submitted to immunohistochemistry for molecular phenotype characterization. Of the nine positive FMCs, six were basal-like and three luminal-like. This study highlights the presence of MMTV-like sequences and protein in FMCs of different geographic areas. The characterization of molecular phenotype could contribute to understand the possible role of MMTV-like virus in FMC biological behaviour. Abstract In the last few years MMTV-like nucleotide sequences were detected in some feline and canine mammary tumours. Due to the confirmed role of cats in the epidemiology of the MMTV-like virus, the aim of this study was to investigate the main pathological features of positive feline mammary carcinomas (FMCs). Twenty-four FMCs were collected at the University of Bologna, submitted to laser microdissection and analysed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, an antibody against MMTV protein 14 (p14) was used. MMTV-like sequences were detected in three out of 24 FMCs (12.5%), one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All PCR-positive tumours were also positive for p14. Multiple nucleotide alignment has shown similarity to MMTV ranging from 98% to 100%. All the 102 examined FMCs were submitted to immunohistochemistry for molecular phenotyping. Of the nine MMTV-like positive FMCs, six were basal-like and three luminal-like. Our results demonstrate MMTV-like sequences and protein in FMCs of different geographic areas. Molecular phenotyping could contribute to understand the possible role of MMTV-like virus in FMC tumor biology.

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) expression are frequently related to multidrug resistance (MDR) in neoplastic cells. Canine inflammatory and grade III noninflammatory mammary carcinomas (IMC and non-IMC) are aggressive tumors that could benefit from chemotherapy. This study describes the immunohistochemical detection of P-gp and BCRP in 20 IMCs and 18 non-IMCs from dogs that had not received chemotherapy. Our aim was to determine if P-gp and BCRP expression was related to the “inflammatory” phenotype, to establish a basis for future studies analyzing the response to chemotherapy in dogs with highly malignant mammary cancer. Immunolabeling was primarily membranous for P-gp with a more intense labeling in emboli, and immunolabeling was membranous and cytoplasmic for BCRP. P-gp was expressed in 17 of 20 (85%) IMCs compared to 7 of 18 (39%) non-IMCs ( P = 0.006). BCRP was expressed within emboli in 15 of 19 (79%) emboli in IMC, 12 of 15 (80%) primary IMCs, and 12 of 18 (67%) non-IMCs, without statistically significant differences ( P > .05). All IMCs and 67% of non-IMCs expressed at least 1 of the 2 transporters, and 63% (12/19) of IMCs and 39% (7/18) of non-IMCs expressed both P-gp and BCRP. P-gp and BCRP evaluation might help select patients for chemotherapy. P-gp, expressed in a significantly higher percentage of IMCs vs non-IMCs, might play a specific role in the chemoresistance of IMC.

Simple Summary The human epidermal growth factor receptor-2 (HER2) has been studied in many human carcinomas. This receptor can be amplified and overexpressed in tumoral cells and serve as a prognostic, therapeutic, and predictive biomarkers. In human medicine there are many target drugs direct against this receptor, and these drugs are able to improve the clinical outcome of the patients. The overexpression of this receptor is mainly evaluated by immunohistochemistry, a technique that allows to evaluate the expression of the receptor on the cell surface. Recently, HER2 expression has been investigated in several canine tumors, but its role in the development of canine carcinoma is an object of debate. In this study we wanted to investigate the expression of HER2 protein in different histotypes of canine carcinomas in order to identify potential tumors that could benefit from the HER2-targeted therapy. We confirmed the hypothesis that HER2 is involved in tumor development of several canine carcinomas, amongst which carcinomas of the intestinal tract predominate. In dogs included in this study, 80% of intestinal carcinomas and a high proportion of rectal carcinomas (42%) were HER2 positive, suggesting that tumors of intestinal origin may potentially benefit from HER2-directed therapy. Abstract HER2 overexpression has been reported in various human and canine tumours. The aim of this study is to investigate the expression of HER2 protein in different histotypes of canine carcinomas in order to identify potential tumours that could benefit from the HER2-targeted therapy. Eighty-two (82) canine carcinomas (squamous cell, gastro-intestinal, rectal, pulmonary, prostatic, urothelial, and ovarian) from paraffin-embedded samp les were immunohistochemically evaluated. The degree of HER2 expression was scored based on the ASCO/CAP 2018 guidelines. Intestinal carcinomas were those with greater HER2 overexpression (3+) with 81% of positive cases, followed by 42% of rectal carcinomas and 28% of squamous cell carcinomas. These observations suggest that HER2 overexpression could be a driver in the oncogenesis of several types of canine carcinomas and lay the foundations for the identification of different types of canine carcinomas that could benefit from HER2-targeted therapy.

Canine liposarcoma is an uncommon soft tissue sarcoma usually arising in the subcutis. While liposarcoma classification in dogs is based solely on histology, in humans it depends on the detection of genetic abnormalities that can lead to specific protein overexpression. This study is an immunohistochemical evaluation of MDM2 and CDK4 expression in canine liposarcoma designed to assess the correlation of these proteins with histologic type, grade, mitotic index and Ki67 labeling index and evaluate their utility in improving tumor classification. Fifty-three liposarcomas were retrospectively collected: 24 were well differentiated liposarcomas (WDL), 16 of which expressed MDM2 and 21 CDK4; 7 were myxoid liposarcomas (ML), 1 of which expressed MDM2 and 5 expressed CDK4; 18 were pleomorphic liposarcomas (PL), all were MDM2 negative and 12 expressed CDK4. Four tumors were morphologically consistent with dedifferentiated liposarcoma (DDL) a subtype described only in humans: 3 expressed MDM2 and 4 expressed CDK4. MDM2 expression correlated with histotype (highly expressed in WDL and DDL) and grade (highly expressed in grade 1 tumors). Histotype correlated with the Ki67 labeling index (lowest in WDL and highest in DDL). A revised classification, considering MDM2 expression, allowed 8 WDL to be reclassified as PL and correlated significantly with mitotic and Ki67 labeling index (both significantly lower in WDL and progressively higher in ML and DDL). These results partially parallel data reported for human liposarcomas, suggesting that WDL and DDL are distinct neoplastic entities characterized by MDM2 expression, which may represent a useful diagnostic and potentially prognostic marker for canine liposarcoma.

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.

Distant metastases represent a major step in the progression and fatal outcome of canine and feline mammary carcinomas. Recent studies have characterized the molecular phenotypes of mammary tumours and provided information on molecules that may allow targeted therapy in sites from which the tumours may not readily be surgically resected. Molecular phenotypes were determined immunohistochemically in three feline and two canine cases of mammary neoplasia, each presenting with multiple distant metastases. These tumours and their metastases often overexpressed the c-erbB-2 phenotype. A basal-like phenotype was found in the distant metastases from two cases. These findings suggest that canine and feline mammary tumours with distant metastases may be amenable to novel targeted therapies.

The expression of tyrosine kinase receptors is attracting major interest in human and veterinary oncological pathology because of their role as targets for adjuvant therapies. Little is known about tyrosine kinase receptor (TKR) expression in canine liposarcoma (LP), a soft tissue sarcoma. The aim of this study was to evaluate the immunohistochemical expression of the TKRs fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptor–β (PDGFRβ); their ligands, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGFB); and c-kit in canine LP. Immunohistochemical labeling was categorized as high or low expression and compared with the mitotic count and MIB-1–based proliferation index. Fifty canine LPs were examined, classified, and graded. Fourteen cases were classified as well differentiated, 7 as myxoid, 25 as pleomorphic, and 4 as dedifferentiated. Seventeen cases were grade 1, 26 were grade 2, and 7 were grade 3. A high expression of FGF2, FGFR1, PDGFB, and PDGFRβ was identified in 62% (31/50), 68% (34/50), 81.6% (40/49), and 70.8% (34/48) of the cases, respectively. c-kit was expressed in 12.5% (6/48) of the cases. Mitotic count negatively correlated with FGF2 ( R = –0.41; P < .01), being lower in cases with high FGF2 expression, and positively correlated with PDGFRβ ( R = 0.33; P < .01), being higher in cases with high PDGFRβ expression. No other statistically significant correlations were identified. These results suggest that the PDGFRβ-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies.

Mammary gland neoplasms in mares are rare. This report describes the clinical and histopathological findings in a mare with mammary gland adenoma, discussing the results of immunohistochemistry undertaken to determine the expressione of hormone receptors.

Non-angiomatous-non-lymphomatous sarcomas (NANLs) represent 23%-34% of canine primary splenic sarcomas. Splenic liposarcomas account for 2%-6% of NANLs but myxoid variants are rarely reported and information on their behaviour is fragmentary. An 8-year-old male crossbreed (case 1), a 12-year-old female French bulldog (case 2), and an 11-year-old crossbreed (case 3) underwent splenectomy after the detection of a splenic nodule. Histology, histochemistry, immunohistochemistry, and transmission electron microscopy (TEM) were performed. Bundles of spindle-to-polygonal cells containing occasional cytoplasmic oil-red-O positive vacuoles embedded in an Alcian blue-positive extracellular matrix were observed. Aggregates of round cells were detected in cases 1 and 3. All tumours were vimentin positive and actin, desmin, Factor VIII, and S100 negative. The TEM evidenced different maturational stages of adipose cells (lipoblasts, intermediate, and undifferentiated). All the cases developed hepatic metastases and were euthanized. Disease free interval was 2 months in cases 1 and 3, and 21 months in case 2. The presence of a neoplastic embolus in case 1 and areas of round cell differentiation in cases 1 and 3 represented the sole prognostic indices.

Summary This report describes a spontaneously arising rhabdomyosarcoma of soft tissues in a brook trout (Salvelinus fontinalis). The lesion was examined by means of histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM). The cross-reactivity of the primary antibodies used in the IHC was investigated in silico using the Protein Blast system. Microscopically, the lesion appeared as a ‘small round cell’ undifferentiated sarcoma with rare myotube formation. IHC identified expression of sarcomeric actin and vimentin and these molecules showed the highest protein sequence identity. Lower protein sequence identity coincided with negative immunolabelling for desmin, MyoD1, myogenin and CD3. TEM revealed myofibrils, but without a defined sarcomeric architecture. The diagnosis of solid alveolar rhabdomyosarcoma of soft tissues was achieved on the basis of histological and ultrastructural findings.

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.

The biological behaviour of canine cutaneous mast cell tumours (MCTs) is highly variable. The aim of this study was to investigate whether intratumoral microvessel density (IMVD), as an indicator of angiogenesis, was predictive of biological behaviour in MCTs of canine skin, and to relate this parameter to histological grade and other histomorphological features. IMVD was evaluated by immunohistochemical labelling of the endothelial marker factor VIII (Von Willebrand’s factor)-related antigen in 32 primary cutaneous MCTs. Computer image analysis was used to measure IMVD, expressed as the number of microvessels per square millimetre. Cases were assigned to low and high vessel density groups, the median value (14.1) being used as the “cut-off”. Kaplan–Meier-estimated survival curves of the groups revealed that IMVD was significantly associated with survival time (P = 0.046). One-way analysis of variance, performed to compare the IMVD values of tumours of different histological grades, showed a significant difference between grades I and III (P = 0.0002). There was a significant difference in IMVD between moderately invasive and highly invasive tumours (P = 0:002) and between tumours with different numbers of mitoses (P = 0.0046). In multivariate analysis, performed to test the combined influence on survival of each parameter, only the mitotic index and IMVD were independent prognostic variables (P = 0.00019). The relation of angiogenesis to biological behaviour and histological parameters of malignancy of cutaneous MCTs was consistent with previous findings in human and animal tumours.

An intraocular mass in the left eye causing chronic severe exophthalmia in an adult female goldfish ( Carassius auratus) is described. The fish shared an aquarium with another goldfish found dead with gross and microscopic lesions consistent with mycobacteriosis. Histological examination of the left eye, histochemical (periodic acid–Schiff [PAS], Alcian blue, Ziehl–Neelsen) and immunohistochemical tests (glial fibrillary acidic protein, human neuronal protein, vimentin, and cytokeratin AE1/AE3) were carried out on the intraocular mass. Neoplastic cells forming an unencapsulated highly cellular proliferation partially covered by an intact corneal epithelium were stained with Alcian blue, which demonstrated an abundant hyaluronic acid–rich extracellular matrix. Multifocally, there were cyst-like dilatations bordered by neuroepithelial cells, which were PAS-positive. The complex neoplastic proliferation was composed of glial-like cells, neuronal-like cells (immunoreactive to glial fibrillary acidic protein and human neuronal protein, respectively) and neuroepithelium, which suggested a retinal origin.

Introduction: Immunohistochemistry (IHC) has played an increasing role as a diagnostic tool for the identification of neoplasms and pathogens. In dogs with mammary tumors (CMTs), there have been an increasing number of studies looking for reliable diagnostic and/or prognostic immunohistochemical biomarkers but no consensus has been reached. These studies cannot be compared because of the differences in how these biomarkers have been used and evaluated. Materials and Methods: Review of the literature including IHC markers on CMTs in order to detect the best immunolabeling and evaluation systems for each marker. Results: After the review process, the authors reached a consensus regarding practical questions related to the methodology and provide guidelines on immunolabeling for CMTs markers along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Conclusion: By standardizing the methods used for labeling and interpretation of these IHC markers, we aim to ensure consistency and reproducibility of future study results.

A Sertoli cell tumour occurred in a cryptorchid testis of a one-year-old cat with no signs of feminisation. The tumour showed intratubular growth without interstitial infiltration and the neoplastic cells appeared polymorphous and vacuolated. Mitotic figures were rare. The diagnosis was based on histopathological, ultrastructural, and immunohistochemical features of the tumour cells.

Background This study aimed to evaluate the relationship between the molecular phenotype of the primary mammary tumor and its related lymph node metastasis in the dog to develop prognostic-predictive models and targeted therapeutic options. Results Twenty mammary tumor samples and their lymph node metastases were selected and stained by immunohistochemistry with anti-estrogen receptor (ER), -progesterone receptor (PR), -human epidermal growth factor receptor 2 (c-erbB-2), -cytokeratin 5/6 (CK 5/6), -cytokeratin 14 (CK14), -cytokeratin 19 (CK 19) and -protein 63 (p63) antibodies. Four phenotypes (luminal A, luminal B, c-erbB2 overexpressing and basal-like) were diagnosed in primary tumors and five (luminal A, luminal B, c-erbB-2 overexpressing, basal-like and normal-like) in the lymph node metastases. Phenotypic concordance was found in 13 of the 20 cases (65%), and seven cases (35%) showed discordance with different lymph node phenotypic profile from the primary tumor. Conclusions The phenotype of the primary tumor assumes a predictive-therapeutic role only in concordant cases, meaning that both the primary tumor and its lymph node metastasis should be evaluated at the same time. A treatment plan based only on the primary tumor phenotype could lead to therapeutic failures if the phenotype of the lymph node metastasis differs from that of the primary tumor.

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen's Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen's Kappa coefficient was 0.72. The high Cohen's Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.

A 6-year-old, intact male Jack Russell terrier was diagnosed with a mass in the caudal abdomen, and ultrasound revealed a large prostatic mass. A total-body computed tomography scan was performed for staging, and lung nodules were detected. Histological examination showed a proliferation of spindle cells arranged in interlacing fascicles. Immunohistochemical staining revealed cells were cytokeratin negative and immunoreactive for vimentin and α-smooth muscle actin; cells stained with desmin and S-100 were negative. A diagnosis of primary prostatic leiomyosarcoma with pulmonary metastases was made. This is one of the rare cases of primary prostatic mesenchymal tumor in the canine species.

Endometrial adenocarcinoma is the most common uterine tumour of domestic rabbits. The present immunohistochemical study examined the expression of cytokeratin 19 (CK19), the progesterone receptor (PR), the proliferation-associated antigen Ki-67 and telomerase in normal rabbit uterine tissue and examples of endometrial hyperplasia, adenoma and adenocarcinoma. Tubulopapillary adenomas and adenocarcinomas were the most common histological subtypes in this series. Cytoplasmic expression of CK19 was recorded in two of three samples of normal endometrium and in one of three samples of endometrial hyperplasia, in all adenomas and five of six adenocarcinomas. PR was expressed within the nucleus of normal endometrial cells and in one of three samples of endometrial hyperplasia, each of four adenomas and in four of six adenocarcinomas. This finding suggests that PR expression is not directly involved in neoplastic transformation of the endometrium and that such expression is not a prognostic indicator. Nuclear labelling of telomerase activity was found in one of three normal uteri, all samples of endometrial hyperplasia, two of four adenomas, but none of the adenocarcinomas. The proliferation index as determined by Ki-67 expression was 9.7+/-2.75% (mean+/- standard-deviation (SD)) for normal endometrium, 11.29+/-2.5% for hyperplastic endometrium, 19.40+/-3.01% for benign tumours and 19.41+/-7.9% for malignant tumours. These findings may be interpreted to suggest that hormonal and anti-proliferative treatment may be more appropriate for the management of uterine carcinomas in rabbits than anti-telomerase treatment.

Background Human breast cancer is classified by gene expression profile into subtypes consisting of two hormone (oestrogen and/or progesterone) receptor-positive types (luminal-like A and luminal-like B) and three hormone receptor-negative types [human epidermal growth factor receptor 2-expressing, basal-like, and unclassified ("normal-like")]. Immunohistochemical surrogate panels are also proposed to potentially identify the molecular-based groups. The present study aimed to apply an immunohistochemical panel (anti-ER, -PR, -ERB-B2, -CK 5/6 and -CK14) in a series of canine malignant mammary tumours to verify the molecular-based classification, its correlation with invasion and grade, and its use as a prognostic aid in veterinary practice. Results Thirty-five tumours with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative. Most luminal-like A and basal-like tumours were grade 1 carcinomas, while the percentage of luminal B tumours was higher in grades 2 and 3 (Pearson Chi-square P = 0.009). No difference in the percentage of molecular subtypes was found between simple and complex/mixed carcinomas (Pearson Chi-square P = 0.47). No significant results were obtained by survival analysis, even if basal-like tumours had a more favourable prognosis than luminal-like lesions. Conclusion The panel of antibodies identified only three tumour groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same carcinoma molecular subtypes in women awaits confirmation. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. Stage and grade showed independent associations with survival in the multivariate regression, while molecular subtype grouping and histological type did not show associations. This suggests that caution should be used when applying this classification to the dog, in which invasion and grade supply the most important prognostic information.

Yersiniosis or red mouth disease is a contagious infectious disease of salmonids caused by the bacterium Yersinia ruckeri, which lead to significant economic losses in trout aquaculture worldwide. Sources of infection are the sick fish and asymptomatic carriers, which eliminates yersiniosis faeces, causing contamination of water and feed. Triggers the disease was in March, the surge in water temperature values of 15-170 C. The fish affected were the younger with low immune system, the sudden departure of the winter period. Histological examinations, immunohistochemical and bacteriological made from liver, spleen, kidney and blood of the trout heart yersiniosis suspects resulted in the identification of the species bacterium Yersinia ruckeri strain, confirming the suspected diagnosis based on clinical investigations (pronounced anemia of mouth and gills mucosa, dark color of skin, bleeding on the lingual mucosa, protruding eye bleeding). Bacteriological confirmation was done by identifying the causative agent, based on morphological characters (gram negative bacilli, mobile) and the distinctive biochemical characters tested using the API rapid tests. Histological examinations revealed colonies of bacteria in liver, spleen and kidneys were confirmed to be colonies of Yersinia ruckeri by immunohistochemistry with anti-Yersinia ruckeri. Antibiogram revealed sensitivity of Yersinia ruckeri at: Oxytetracycline, Flumequin, Trimethoprim and Ceftiofur, and moderate susceptibility to Amoxicillin and Enrofloxacin.

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