HER2 Overexpression and Cytogenetical Patterns in Canine Mammary Carcinomas.

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.

Standardized approach for evaluating tumor infiltrating lymphocytes in canine mammary carcinoma: Spatial distribution and score as relevant features of tumor malignancy.

Neoplastic cells, through immunoediting mechanisms, can establish a state of immunosuppression to evade host immune defenses. The aims of this study were: (1) to validate a standard method for assessing tumor infiltrating lymphocytes (TILs) in canine mammary carcinoma by applying international human breast cancer guidelines; (2) to investigate if the TILs population was composed of a subset of regulatory T lymphocytes (Tregs); and (3) to evaluate the relationship between the number of TILs and Tregs and the biological behavior of the tumors. One hundred and twenty-nine canine mammary tumors were retrospectively selected for this study. Histological diagnosis, grading and histological evaluation of TILs was performed on hematoxylin and eosin-stained sections. TILs were evaluated using a three-tier semiquantitative method, previously validated in human medicine, based on the percentage of TILs (0-10%, 11-40% and 41-90%). Lymphocyte immunophenotype was confirmed by CD3 and CD79, while an anti-FoxP3 antibody was used to determine the presence of Tregs. The number of stromal TILs and invasive front TILs significantly correlated with each other (P < 0.0001) and increased with increasing histological grade (P = 0.002 and P = 0.004, respectively). A subset of TILs was composed of FOXP3+ Tregs. Stromal Tregs and invasive front Tregs were associated with stromal TILs and invasive front TILs (P = 0.03; P = 0.01 and P = 0.003; P = 0.007, respectively). In conclusion, in canine mammary carcinomas, an increased number of stromal and invasive front TILs is associated with increased malignancy and significant increase of Tregs that could lead to immunosuppression and evasion of the host immune system.

Red mark syndrome of trout (Oncorhynchus mykiss; Walbaum, 1792): Histopathological scoring and correlation with gross lesions.

Red mark syndrome (RMS) is a skin disorder affecting rainbow trout (Oncorhynchus mykiss). The present work aimed to correlate the gross skin lesions affecting 46 fish sampled from farms surveyed for RMS with their microscopic features, identifying histological parameters that may be suggestive of disease progression. Skin lesions were grossly included in one of three categories (types I, II and III) according to the progressive degree of severity. Histological parameters and anti-proliferating cell nuclear antigen (PCNA) tissue immunoreactivity were semi-quantitatively assessed. In the dermis, PCNA-positive lymphocytes, fibroblasts and endothelial cells were indicative of active phlogosis. A significant increase in PCNA-immunoreactive lymphocytes, from gross type I to type III cases, was found only in the hypodermis. The histological parameters significantly associated with the gross lesion severity were progressive loss of the epithelium and scales, recruitment of inflammatory cells in the stratum compactum, loss of architecture of the stratum compactum, perivascular and perineural granulomatous inflammation and increase in lymphocyte infiltration of the muscular layer. In the type II and type III categories, inflammation in the hypodermis and muscle displayed a granulomatous pattern, reinforcing the hypothesis of an immunopathological mechanism. The morphological diagnosis of "deep chronic dermatitis associated to panniculitis and myositis, characterised by lympho-histiocytic and granulomatous reaction" is suggested.

Ongoing Research Protocols for the Pharmacological Treatment of Neuropsychiatric Symptoms in Dementia.

The implementation of effective interventions for neuropsychiatric symptoms (NPS) is perceived as one of the most pressing research priorities in the field of dementia and one of the main unmet needs from the perspective of affected individuals and their caregivers and relatives. Nevertheless, to date, only a relatively marginal part of dementia research has focused on NPS. This study aimed to describe and discuss the state of the art concerning the identification and development of new pharmacological treatments for NPS in dementia. A review of 320 ongoing phase 1, 2, 3, and 4 protocols registered in the clinicaltrials.gov database was performed. All the trials enrolling patients with dementia were selected. Only studies adopting clinical measures of NPS frequency and/or severity as primary outcome were retained and analyzed. Overall, only a minority of ongoing phase 1, 2, 3 and 4 protocols on dementia (i.e., 9.0%) is primarily targeting NPS. Most of these studies are adopting a placebo-controlled parallel assignment design, testing oral compounds, and targeting specific NPS (mostly agitation and/or aggression). A total of 3,445 subjects with dementia will tentatively be recruited in these trials. The methodologies adopted in these studies, the characteristics of the tested interventions, the eligibility criteria, and the operational definitions of NPS are presented and discussed. The relevance of NPS is not yet matched by an adequate research effort. The current tendency at privileging disease-modifying approaches and other symptoms of dementia and the methodological complexity of studying NPS are still substantially contributing to the gap between research activities and clinical needs.

Tyrosine Kinase Receptor Expression in Canine Liposarcoma.

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-ß (PDGFRß); 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 PDGFRß 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 PDGFRß ( R = 0.33; P < .01), being higher in cases with high PDGFRß expression. No other statistically significant correlations were identified. These results suggest that the PDGFRß-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies.

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.

Histological Classification and Immunohistochemical Evaluation of MDM2 and CDK4 Expression in Canine Liposarcoma.

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.

Cryptosporidium parvum: From foal to veterinary students.

This paper describes the transmission of a zoonotic subtype of Cryptosporidium parvum between two foals hospitalized in an Equine Perinatology Unit (EPU) linked to an outbreak of cryptosporidiosis in veterinary students. Fecal specimens of 36 mares (105 samples) and 28 foals (122 samples) were subjected to Ziehl-Neelsen staining, nested PCR of 18S rDNA. Two foals tested positive for Cryptosporidium; PCR restriction fragment length polymorphism (PCR-RFLP) analysis and subtyping by nested PCR of the 60kDa glycoprotein (gp60) gene revealed C. parvum subtype IIdA23G1. The introduction of Cryptosporidium into the EPU is suspected to be in a foal showing no initial clinical signs that tested positive for C. parvum during an asymptomatic phase. A second foal, hospitalized afterwards for perinatal asphyxia syndrome complicated with failure of passive transfer and sepsis, showed severe watery diarrhea after 4 days of hospitalization and was positive for the same subtype. During this period, six students attending the EPU complained of abdominal pain and diarrhea and were positive for the same subtype of C. parvum. To the authors' knowledge, this is the first description of this subtype in foals and the first report of evidence of zoonotic transmission of cryptosporidiosis from foals to human.

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.

Rhabdomyosarcoma of Soft Tissues in an Adult Brook Trout (Salvelinus fontinalis).

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.

Pathological characterization of primary splenic myxoid liposarcomas in three dogs.

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.

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.

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.

Quantitative immunohistochemical assessment of IgA, IgM, IgG and antigen-specific immunoglobulin secreting plasma cells in pig small intestinal lamina propria.

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa.

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.

Molecular phenotype of primary mammary tumours and distant metastases in female dogs and cats.

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.

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.

Effects of two water disinfectants (chloramine T and peracetic acid) on the epidermis and gills of Garra rufa used in human ichthyotherapy.

Doctor fish (Garra rufa) have recently been used for aesthetic purposes and as a medical treatment in patients with psoriasis (ichthyotherapy). For this particular kind of human therapy it is essential to guarantee adequate hygienic conditions for both people and fish. The aim of this study was to test two concentrations of water disinfectants, chloramine T and peracetic acid, on Garra rufa to ascertain possible exposure damage to the epidermis and gills. Fish were exposed to 2 mg/l and 10 mg/l of chloramine T and to 15 microl/l and 45 microl/l of peracetic acid in a 40-minute static bath up to six times a day for one week. The epidermis and gills were checked for histological changes and the number of epidermal mucous cells, club cells and taste buds were quantified; mucous cells were also characterized histochemically to detect alterations in mucin production. No mortality or severe histological changes were found in treated or control fish. Cell count showed a significant increase (p < 0.05) in mucous cells (mean 49.1 +/- 6.7 vs 37.0 +/- 13.1 of controls) in animals treated with peracetic acid independently of the dose. Club cell number showed a significant (p < 0.05) decrease in fish treated with 2 mg/l of chloramine T (mean 74.3 +/- 15.6) and with 45 microl/1 of peracetic acid (mean 78.17 +/- 10.5) compared to controls (mean 107.0 +/- 19.2). Histochemical evaluation of mucous cells did not reveal changes in mucin type in fish exposed to the two disinfectants. The results suggest a good tolerability of Garra rufa to the two disinfectants at the concentrations tested.

Molecular phenotype in mammary tumours of queens: correlation between primary tumour and lymph node metastasis.

The molecular characterization of mammary tumours represents a new stage in the development of effective predictive models and targeted therapies. The aim of this study was to evaluate the relationship between the molecular phenotype of a primary feline mammary tumour and that of a related lymph node metastasis. Twenty-one mammary tumour samples and their lymph node metastases were selected and evaluated immunohistochemically for expression of oestrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (c-erbB-2), cytokeratin 5/6, cytokeratin 14, cytokeratin 19 and protein 63. Mammary tumours were classified into five subtypes: luminal A, luminal B, c-erbB-2 overexpressing, basal-like and normal-like, based on an algorithm applied in both human and veterinary medicine. Concordance between the primary tumour and its lymph node metastasis was detected in 12 of 21 cases (57.1%). In the remaining nine cases (42.9%) there was discordance in the molecular profile at the two sites. Therefore, the tumour molecular profile must be evaluated in both sites in order to obtain definitive identification of the tumour profile (or profiles) and to plan an appropriate therapy.