Massimo Castagnaro

The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.

Guidelines for treatment of leishmaniasis in dogs

rug treatment of leishmaniasis in dogs is a chal-lenge for veterinary practitioners. Because of its complex pathogenesis, leishmaniasis may manifest with various clinical signs, ranging from mild and nonspe-cific to those reflecting severe involvement of several organs. The immune response plays an important role in the development, outcome, and response to treat-ment of

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.

Canine Mammary Tumors

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.

An Immunohistochemical study of HER-2 expression in feline mammary tumours.

The aim of the present study was to evaluate HER-2 expression in feline mammary tumours. Five different immunohistochemical protocols were tested with 73 feline mammary carcinomas (MCs), 10 mammary adenomas and 73 hyperplastic or dysplastic mammary lesions. The histological features of these lesions, clinical follow-up and expression of Ki-67 and p53 were also examined. With an optimized immunohistochemical protocol, HER-2 overexpression was detected in only four of the 73 (5.5%) MCs and did not correlate with histological classification or with the 1 year post-surgical clinical outcome. No correlation was found between the expression of Ki-67 or p53 and HER-2. Five of the 73 (6.8%) hyperplastic or dysplastic lesions and one of the 10 (10%) mammary adenomas were HER-2 positive. These results suggest that HER-2 may not play as significant role in mammary carcinogenesis and prognosis in cats as it does in human patients.

Proliferation activity in oral and cutaneous canine melanocytic tumours: correlation with histological parameters, location, and clinical behaviour

A total of 62 canine melanocytic tumours (10 melanocytomas and 52 primary malignant melanomas) were investigated to compare the accuracy of prognosis provided by MIB-1 proliferation index (MIB-1-PI) with classical histological criteria and location. MIB-1-PI was assessed by means of quantitative image analysis of sections immunostained with MIB-1 monoclonal antibody. Tumour location, histological cell type, stromal or lymphatic vessel invasion, maximum tumour thickness, and presence of inflammation or necrosis were recorded for each case. Thirty-eight dogs were submitted to a 1-year follow-up and the clinical outcome of the disease determined. MIB-1-PI in melanocytomas differed significantly from that detected in primary malignant melanomas (P=0.0001). A significant difference in MIB-1-PI was revealed between oral and cutaneous malignant melanomas (P=0.015), and between presence and absence of lymphatic vessel invasion (P=0.05). MIB-1-PI was not correlated with the other parameters. In univariate analysis, only tumour location (oral vs cutaneous), presence of lymphatic vessel invasion, and MIB-1-PI were associated with decreased overall survival (P=0.0001,P=0.0144, and P=0.0489, respectively). In conclusion, the results of our study confirm that the assessment of the MIB-1-PI may be of additional prognostic value for dogs with primary malignant melanomas.

Ki-7 index as indicator of the post-surgical prognosis infeline mammary carcinomas

Abstract Forty-eight feline mammary carcinomas ( fmc ) were resected surgically from 48 cats to determine whether the Ki-67 index (Ki-67I) would provide an indication of the post-surgical survival time ( pst ). Twenty-four cats (50 per cent) were still alive (group A) one year after surgery, whilst 24 (50 per cent) (group B) had died. Formalin-fixed, paraffin wax-embedded histological sections were immunostained with a monoclonal antibody to Ki-67 (MIB-1) and at least 1000 nuclei in eight to 10 representative fields were counted. The Ki-67I was expressed as the percentage of positive nuclei. In fmc , the IC-671 ranged from 7·5 to 49·7 (24·8±9·5). A statistically significant difference (P = 0000006) in the Ki-671 was found between group A and group B cats. No other statistically significant differences were found between these groups. The Ki-671 did not correlate with age or different histological type, according to the WHO classification. A Ki-671 cut-off of 25-2 represents a useful tool for identifying fmc with a more aggressive course.

Expression profiling of skeletal muscle in young bulls treated with steroidal growth promoters

Dexamethasone (Dex), alone or in association with estrogens, is often illegally administered per os at very low dosage as a growth promoter in beef cattle, with effects that are opposite to the muscle wasting and atrophy induced by repeated administration at therapeutic dosages. In vitro and in vivo studies have investigated the catabolic effects of Dex at therapeutic doses on skeletal muscle, demonstrating an increase in the expression of GDF8 (myostatin) gene, a well-known negative regulator of skeletal muscle mass, in a dose-dependent way. This suggested a direct role of myostatin in Dex-induced muscle wasting. In the present study, an oligonucleotide microarray platform was used to compare expression profiles of beef cattle muscle in animals treated with either Dex or Dex plus 17-beta estradiol (Estr) administered at subtherapeutic dosage, against untreated controls. Data analysis demonstrates that the expression profiles were strongly affected by Dex treatment with hundreds of genes upregulated with relevant fold-change, whereas seven genes were downregulated including the myostatin gene. On the contrary, the number of differentially regulated genes was lower in response to the addition of Estr to the Dex treatment. Differentially regulated genes were analyzed to describe the effects of these treatments on muscle physiology, highlighting the importance of specific pathways (e.g., Wnt or cytokine signaling) and cellular processes (e.g., cell shape and motility). Finally, the observed differences in the expression profile will allow the development of indirect bio-markers to detect illegal Dex treatments in beef cattle using quantitative RT-PCR.

Fatal necrotizing fasciitis and myositis in a captive common bottlenose dolphin (Tursiops truncatus) associated with Streptococcus agalactiae

A common bottlenose dolphin (Tursiops truncatus) was presented for necropsy after acute onset of gastrointestinal signs and cutaneous lesions that rapidly progressed to death. Gross and microscopic findings were characterized by locally extensive severe necrohemorrhagic fasciitis and cellulitis, and severe necrotizing myositis in the head and dorsocranial thorax, with numerous disseminated gram-positive cocci. Streptococcus agalactiae was isolated from the lesions and from visceral organs (liver and lung), and it was identified by standard microbiology techniques. This communication is the first report of necrotizing fasciitis in a marine mammal associated with S. agalactiae.

Feline mammary tumours in comparative oncology

Naturally occurring tumours in domestic animals have been recognized as an interesting opportunity for comparative oncology (MacEwen, 1990; Vail & MacEwen, 2000). Cancer is the second most frequent cause of death in humans and the first one in dogs and cats (Jemal et al. 2003). The age-adjusted overall cancer incidence per 100000 individuals per year is comparable in humans and domestic animals, being approximately 300 in humans, 381 in dogs and 264 in cats (Vail & MacEwen, 2000). When analysing the incidence by site, breast cancer is the most frequent (32%) in women, the first of all neoplasia (52%) occurring in bitches and the third (17%) in queens after lymphohaemopoietic and skin tumours (Hayes et al. 1981; Hayes & Mooney, 1985; MacEwen, 1990; MacEwen & Withrow, 1996; Jemal et al. 2003). Several other aspects contribute to the value of domestic animals as models for human cancers (MacEwen, 1990; Vail & MacEwen, 2000). Tumours occur spontaneously in companion animals that share a similar environment with humans and therefore might be exposed to similar risk factors. The high incidence of some tumour types offers large population samples. The shorter overall lifespan of domestic animals associated with a more rapid progression of cancer allows adequate comparison of response time with humans. Biological, anatomical, histopathological, genetic, and molecular similarities between some animal and human tumours are also well established (Hansen & Khanna, 2004). Finally, testing novel therapies is more ethically acceptable when treating spontaneous diseases in companion animals rather than experimentally induced pathologies in animal models. At the same time, there is an increasing interest of owners towards the use of the most advanced therapeutic tools for companion animals despite the higher economic costs associated with these therapies.