Completeness of the dog registry and estimation of the dog population size in a densely populated area of Rome

In most European countries, registration and identification of dogs is compulsory. In Italy, the national dog registry is composed of regional dog registries. Although dog registries have been established for many years, the issue related to completeness of data has not been addressed so far. The objective of this study was twofold: first to assess the completeness of data of the dog registry through telephone interview of a sample of dog owners drawn from the dog registry, then to estimate the total owned dog population in 4 boroughs of Rome. For the second objective, a capture–recapture method was applied using data from the dog registry and data from a face-to-face questionnaire submitted to people waiting in the sitting room of 5 points of access for booking and payment of primary and specialist care. Different scenarios are proposed to verify the assumptions of the estimation procedure and potential biases are discussed. The completeness of data of the dog registry was 88.9% (95% CI: 85.8–91.9%) and the owned-dog population was estimated at 26,244 dogs (95% CI: 24,110–28,383). The dog registry is an important source of information especially when it is properly updated and completeness of data is known.     

Veterinary cancer registries in companion animal cancer: a review

Current and prior veterinary cancer registries are few in number and scattered. Different inclusion criteria, dissimilar collection methods and variable reference population estimation methods pose obstacles in the comparisons between veterinary and human cancer registries. Veterinary cancer registries have yielded information on the risk and incidence of different cancer types in certain breeds and geographical regions, as well as provided information on genetic and environmental risk factors in some cancers. The objective of this article is to review the prior and current veterinary cancer registries, the information they have contributed and to discuss different issues relating to their structure including inclusion criteria, study populations, reference populations utilized in evaluations, recorded variables and the outcome from these.     

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.     

miR‐497 induces apoptosis by the IRAK2/NF‐κB axis in the canine mammary tumour

Since companion dogs have the same living environment as humans, they are a good animal model for the study of human diseases; this is especially true of canine spontaneous mammary tumours models. A better understanding of the natural history and molecular mechanisms of canine mammary tumour is of great significance in comparative medicine. Here, we collected canine mammary tumour cases and then assayed the clinical cases by pathological examination and classification by HE staining and IHC. miRNA‐497 family members (miR‐497, miR‐16, miR‐195 and miR‐15) were positively correlated with the breast cancer marker genes p63 and PTEN. Modulation of the expression of miR‐497 in the canine mammary tumour cell lines CMT1211 and CMT 7364 induced apoptosis and inhibited cell proliferation. Mechanistically, IRAK2 was shown to be a functional target of miR‐497 that affects the characteristics of cancer cells by inhibiting the activity of the NF‐κB pathway. Overall, our work reveals the miR‐497/IRAK2/NF‐κB axis as a vital mechanism of canine mammary tumour progression and suggests this axis as a target in breast cancer.     

Pevonedistat targeted therapy inhibits canine melanoma cell growth through induction of DNA re‐replication and senescence

MLN4924 (pevonedistat) is a potent and selective NEDD8‐activating enzyme (NAE) inhibitor. The NEDD8‐regulated neddylation system is responsible for the regulated degradation of intracellular proteins with important cellular functions in cancer cell growth, apoptosis, angiogenesis and metastasis. In human melanoma, inhibition of NAE results in induction of DNA re‐replication, S phase cell cycle arrest, DNA damage and apoptosis. The study aimed to assess the anti‐cancer effect of MLN4924 on canine malignant melanoma cell lines and patient samples and to elucidate the underlying mechanisms. Canine melanoma cell lines and primary patient samples were evaluated for cell viability after incubation with varying concentrations of MLN4924 or dimethyl sulfoxide. Apoptosis, cell proliferation and senescence assays were performed to address underlying mechanisms of MLN4924‐mediated anti‐tumour effects. Gene expression of seven previously identified deregulated genes in human melanoma was compared in sensitive vs resistant samples. MLN4924 treatment significantly reduced the viability of canine melanoma cell lines and primary samples in a dose‐ and time‐dependent manners. MLN4924 promoted cell apoptosis and inhibited cell growth through induction of DNA re‐replication and cell senescence. While the majority of canine melanoma samples demonstrated sensitivity at nanomolar ranges, some samples were resistant to the treatment. Modulation of P21 levels correlated with canine melanoma cell sensitivity. These results provided justification for further exploration of MLN4924 as a treatment of canine melanoma.     

Standing in the canine precision medicine knowledge gap: Improving annotation of canine cancer genomic biomarkers through systematic comparative analysis of human cancer mutations in COSMIC

The accrual of cancer mutation data and related functional and clinical associations have revolutionised human oncology, enabling the advancement of precision medicine and biomarker-guided clinical management. The catalogue of cancer mutations is also growing in canine cancers. However, without direct high-powered functional data in dogs, it remains challenging to interpret and utilise them in research and clinical settings. It is well-recognised that canine and human cancers share genetic, molecular and phenotypic similarities. Therefore, leveraging the massive wealth of human mutation data may help advance canine oncology. Here, we present a structured analysis of sequence conservation and conversion of human mutations to the canine genome through a ‘caninisation’ process. We applied this analysis to COSMIC, the Catalogue of Somatic Mutations in Cancer, the most prominent human cancer mutation database. For the project's initial phase, we focused on the subset of the COSMIC data corresponding to Cancer Gene Census (CGC) genes. A total of 670 canine orthologs were found for 721 CGC genes. In these genes, 365 K unique mutations across 160 tumour types were converted successfully to canine coordinates. We identified shared putative cancer-driving mutations, including pathogenic and hotspot mutations and mutations bearing similar biomarker associations with diagnostic, prognostic and therapeutic utility. Thus, this structured caninisation of human cancer mutations facilitates the interpretation and annotation of canine mutations and helps bridge the knowledge gap to enable canine precision medicine.     

Molecular carcinogenesis of canine mammary tumors: news from an old disease

Studies focusing on the molecular basis of canine mammary tumors (CMT) have long been hampered by limited numbers of molecular tools specific to the canine species. The lack of molecular information for CMT has impeded the identification of clinically relevant tumor markers beyond histopathology and the introduction of new therapeutic concepts. Additionally, the potential use for the dog as a model for human breast cancer is debatable until questions are answered regarding cellular origin, mechanisms, and cellular pathways. During the past years, increasing numbers of canine molecular tools have been developed on the genomic, RNA, and protein levels, and an increasing number of studies have shed light on specific aspects of canine carcinogenesis, particularly of the mammary gland. This review summarizes current knowledge on the molecular carcinogenesis of CMT, including the role of specific oncogenes, tumor suppressors, regulators of apoptosis and DNA repair, proliferation indices, adhesion molecules, circulating tumor cells, and mediators of angiogenesis in CMT progression and clinical behavior. Whereas the data available are far from complete, knowledge of molecular pathways has a significant potential to complement and refine the current diagnostic and therapeutic approach to this tumor type. Furthermore, current data show that significant similarities and differences exist between canine and human mammary tumors at the molecular level. Clearly, this is only the beginning of an understanding of the molecular mechanisms of CMT and their application in clinical patient management.     

The Flint Animal Cancer Center (FACC) Canine Tumour Cell Line Panel: a resource for veterinary drug discovery,comparative oncology and translational medicine

Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth and response to therapies. The current use of large panels of cell lines with associated phenotypic and genotypic information now allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. Current cell line panels with large amounts of associated drug sensitivity and genomics data are comprised of human cancer cell lines (i.e. NCI60 and GDSC). There is increased recognition of the contribution of canine cancer to comparative cancer research as a spontaneous large animal model with application in basic and translational studies. We have assembled a panel of canine cancer cell lines to facilitate studies in canine cancer and report here phenotypic and genotypic data associated with these cells.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

Inhibition of canine telomerase in vitro and in vivo using RNAi: further development of a natural canine model for telomerase-based cancer therapies

Despite advances in cancer therapy, cancer related morbidity and mortality among humans and companion animals remains high, and there is a clear need to develop novel targeted therapies. Expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of canine cancer cells and has thus become a candidate for targeted molecular therapies. In this study, the value of telomerase inhibition to target telomerase expressing cancer cells was explored using the novel mechanism of RNA interference (RNAi). Using a Lentiviral expression construct, targeting the RNA component of canine telomerase was effective at inhibiting telomerase in vitro and tumour growth in vivo, but possible resistance mechanisms are highlighted. As canine telomerase biology is more closely related to human telomerase biology than the murine system, it is proposed that this study highlights the value of natural canine models to study anti-telomerase therapies for human patients.     

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle‐shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.     

USE OF CONE‐BEAM COMPUTED TOMOGRAPHY TO CHARACTERIZE DAILY URINARY BLADDER VARIATIONS DURING FRACTIONATED RADIOTHERAPY FOR CANINE BLADDER CANCER

Urinary bladder cancer is difficult to treat accurately with fractionated radiation therapy (RT) due to daily positional changes of the bladder and surrounding soft‐tissue structures. We quantified the daily motion experienced by the canine bladder with patients in dorsal vs. sternal vs. lateral recumbency. We also described the dose distribution for three different planning target volume expansions (5, 10, and 15 mm) for each of the three positions to ensure adequate bladder dose and minimize irradiation of nearby healthy tissues. Analysis was based on data from retrospective daily cone‐beam computed tomography (CT) (CBCT) images obtained for positioning of canine patients undergoing routine RT. Organs of interest were contoured on each CBCT data set and the images, along with the contours, were registered to the original planning CT. All measurements were made relative to the planning CT and dosimetric data for the organs of interest was determined using a dose volume histogram generated from sample parallel‐opposed beam configuration. There was a wide range in bladder position throughout treatment. The least amount of bladder variation and the lowest rectal dose was with dogs in lateral recumbency. It was also determined that a margin of 10 mm would allow for sufficient dose to be delivered to the bladder while minimizing rectal dose.     

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan‐1) for nuclear medicine preclinical trials on spontaneous tumours

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B‐cell lymphoma, CD138 expression is associated with the ‘non‐germinal center’ phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour‐bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio‐immunotherapy.     

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA‐microarray assay and quantitative RT‐PCR. Importantly, a decreased expression of miR‐203 was significantly associated with a shorter survival time. Also, miR‐203 and ‐205 were markedly down‐regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR‐205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR‐203 is a new prognostic factor in canine oral MMs and that miR‐205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

Canine leishmaniasis. A review based on 95 cases in The Netherlands

The clinically relevant aspects of canine leishmaniasis are reviewed. Included are data from 92 dogs imported from the Mediterranean basin and at least 2 autochthonous cases. New aspects on pathogenesis are presented, including evidence that canine leishmaniasis is an immune complex disease. Therapy with meglumine antimonate (Glucantime) is evaluated. The hazards of imported canine leishmaniasis for public health are discussed.