Targeting SCD triggers lipotoxicity of cancer cells and enhances anti-tumor immunity in breast cancer brain metastasis mouse models.

Breast cancer brain metastases (BCBM) are a significant cause of mortality and are incurable. Thus, identifying BCBM targets that reduce morbidity and mortality is critical. BCBM upregulate Stearoyl-CoA Desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, suggesting a potential metabolic vulnerability of BCBM. In this study, we tested the effect of a brain-penetrant clinical-stage inhibitor of SCD (SCDi), on breast cancer cells and mouse models of BCBM. Lipidomics, qPCR, and western blot were used to study the in vitro effects of SCDi. Single-cell RNA sequencing was used to explore the effects of SCDi on cancer and immune cells in a BCBM mouse model. Pharmacological inhibition of SCD markedly reshaped the lipidome of breast cancer cells and resulted in endoplasmic reticulum stress, DNA damage, loss of DNA damage repair, and cytotoxicity. Importantly, SCDi alone or combined with a PARP inhibitor prolonged the survival of BCBM-bearing mice. When tested in a syngeneic mouse model of BCBM, scRNAseq revealed that pharmacological inhibition of SCD enhanced antigen presentation by dendritic cells, was associated with a higher interferon signaling, increased the infiltration of cytotoxic T cells, and decreased the proportion of exhausted T cells and regulatory T cells in the tumor microenvironment (TME). Additionally, pharmacological inhibition of SCD decreased engagement of immunosuppressive pathways, including the PD-1:PD-L1/PD-L2 and PVR/TIGIT axes. These findings suggest that SCD inhibition could be an effective strategy to intrinsically reduce tumor growth and reprogram anti-tumor immunity in the brain microenvironment to treat BCBM.

Tumor Immune Microenvironment and Its Clinicopathological and Prognostic Associations in Canine Splenic Hemangiosarcoma.

Tumor cells can induce important cellular and molecular modifications in the tissue or host where they grow. The idea that the host and tumor interact with each other has led to the concept of a tumor microenvironment, composed of immune cells, stromal cells, blood vessels, and extracellular matrix, representing a unique environment participating and, in some cases, promoting cancer progression. The study of the tumor immune microenvironment, particularly focusing on the role of tumor-infiltrating lymphocytes (TILs), is highly relevant in oncology due to the prognostic and therapeutic significance of TILs in various tumors and their identification as targets for therapeutic intervention. Canine splenic hemangiosarcoma (HSA) is a common tumor; however, its immune microenvironment remains poorly understood. This retrospective study aimed to characterize the histological and immunohistochemical features of 56 cases of canine splenic HSA, focusing particularly on tumor-infiltrating lymphocytes (TILs). We assessed the correlations between the lymphocytic response, the macroscopic and histological characteristics of the tumor, and the survival data. Our study demonstrated that FoxP3 distribution was associated with tumor-related death and survival, while the CD20 count was associated with metastasis. This study provides an in-depth characterization of the tumor immune microenvironment in canine splenic HSA and describes potential prognostic factors.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/ß-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/ß-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Epithelial-to-Mesenchymal Transition and Phenotypic Marker Evaluation in Human, Canine, and Feline Mammary Gland Tumors.

Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. EMT has been closely associated with cancer cell aggressiveness. The aim of this study was to evaluate the mRNA and protein expression of EMT-associated markers in mammary tumors of humans (HBC), dogs (CMT), and cats (FMT). Real-time qPCR for SNAIL, TWIST, and ZEB, and immunohistochemistry for E-cadherin, vimentin, CD44, estrogen receptor (ER), progesterone receptor (PR), ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, and CK14 were performed. Overall, SNAIL, TWIST, and ZEB mRNA was lower in tumors than in healthy tissues. Vimentin was higher in triple-negative HBC (TNBC) and FMTs than in ER+ HBC and CMTs (p < 0.001). Membranous E-cadherin was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin was higher in TNBCs when compared with ER+ HBC (p < 0.001). A negative correlation between membranous and cytoplasmic E-cadherin was found in all three species. Ki-67 was higher in FMTs than in CMTs (p < 0.001), whereas CD44 was higher in CMTs than in FMTs (p < 0.001). These results confirmed a potential role of some markers as indicators of EMT, and suggested similarities between ER+ HBC and CMTs, and between TNBC and FMTs.

Characterization and function of extracellular vesicles in a canine mammary tumour cell line: Ultracentrifugation versus size exclusion chromatography.

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50-1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC-isolated EVs slightly enhanced cell migration compared to UC-isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV-product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.

The Italian Network of Laboratories for Veterinary Oncology (NILOV) 2.0: Improving Knowledge on Canine Tumours.

Advances in tumour research are crucial, and comparative oncology can improve the knowledge in several ways. Dogs are not only models of specific naturally occurring tumours but can also be sentinels of environmental exposures to carcinogens, as they share the same environment with their owners. The purpose of this work was to describe the data collected by The Italian Network of Laboratories for Veterinary Oncology in the first 9 years of activity (2013-2021) and to evaluate their potential epidemiological significance. Frequencies of tumour topographies and main morphologies in dogs were described, analysed and compared, calculating age-adjusted proportional morbidity ratios and considering several risk factors (breed, sex, period and region of residence). These observations allowed us to highlight differences not only in morphology and topography of some tumours but also to formulate hypotheses on the potential role of some risk factors, e.g., neutering/spaying or geographical location. In our opinion, the results of this case series confirm the importance of initiating and consolidating animal cancer registration initiatives that would facilitate the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumours in dogs from a comparative perspective.

Reproducibility and Feasibility of Classification and National Guidelines for Histological Diagnosis of Canine Mammary Gland Tumours: A Multi-Institutional Ring Study.

Histological diagnosis of Canine Mammary Tumours (CMTs) provides the basis for proper treatment and follow-up. Nowadays, its accuracy is poorly understood and variable interpretation of histological criteria leads to a lack of standardisation and impossibility to compare studies. This study aimed to quantify the reproducibility of histological diagnosis and grading in CMTs. A blinded ring test on 36 CMTs was performed by 15 veterinary pathologists with different levels of education, after discussion of critical points on the Davis-Thompson Foundation Classification and providing consensus guidelines. Kappa statistics were used to compare the interobserver variability. The overall concordance rate of diagnostic interpretations of WP on identification of hyperplasia-dysplasia/benign/malignant lesions showed a substantial agreement (average k ranging from 0.66 to 0.82, with a k-combined of 0.76). Instead, outcomes on ICD-O-3.2 morphological code /diagnosis of histotype had only a moderate agreement (average k ranging from 0.44 and 0.64, with a k-combined of 0.54). The results demonstrated that standardised classification and consensus guidelines can produce moderate to substantial agreement; however, further efforts are needed to increase this agreement in distinguishing benign versus malignant lesions and in histological grading.

Canine and feline in situ mammary carcinoma: A comparative review.

Carcinoma in situ of the breast is a well-known entity in humans. In veterinary medicine, particularly in canine and feline mammary literature, there is no agreement whether the term in situ should be used to indicate a specific carcinoma histotype or the noninvasive status of a carcinoma of any histotype. Moreover, in the most recent histologic classification of mammary tumors published by the Davis-Thompson Foundation, it is suggested to abandon the term carcinoma in situ given the lack of standardized criteria defining this entity, replacing it with epitheliosis or ductal/lobular hyperplasia with severe atypia. This publication presents a critical review of the term in situ in human and veterinary medicine considering the evolution of the term over the years and its heterogeneous use by different authors, including variations in immunohistochemical markers for classification. This review aims to point out the lack of uniformity in the nomenclature and classification issues in veterinary medicine regarding the use of the term in situ, laying the ground for a process of standardization in future publications.

Mammary Fibroadenoma in Cats: A Matter of Classification.

Benign mammary lesions are infrequent in cats. Among these, the most common is feline fibroadenomatous change, a hyperplastic/dysplastic change associated with hormonal imbalances. Although never thoroughly described in scientific literature, feline fibroadenomas, which share some morphological features with fibroadenomatous change, have been variably included in classification systems. The aim of this study was to characterise feline mammary fibroadenomas from a histological and immunophenotypical point of view in order to allow the standardisation of classification. Nine cases were retrospectively collected from eight female and one male cat with no history of hormonal stimulation. Diagnostic inclusion criteria were defined and immunohistochemistry was performed. Histologically, nodules were composed of neoplastic epithelial cells arranged in arborizing lobular-like structures surrounded by abundant proliferating stroma. In all analysed cases, epithelial elements showed immunolabelling for pancytokeratin, cytokeratin19, and ß-catenin. Interestingly, five cases showed multifocal epithelial vimentin positivity. Epithelial nuclear oestrogen receptor positivity was observed in three of the nine samples. In all cases, myoepithelial cells did not extend into the interstitium. Stromal cells expressed vimentin, calponin, and mild ß-catenin. The median Ki67 scores were 18% and 8.3% in the epithelial and stromal components, respectively. This study describes, for the first time, the morphological and immunophenotypical features of feline mammary fibroadenoma, highlighting its existence as a separate entity from fibroadenomatous change.

Teat sinus and duct adenomatous hyperplasia in dogs.

Seventeen lesions diagnosed as teat sinus and duct adenomatous hyperplasia were identified in 10 dogs. All of the dogs were small breeds. Six were spayed female and 4 were male, 3 castrated and 1 intact. In 5 cases, the lesions involved multiple teats. They were pink to black, flattened to round, and sometimes crusted. Histologically, the lesions were usually pigmented (16/17), plaque-like to nodular masses composed of polygonal cells arranged in anastomosing trabeculae and bilayered ducts and/or cysts, with a fibrous to mucinous (Alcian blue-positive) stroma and squamous cysts (12/17). Scattered epithelial cells contained single, discrete, clear cytoplasmic vacuoles. Atypia was mild, and the mitotic count per 2.37 mm2 varied from 0 to 15 (average 2.7). Immunohistochemistry was performed on 14 of the lesions from 8 dogs. Epithelial cells were 100% panCK+ and included basally located CK14+/CK5_6+/p63+/calponin- cells and nonbasal CK19+/CK7+ cells. Cells manifesting squamous differentiation were usually panCK+/CK14+/CK5_6+/CK19-/CK7-/p63±/calponin-. In addition to fibroblasts, vimentin positivity was found in disseminated, round to stellate stromal and intraepithelial cells that often had black, granular, cytoplasmic pigment (consistent with dendritic/phagocytic cells and/or melanocytes). Of the 8 dogs for which clinical follow-up information was available, all were still alive and well, with no significant teat changes, development of mammary lesions or other masses 4 to 22 months (median 12.5) after biopsy. The histologic, immunohistochemical, and clinical findings were consistent with teat duct and sinus adenomatous hyperplasia. This is an uncommon, benign proliferative lesion that can involve multiple teats of female and male, small breed dogs.

Microfluidic Strategies for Extracellular Vesicle Isolation: Towards Clinical Applications.

Extracellular vesicles (EVs) are double-layered lipid membrane vesicles released by cells. Currently, EVs are attracting a lot of attention in the biological and medical fields due to their role as natural carriers of proteins, lipids, and nucleic acids. Thus, they can transport useful genomic information from their parental cell through body fluids, promoting cell-to-cell communication even between different organs. Due to their functionality as cargo carriers and their protein expression, they can play an important role as possible diagnostic and prognostic biomarkers in various types of diseases, e.g., cancers, neurodegenerative, and autoimmune diseases. Today, given the invaluable importance of EVs, there are some pivotal challenges to overcome in terms of their isolation. Conventional methods have some limitations: they are influenced by the starting sample, might present low throughput and low purity, and sometimes a lack of reproducibility, being operator dependent. During the past few years, several microfluidic approaches have been proposed to address these issues. In this review, we summarize the most important microfluidic-based devices for EV isolation, highlighting their advantages and disadvantages compared to existing technology, as well as the current state of the art from the perspective of the use of these devices in clinical applications.

A Recurrent Inflammatory Myofibroblastic Tumor-like Lesion of the Splenic Capsule in a Kitten: Clinical, Microscopic and Ultrastructural Description.

OBJECTIVE: To describe the findings of an unusual splenic tumor in a kitten. METHODS: A grossly multinodular mass arising from the splenic capsule of a 7-month-old male Havana kitten was echographically detected and surgically removed by splenectomy, then analyzed microscopically and ultrastructurally. RESULTS: The mass showed features of an inflammatory myofibroblastic tumor. TREATMENT AND OUTCOME: Two months after surgical excision, the mass recurred in the same intra-abdominal area but disappeared after 2 months of anti-inflammatory therapy. Follow-up at 18 months after surgery revealed resolution of the disease. CLINICAL RELEVANCE: Inflammatory myofibroblastic tumor in cats have been rarely reported and are usually in the orbital region. In the present report, an unusual multinodular gross presentation, a recurrence over time, and a favorable clinical course, are described.

Evaluation of TFR-1 Expression in Feline Mammary Cancer and In Vitro Antitumor Efficacy Study of Doxorubicin-Loaded H-Ferritin Nanocages.

The transferrin receptor 1 (TFR-1) has been found overexpressed in a broad range of solid tumors in humans and is, therefore, attracting great interest in clinical oncology for innovative targeted therapies, including nanomedicine. TFR-1 is recognized by H-Ferritin (HFn) and has been exploited to allow selective binding and drug internalization, applying an HFn nanocage loaded with doxorubicin (HFn(DOX)). In veterinary medicine, the role of TFR-1 in animal cancers remains poorly explored, and no attempts to use TFR-1 as a target for drug delivery have been conducted so far. In this study, we determined the TFR-1 expression both in feline mammary carcinomas during tumor progression, as compared to healthy tissue, and, in vitro, in a feline metastatic mammary cancer cell line. The efficacy of HFn(DOX) was compared to treatment with conventional doxorubicin in feline mammary cancer cells. Our results highlighted an increased TFR-1 expression associated with tumor metastatic progression, indicating a more aggressive behavior. Furthermore, it was demonstrated that the use of HFn(DOX) resulted in less proliferation of cells and increased apoptosis when compared to the drug alone. The results of this preliminary study suggest that the use of engineered bionanocages also offers unprecedented opportunities for selective targeted chemotherapy of solid tumors in veterinary medicine.

Case Report of a Mare Diagnosed with a Metastatic Mammary Carcinoma after the Excision of a Recurrent Intraocular Neuroepithelial Tumor.

A 24-year-old Irish Cob mare was presented with a peripheral iris mass, which was surgically resected and diagnosed as an undifferentiated neuroepithelial tumor. A few months later, a relapse occurred with histological features characterized by a more solid appearance and squamous differentiation. Subsequently, the mare was presented with rapidly spreading multiple subcutaneous masses and, at the onset of neurological signs, was humanely euthanized and subjected to a complete post mortem examination. The necropsy confirmed the presence of numerous widespread masses in the subcutaneous tissue, several internal organs, and mammary gland. Histological and immunohistochemical (IHC) examinations were performed on all masses, allowing the diagnosis of mammary carcinoma with several visceral and subcutaneous metastases. Considering the post mortem findings, the second intraocular mass was submitted to histological and IHC re-evaluation to differentiate it from an intraocular metastasis of the mammary carcinoma. The results of the histological and IHC analyses confirmed the diagnosis of neuroepithelial tumor relapse. This is the first case of a metastatic mammary carcinoma concurrent with a recurrent intraocular neuroepithelial tumor in a mare. This case was a challenge for both clinicians and pathologists involved and highlighted the importance of post mortem and IHC evaluations.

A Statistical Analysis of Risk Factors and Biological Behavior in Canine Mammary Tumors: A Multicenter Study.

Canine mammary tumors (CMTs) represent a serious issue in worldwide veterinary practice and several risk factors are variably implicated in the biology of CMTs. The present study examines the relationship between risk factors and histological diagnosis of a large CMT dataset from three academic institutions by classical statistical analysis and supervised machine learning methods. Epidemiological, clinical, and histopathological data of 1866 CMTs were included. Dogs with malignant tumors were significantly older than dogs with benign tumors (9.6 versus 8.7 years, P < 0.001). Malignant tumors were significantly larger than benign counterparts (2.69 versus 1.7 cm, P < 0.001). Interestingly, 18% of malignant tumors were smaller than 1 cm in diameter, providing compelling evidence that the size of the tumor should be reconsidered during the assessment of the TNM-WHO clinical staging. The application of the logistic regression and the machine learning model identified the age and the tumor's size as the best predictors with an overall diagnostic accuracy of 0.63, suggesting that these risk factors are sufficient but not exhaustive indicators of the malignancy of CMTs. This multicenter study increases the general knowledge of the main epidemiologica-clinical risk factors involved in the onset of CMTs and paves the way for further investigations of these factors in association with CMTs and in the application of machine learning technology.

Wnt/ß-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats.

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/ß-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for ß-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and ß-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, ß-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active ß-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of ß-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of ß-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/ß-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/ß-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

Biphasic Feline Mammary Carcinomas Including Carcinoma and Malignant Myoepithelioma.

Feline mammary tumors are usually malignant and aggressive carcinomas. Most cases are simple monophasic carcinomas (1 epithelial population), and additional phenotyping is usually not needed. In this study, we describe 10 malignant mammary tumors from 9 female cats that had unusual histomorphology: they appeared biphasic, with 2 distinct cell populations. Initially, they were morphologically diagnosed as either carcinosarcoma (1/10) or malignant pleomorphic tumor (9/10) of the mammary gland, as the latter did not match any previously described histological subtype. Immunohistochemistry (IHC) was performed for pancytokeratin, cytokeratins 8 and 18, cytokeratin 14, cytokeratins 5 and 6, vimentin, p63, calponin, alpha-smooth muscle actin, Ki-67, ERBB2, estrogen receptor alpha, and progesterone receptor. In 7 of 10 cases, the biphasic nature was confirmed and, on the basis of the IHC results, they were classified as carcinoma and malignant myoepithelioma (4/10), ductal carcinoma (1/10), and carcinosarcoma (2/10). The other 3 of 10 cases were monophasic based on IHC. In the cases of carcinoma and malignant myoepithelioma, the malignant myoepithelial cells were 100% positive for vimentin (4/4) and variably positive for p63, calponin, and cytokeratins (4/4). These findings show that, although rare, biphasic mammary carcinomas do occur in cats. In dogs and humans, tumors composed of malignant epithelial and myoepithelial cells have a less aggressive behavior than certain simple carcinomas, and therefore, their identification might also be clinically significant in the cat.

Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells.

Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers.

Tumours in European Shorthair cats: a retrospective study of 680 cases.

OBJECTIVES: Naturally occurring tumours in domestic cats are less common than in dogs and represent the leading cause of death among older animals. The main objective of this study was to analyse a large data set of histologically diagnosed tumours to highlight the most common World Health Organization (WHO) tumour histotypes, the effect of age and sex, and the International Classification of Diseases for Oncology (ICD-O) topographical site predilections of feline breed-specific tumours. METHODS: A total of 680 feline tumours diagnosed in European Shorthair cats by three veterinary diagnostic laboratories located in central Italy from 2013 to 2019 were collected. Data on age, sex and topography of lesions were recorded. Samples were morphologically and topographically coded using the WHO and the ICD-O-3 classification system. RESULTS: Skin and soft tissue neoplasms comprised 55.9% of all tumours, followed by mammary gland (11%), alimentary tract (7.9%), oral cavity and tongue (7.3%), nasal cavity and middle ear (6%), lymph node (3.1%), bone (1.8%) and liver/intrahepatic bile duct (1.3%) tumours. Squamous cell carcinoma (SCC), sarcoma, lymphoma and basal cell tumours were the most diagnosed neoplasms. Malignant tumours were 82.9% of the total and the topographical sites mainly involved were skin (C44), connective/subcutaneous/other soft tissues (C49), mammary gland (C50), small intestine (C17), nasal cavity and middle ear (C30), and gum (C03). CONCLUSIONS AND RELEVANCE: This study aimed to provide an in-depth evaluation of spontaneous feline tumours in the European Shorthair cat breed. Results identify SCC as the most commonly represented skin neoplasm. It is likely that the analysed feline population, living in southern latitudes, was more subject to prolonged exposure to ultraviolet light, explaining the discrepancy with previous studies in which SCC was less represented.

Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma.

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non-UV induced melanomas. Twenty-eight cases of COM were retrieved from paraffin-blocks archives. A total of 4 µm thick sections were immunostained with an antibody against human Cyclin D1 and Ki-67. Cyclin D1 and Ki-67 expressions were scored through two counting methods. DNA was extracted from 20 µm thick sections of formalin-fixed paraffin-embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki-67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki-67 (r = 0.56; P = .003). The correlation found between Ki-67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single-nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.