PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

CanISO: a database of genomic and transcriptomic variations in domestic dog (Canis lupus familiaris).

BACKGROUND: The domestic dog, Canis lupus familiaris, is a companion animal for humans as well as an animal model in cancer research due to similar spontaneous occurrence of cancers as humans. Despite the social and biological importance of dogs, the catalogue of genomic variations and transcripts for dogs is relatively incomplete. RESULTS: We developed CanISO, a new database to hold a large collection of transcriptome profiles and genomic variations for domestic dogs. CanISO provides 87,692 novel transcript isoforms and 60,992 known isoforms from whole transcriptome sequencing of canine tumors (N = 157) and their matched normal tissues (N = 64). CanISO also provides genomic variation information for 210,444 unique germline single nucleotide polymorphisms (SNPs) from the whole exome sequencing of 183 dogs, with a query system that searches gene- and transcript-level information as well as covered SNPs. Transcriptome profiles can be compared with corresponding human transcript isoforms at a tissue level, or between sample groups to identify tumor-specific gene expression and alternative splicing patterns. CONCLUSIONS: CanISO is expected to increase understanding of the dog genome and transcriptome, as well as its functional associations with humans, such as shared/distinct mechanisms of cancer. CanISO is publicly available at https://www.kobic.re.kr/caniso/ .

1H NMR based urinary metabolites profiling dataset of canine mammary tumors.

The identification of efficient and sensitive biomarkers for non-invasive tests is one of the major challenges in cancer diagnosis. To address this challenge, metabolomics is widely applied for identifying biomarkers that detect abnormal changes in cancer patients. Canine mammary tumors exhibit physiological characteristics identical to those in human breast cancer and serve as a useful animal model to conduct breast cancer research. Here, we aimed to provide a reliable large-scale metabolite dataset collected from dogs with mammary tumors, using proton nuclear magnetic resonance spectroscopy. We identified 55 metabolites in urine samples from 20 benign, 87 malignant, and 49 healthy control subjects. This dataset provides details of mammary tumor-specific metabolites in dogs and insights into cancer-specific metabolic alterations that share similar molecular characteristics.

Canine mammary cancer in overweight or obese female dogs is associated with intratumoral microvessel density and macrophage counts.

Obesity is a major health condition owing to its effects on chronic diseases and cancers in humans, but little information is available regarding the role of obesity in canine mammary cancer (CMC). In the present study, we performed immunohistochemistry to investigate the effect of obesity on CMC by analyzing the number of tumor-associated macrophages, intratumoral microvessel density (iMVD), and the expression of prognostic factors including epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX-2), and Ki67 in CMC specimens. These data were compared in CMC specimens from lean or ideal body weight (Group 1) versus overweight or obese (Group 2) female dogs (n = 60 for each group). Associations between obesity status and histologic characteristics, such as histologic subtype, grading, and lymphatic invasion, were also investigated. Compared with lean or ideal body weight dogs, TAM (tumor-associated macrophage) counts (P < .005) and iMVD (P < .001) were significantly higher in overweight or obese dogs. CMC specimens of dogs in the overweight or obese group also showed higher histologic grade (P < .001). In addition, although no association was found between obesity status and either COX-2 or EGFR expression, Ki67 expression was greater in CMC specimens of overweight or obese dogs (P < .005). The results of this study suggest that obesity may influence CMC development and progression, being associated with higher histologic grade, greater infiltration of TAMs, and increased tumor angiogenesis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) loss in canine mammary carcinoma.

Escaping apoptosis is a hallmark of cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a central molecule that regulates the extrinsic apoptotic pathway, has been widely investigated in human oncology; however, investigations focusing on the endogenous expression of TRAIL in canine tumours are lacking. Therefore, we aimed to examine the expression of endogenous TRAIL in canine mammary tumours and analysed its correlation with downstream molecules Fas-associated protein with death domain (FADD) and caspase-3, and to the apoptotic index. A total of 147 samples, classified as normal mammary gland (n = 9), mammary adenoma (n = 30), low-grade carcinoma (n = 42) and high-grade carcinoma (n = 66), were included in the immunohistochemical analyses, and 43 samples with sufficient levels of RNA were analysed via RNA in situ hybridization and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. In immunohistochemistry, TRAIL protein expression was significantly decreased in high-grade carcinoma compared to those in normal mammary gland and adenoma, with similar downregulation of TRAIL mRNA expression. Also, FADD and caspase-3 expression positively correlated with TRAIL expression. However, the apoptotic index was paradoxically elevated in high-grade tumours. Overall, these results suggest that the loss of TRAIL accompanied by dysregulation of TRAIL-induced extrinsic apoptotic pathway molecules could affect malignant progression of canine mammary tumours.

Evaluation of circulating IGF-I and IGFBP-3 as biomarkers for tumors in dogs.

BACKGROUND: Serum-based parameters are considered non-invasive biomarkers for cancer detection. In human studies, insulin-like growth factor-I and II (IGF-I and IGF-II) and insulin-like growth factor binding protein-3 (IGFBP-3) are useful as diagnostic or prognostic markers and potential therapeutic targets. OBJECTIVES: This study examined the diagnostic utility of circulating IGF-I, IGF-II, and IGFBP-3 levels in healthy dogs and dogs with tumors. METHODS: The serum concentrations of these biomarkers in 86 dogs with tumors were compared with those in 30 healthy dogs using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA results showed no difference between healthy dogs and dogs with tumors in the serum IGF-II concentrations. On the other hand, there was a significant difference in the circulating IGF-I and IGFBP-3 levels between healthy dogs and dogs with tumors. The concentrations of serum IGF-I (median [interquartile range], 103.4 [59.5-175] ng/mL) in dogs with epithelial tumors were higher than those (58.4 ng/mL [43.5-79.9]) in healthy dogs. Thus, the concentrations of serum IGFBP-3 (43.4 ng/mL [33.2-57.2]) in dogs with malignant mesenchymal tumors were lower than those (60.8 ng/mL [47.6-70.5]) in healthy dogs. CONCLUSIONS: The serum IGF-I and IGFBP-3 levels can be used as diagnostic biomarkers in dogs with tumors.

Arginase-1 and P-glycoprotein are downregulated in canine hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the most common primary hepatic malignancy in humans and dogs. Several differentially expressed molecules have been studied and reported in human hepatocellular carcinoma and non-neoplastic liver lesions. However, studies on the features of canine hepatocellular carcinoma are limited, especially related to the differential characteristics of neoplastic and non-neoplastic lesions. OBJECTIVES: The study's objective was 1) to examine and evaluate the expression of arginase-1, P-glycoprotein, and cytokeratin 19 in canine liver tissues and 2) to investigate the differential features of hepatocellular carcinomas, liver tissue with non-neoplastic lesions, and paracancerous liver tissues in dogs. METHODS: The expression levels of three markers underwent immunohistochemical analysis in 40 non-neoplastic liver tissues, 32 hepatocellular carcinoma tissues, and 11 paracancerous liver tissues. Scoring of each marker was performed semi-quantitatively. RESULTS: Arginase-1 and P-glycoprotein were significantly downregulated in hepatocellular carcinoma, compared with hepatic tissues with non-neoplastic diseases (p < 0.001). Expression levels of arginase-1 and P-glycoprotein were also significantly lower in hepatocellular carcinoma than in paracancerous liver tissues (arginase-1, p = 0.0195; P-glycoprotein, p = 0.047). Few cytokeratin 19-positive hepatocytes were detected and only in one hepatocellular carcinoma and one cirrhotic liver sample. CONCLUSIONS: The results of this study suggest that downregulation of arginase-1 and P-glycoprotein is a feature of canine hepatocellular carcinoma; thus, those markers are potential candidates for use in differentiating hepatocellular carcinomas from non-neoplastic liver lesions in dogs.

Dysregulation of PI3K/Akt/PTEN Pathway in Canine Mammary Tumor.

The PI3K/Akt/PTEN axis is one of the most important signaling pathways in tumorigenesis. Recently, mutation of PIK3CA has been highlighted due to the similarities of mutational hotspots in both dogs and humans. PIK3CA H1047R (c.3140A > G) has been discovered as the most common mutational hot spot in canine mammary tumor in recent studies, while the feature of PIK3CA-mutated canine mammary tumor is obscure. METHODS: A total of 83 mammary samples classified as normal (n = 13), adenoma (n = 25), low-grade carcinoma (n = 21), and high-grade carcinoma (n = 24) were included in this study. Genomic DNA from each sample was extracted, amplified by conventional PCR, and analyzed through Sanger sequencing. Analysis for the expression of PIK3CA, Akt, p-Akt, and PTEN was performed by immunohistochemistry, and of Akt2 by RNA in situ hybridization. RESULTS: PIK3CA H1047R mutation was detected in 14.3% (10/70) of tumor samples. Dysregulation of p-Akt, Akt2, and PTEN was observed in mammary tumor samples, but only PTEN dysregulation was associated with PIK3CA H1047R mutation. CONCLUSIONS: The present study showed that dysregulation of components in the PI3K/Akt/PTEN pathway is a feature of canine mammary tumors, but this dysregulation is not directly correlated to the PIK3CA H1047R mutation except for PTEN expression.

Impact of Histological Subtype on Survival in Canine Mammary Carcinomas: a Retrospective Analysis of 155 Cases.

Canine mammary carcinoma (CMC) is the most common type of neoplasm in intact female dogs. While a previous study in Western countries validated the 2011 classification as an independent prognostic indicator in CMC, its role in CMC prognostication in Asian countries such as Korea remains unclear. In the present study, we estimate the survival rates in CMC types defined by the 2011 classification, elucidate the prognostic significance of the histological subtype and grade and that of the lymphatic invasion status in CMC, and validate the 2011 classification as an independent prognostic indicator in a large cohort of CMCs (excluding cases of multicentric CMCs). A total of 155 CMC cases retrieved from archived formalin-fixed, paraffin-embedded tissues, along with 2-year follow-up data, were retrospectively analysed. A significant association was found between the histological subtype of the 2011 classification and the tumour-specific survival. Carcinosarcoma, adenosquamous carcinoma and anaplastic carcinoma subtypes were associated with the poorest prognosis. Dogs with comedocarcinoma and solid carcinoma followed a disease course that was more aggressive than that observed in dogs with a carcinoma arising in a benign mixed tumour. Moreover, age, histological grade and lymphatic invasion status significantly correlated with tumour-specific survival in univariate analysis. In multivariate analysis, histological subtype, age and lymphatic invasion status remained independent prognostic factors for CMC.

EGFR Overexpression and Sequence Analysis of KRAS, BRAF, and EGFR Mutation Hot Spots in Canine Intestinal Adenocarcinoma.

Epidermal growth factor receptor (EGFR) is overexpressed in many human colorectal cancers and anti-EGFR agents are employed as immunotherapies. However, KRAS, EGFR, and BRAF gene mutations can influence the activity of the anti-EGFR agents. We evaluated EGFR expression at protein and mRNA levels in canine intestinal adenocarcinomas using immunohistochemistry (IHC) and RNA in situ hybridization (RNA-ISH). We also investigated the mutation status of EGFR, KRAS, and BRAF to aid the development of anti-EGFR agents for canine intestinal adenocarcinoma. EGFR expression was highest in adenocarcinoma, followed by intramucosal neoplasia (adenoma and in situ carcinoma), and nonneoplastic canine intestinal tissue, at both protein (P = .000) and mRNA (P = .005) levels. The EGFR, KRAS, and BRAF genes showed wild-type sequences at the mutation hot spots in all 13 specimens. Thus, EGFR might serve as a promising diagnostic marker in canine intestinal adenocarcinoma, and further studies would be needed to develop EGFR-targeted anticancer therapies.

CDX-2 Protein and mRNA Expression in Canine Intestinal Adenocarcinoma.

Caudal-related homeobox transcription factor 2 (CDX-2) is a specific cell marker employed in the diagnosis of human colorectal cancer. Reduced CDX-2 expression is associated with several indicators of poor prognosis in human colorectal cancer. In the present study, CDX-2 protein levels were evaluated and patterns of CDX-2 mRNA accumulation are described for the first time in canine intestinal adenocarcinoma (CIA). Canine intestinal epithelial biopsies from 21 CIAs and 14 non-neoplastic control tissues were retrospectively evaluated for CDX-2 expression and CDX-2 mRNA levels by immunohistochemistry and RNA in-situ hybridization (RNA-ISH), respectively. The mean percentage or intensity of expression was decreased in the CIA group (P = 0.000). RNA-ISH demonstrated a significant correlation between the decrease in CDX-2 mRNA levels and CDX-2 protein expression (P = 0.000). CDX-2 downregulation, in terms of protein as well as mRNA levels, may serve as a diagnostic marker in CIA.

Ovarian adenocarcinoma with metastases in a white rhinoceros.

A 36-y-old white rhinoceros (Ceratotherium simum) was presented with respiratory distress, sanguineous vaginal exudate, and anorexia. The clinical signs progressed over 40 d, and the rhinoceros died. Autopsy revealed significant ascites; a unilateral, 12.5-cm diameter, polypoid mass in the left ovary; a white, firm transmural mass in the right uterine horn; a white, friable mass in the lung; and white-to-tan, friable small nodules in the diaphragm. Histologic examination revealed similar neoplastic cells in the masses in all 4 locations, composed predominantly of epithelial cells proliferating in a tubulopapillary pattern with significant nuclear atypia and numerous atypical mitotic figures (18-42 per 2.37 mm2). Immunohistochemistry for CK7 (cytokeratin 7) and CK20 (cytokeratin 20) suggest that the ovarian, pulmonary, and diaphragmatic lesions were of ovarian origin and that the ovary was the primary tumor site.

In situ c-KIT mRNA quantification of canine cutaneous mast cell tumours and its relationship to prognostic factors.

Cutaneous mast cell tumours (MCTs) are the most frequent malignant skin tumours in dogs. Mutations in the c-KIT proto-oncogene are correlated with the pathogenesis and aggressiveness of MCTs. To date, studies have focused on c-KIT mutations and KIT protein localization, with a general lack of mRNA-level analyses. In this study, c-KIT mRNA expression was investigated in canine MCTs by RNA in situ hybridization (RNA-ISH). Furthermore, we evaluated associations between c-KIT mRNA expression and the histological grade, KIT immunohistochemical staining pattern and other clinicopathological parameters. c-KIT mRNA expression was observed in all MCT samples, appearing as clusters of dots in the cytoplasm of neoplastic cells. A significant correlation was detected between c-KIT mRNA expression (quantified according to the H-score and the percentage of positive cells) and the histological grade (determined using two-and three-tier grading systems; P < .05). We also found a significant positive correlation (all P < .05) between c-KIT mRNA expression and the proliferation indices (mitotic index, Ki-67, and Ag67). However, no significant associations with c-KIT expression from RNA-ISH were found with respect to different KIT staining patterns. Overall, these results demonstrate that c-KIT mRNA expression might be an additional tool for measuring the c-KIT status in canine cutaneous MCTs and could serve as a potential prognostic factor. Further studies should evaluate the prognostic significance of c-KIT mRNA expression in a large and uniform cohort of canine MCTs.

Cross-species oncogenic signatures of breast cancer in canine mammary tumors.

Genomic and precision medicine research has afforded notable advances in human cancer treatment, yet applicability to other species remains uncertain. Through whole-exome and transcriptome analyses of 191 spontaneous canine mammary tumors (CMTs) that exhibit the archetypal features of human breast cancers, we found a striking resemblance of genomic characteristics including frequent PIK3CA mutations (43.1%), aberrations of the PI3K-Akt pathway (61.7%), and key genes involved in cancer initiation and progression. We also identified three gene expression-based CMT subtypes, one of which segregated with basal-like human breast cancer subtypes with activated epithelial-to-mesenchymal transition, low claudin expression, and unfavorable disease prognosis. A relative lack of ERBB2 amplification and Her2-enrichment subtype in CMT denoted species-specific molecular mechanisms. Taken together, our results elucidate cross-species oncogenic signatures for a better understanding of universal and context-dependent mechanisms in breast cancer development and provide a basis for precision diagnostics and therapeutics for domestic dogs.

Expression of platelet-derived growth factor receptor-α/ß, vascular endothelial growth factor receptor-2, c-Abl, and c-Kit in canine granulomatous meningoencephalitis and necrotizing encephalitis.

BACKGROUND: Given the active research on targeted therapy using tyrosine kinase (TK) inhibitors (TKIs) in the field of oncology, further studies have recently been conducted to evaluate their use in autoimmune disorders. Based on immunological investigations, previous studies have suggested that granulomatous meningoencephalomyelitis (GME) and necrotizing encephalomyelitis (NE) are similar to multiple sclerosis (MS), which is a human autoimmune demyelinating central nervous system disease. OBJECTIVES: Considering this perspective, we hypothesized that canine GME and NE have significant expression of one or more TKs, which are associated with human MS pathogenesis. METHODS: To determine the possible use of conventional multi-targeted TKIs as a treatment for canine GME and NE, we characterized the immunohistochemical expression of platelet-derived growth factor receptor (PDGFR)-α, PDGFR-ß, vascular endothelial growth factor receptor (VEGFR)-2, c-Abl and c-Kit in GME and NE samples. RESULTS: Histological samples from four dogs with GME and three with NE were retrieved. All samples stained positive for PDGFR-ß (7/7 [100%]). PDGFR-α and c-Kit were expressed in 3/7 (42.8%) samples each. c-Abl was identified in 2/7 (28.5%) samples; no sample showed VEGFR-2 (0%) expression. Co-expression of TKs was identified in 6/7 (85.7%) dogs. CONCLUSIONS: All samples were positive for at least one or more of PDGFR-α, PDGFR-ß, c-Kit and c-Abl, which are known as the target TKs of conventional multi-targeted TKIs. Their presence does suggest that these TKs may play a role in the pathogenesis of GME and NE. Therefore, multi-targeted TKIs may provide benefits in the treatment of canine GME and NE by suppressing the activity of these TKs.

Quantitative analysis of HER2 mRNA expression by RNA in situ hybridization in canine mammary gland tumors: Comparison with immunohistochemistry analysis.

Spontaneously occurring canine mammary gland tumors share many features with human breast cancer, including biological behavior and histologic features. Compared to transgenic murine model, canine models have advantages including naturally occurring models of human diseases and cancer. In humans, breast cancer is divided into molecular subtypes based on ER, PR, and HER2 expression. In contrast with humans, few studies have evaluated these subtypes in canine mammary gland tumors, including expression of HER2. HER2 expression in canine mammary tissues has been further complicated by controversy regarding the antibody's specificity. This study aimed to investigate c-erbB2 mRNA expression in retrospective formalin-fixed paraffin embedded samples, using RNA in situ hybridization with a novel quantitative assay and to compare this method with immunohistochemistry. Using 48 canine mammary tumor samples and 14 non-neoplastic canine mammary tissues, RNA in situ hybridization was performed with RNAscope® using a canine-specific target gene probe (ERBB2), and quantitative measurement was performed using the housekeeping gene (POLR2A) to calculate the target gene/housekeeping gene ratio. The ratio of ERBB2/POLR2A was quantified using open-source image analysis programs and compared with the immunohistochemistry results. A significant correlation was observed between the HER2 immunohistochemistry score and ERBB2/POLR2A RNA in situ hybridization (P < 0.001). When the HER2 immunohistochemistry score was 3+, significantly higher expression of HER2 mRNA was observed by RNA in situ hybridization. Interestingly, HER2 mRNA was also observed in non-neoplastic mammary tissues by RNA in situ hybridization. This assay potentially facilitates the reliable quantification of mRNA expression levels in retrospective formalin-fixed paraffin-embedded samples. Further studies are required to elucidate the role of HER2 in canine mammary gland tumors and to implement clinical trials in dogs.

Renal interstitial cell tumor in a dog: clinicopathologic, imaging, and histologic features.

Renal interstitial cell tumors are benign tumors of renomedullary origin; however, malignant features have not been reported in dogs, to our knowledge. A 17-y-old spayed female Maltese dog was presented to a local animal hospital with a mass in the right abdomen. Clinicopathologic findings prior to surgery revealed renal insufficiency and anemia. Imaging revealed that the right kidney was enlarged by an amorphous mass with opaque areas, indicative of mineralization. Upon histologic examination, the mass was comprised of malignant mesenchymal cells that produced mucinous matrix. The tumor cells were positive for vimentin and COX-2, but negative for pancytokeratin; the matrix stained positively with alcian blue. Therefore, the mass was diagnosed as a renal interstitial cell tumor, with malignant features. COX-2 may be useful in the diagnosis of canine renal interstitial cell tumors, similar to its diagnostic role in humans.

Whole-exome and whole-transcriptome sequencing of canine mammary gland tumors.

Studies of naturally occurring cancers in dogs, which share many genetic and environmental factors with humans, provide valuable information as a comparative model for studying the mechanisms of human cancer pathogenesis. While individual and small-scale studies of canine cancers are underway, more generalized multi-omics studies have not been attempted due to the lack of large-scale and well-controlled genomic data. Here, we produced reliable whole-exome and whole-transcriptome sequencing data of 197 canine mammary cancers and their matched controls, annotated with rich clinical and biological features. Our dataset provides useful reference points for comparative analysis with human cancers and for developing novel diagnostic and therapeutic technologies for cancers in pet dogs.

Classification, bacteriological findings, and analysis of sex hormone receptors and cytokine expression in mammary lesions of abattoir sows.

Mammary lesions in sows can prevent suckling piglets from consuming colostrum that provides fundamental nutrients and protective immunity. Although mammary gross lesions are frequently found in sows at farms or slaughterhouses, with the exception of mastitis, they have received little research attention. In this study, we investigated mammary lesions observed in South Korean sows between 2015 and 2016. Mammary tissue samples of 82 sows showing gross lesions during meat inspection were histologically classified and immunohistochemical analysis was conducted to assess the expression of estrogen receptor (ER)-α, ER-ß, and progesterone receptor (PR) for mammary hyperplastic lesions as well as that of cluster of differentiation (CD) 3, CD79a, interleukin (IL)-1α, IL-1ß, IL-6, and IL-8 for mastitis. Furthermore, 20 swab samples were cultured, and the isolated bacteria were identified using polymerase chain reactions for 16S ribosomal RNA genes. The lesions were classified as hyperplasia, mastitis, or hyperplasia with mastitis. Immunohistochemistry results revealed that there was neither expression of ER-α nor of ER-ß, but all examined hyperplastic samples expressed PR. In addition, there was a significant correlation between CD3 and IL-1ß expressions, as well as between IL-1ß and IL-6 expressions. Regarding the identity of the isolated bacteria, Pseudomonas spp. were most frequently detected. The results of this study have revealed the incidence and characteristics of porcine mammary lesions.

Increased p63 Expression in Canine Perianal Gland Tumours.

INTRODUCTION: p63 is a homologous molecule of p53 and was recently identified as playing important roles in several key cellular processes, including epithelial development and proliferation. Since then, several studies have demonstrated altered p63 expression in various cancers of epithelial origin. Canine perianal gland tumour is one of the most common skin neoplasms in dogs; however, the molecular characteristics of this tumour remain poorly understood. The objective of the present study was to analyse and compare the expression of p63 in canine perianal gland adenomas and carcinomas. MATERIAL AND METHODS: Haematoxylin and eosin-stained slides were examined and immunohistochemistry was conducted for a total of 65 samples. Immunohistochemical data for p63 expressions were compared between groups using the Mann-Whitney U test. RESULTS: The p63 expression level was increased in perianal gland carcinomas compared to that in the adenoma samples (P < 0.0001). The percentage of cells expressing p63 was higher in perianal gland carcinomas than in adenomas, although the intensity of immunostaining did not differ significantly between the two groups. CONCLUSION: p63 is a candidate factor contributing to the malignant transformation and progression of canine perianal gland tumours.