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/ .

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.

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.

CD204-Expressing Tumor-Associated Macrophages Are Associated With Malignant, High-Grade, and Hormone Receptor-Negative Canine Mammary Gland Tumors.

Tumor-associated macrophages (TAMs) are an important component of leukocyte infiltration in tumors. TAMs can be classified into M1 and M2 phenotypes. In the present study, the expression of CD204, an M2-polarized macrophage receptor, was investigated by immunohistochemistry in the area surrounding TAMs in 101 cases of canine mammary gland tumor (CMT). We examined the relationship between M2-polarized TAMs and malignancy, histological subtype, histological grade, molecular subtype, hormone receptor (HR) status, and clinical obesity indices. The mean number of CD204-positive macrophages was significantly higher in malignant CMTs than in benign CMTs ( P = .000). The number of CD204-positive macrophages differed significantly between histological grades ( P = .000) and were significantly higher in grade III than in grades I and II. Moreover, the mean number of CD204-positive macrophages was significantly higher in HR-negative malignant CMTs than in HR-positive malignant CMTs ( P = .035) and in malignant CMTs with lymphatic invasion compared to malignant CMTs without lymphatic invasion ( P = .000). These findings suggest that CD204-positive macrophages might affect the development and behavior of CMTs and highlight the potential of CD204 as a prognostic factor.

Canine nail bed keratoacanthoma diagnosed by immunohistochemical analysis.

A 10-year-old, Shih Tzu dog was presented with an enlarged, curled 2nd nail in the left forelimb. Digital amputation was performed and the mass was diagnosed as a nail bed keratoacanthoma (infundibular keratinizing acanthoma) histopathologically. There was no recurrence postoperatively. This is the first case report of a canine nail bed keratoacanthoma diagnosed by histologic and immunohistochemical examination including Ki-67 and p53 expression.

Kératocanthome d'un lit d'ongle canin diagnostiqué à l'aide d'une analyse immunohistochimique. Un chien Shih Tzu âgé de 10 ans a été présenté avec un deuxième ongle recourbé et hypertrophié sur la jambe avant gauche. Une amputation du doigt a été réalisée et la masse a été diagnostiquée en histopathologie comme un kératocanthome de lit d'ongle (acanthome kératinisant infundibulaire). Il n'y a pas eu de récurrence après l'opération. Il s'agit du premier rapport de kératocanthome de lit d'ongle canin diagnostiqué à l'aide d'un examen histologique et immunohistochimique incluant l'expression Ki-67 et p53.(Traduit par Isabelle Vallières).

Staphylococcus pseudintermedius infection associated with nodular skin lesions and systemic inflammatory response syndrome in a dog.

A 10-year-old Pekingese dog with atopic dermatitis was referred due to pyrexia, multiple skin nodules, anorexia, and depression. The dog was diagnosed as having systemic inflammatory response syndrome (SIRS) induced by bacterial dermatitis. This case presents diagnosis and treatment of SIRS with staphylococcal skin infection in a dog that was immunosuppressed due to long-term use of corticosteroid.

Infection parStaphylococcus pseudintermediusassociée à des lésions cutanées nodulaires et à un syndrome de réaction inflammatoire systémique chez un chien. Un chien Pékinois âgé de 10 ans atteint de dermatite atopique a été référé en raison d'une pyrexie, de nodules cutanés multiples, d'anorexie et de dépression. Le chien a été diagnostiqué comme ayant un syndrome de réaction inflammatoire systémique (SRIS) induit par une dermatite bactérienne. Ce cas présente le diagnostic et le traitement du SRIS avec une infection aux staphylocoques cutanés chez un chien ayant une immunodépression causée par l'utilisation à long terme de corticostéroïdes.(Traduit par Isabelle Vallières).

Analysis of the Prevalence of Canine Splenic Mass Lesions in Republic of Korea via Histopathological Diagnosis with Immunohistochemistry.

The histopathological diagnosis of canine splenic mass lesions is crucial for prognostication. However, thus far, no study has been conducted on the histopathology of canine splenic mass lesions in Republic of Korea. Herein, the prevalence of splenic diseases was analyzed in 137 canine splenic mass lesions via histopathological diagnosis, and the microscopic pattern associated with each disorder was described. Immunohistochemistry was performed for CD31, CD3, PAX5, Iba1, and C-kit for a more accurate diagnosis of splenic tumors. The proportion of non-neoplastic disorders, including nodular hyperplasia (48.2%, n = 66) and hematoma (24.1%, n = 33), was 72.3%. Splenic tumors, including splenic hemangiosarcoma (10.2%, n = 14), splenic lymphoma (nodular and diffuse types, 8.0%, n = 11), splenic stromal sarcoma (7.3%, n = 10), myelolipoma (1.5%, n = 2), and mast cell tumors (0.7%, n = 1), accounted for 27.7% of cases. The results of this study will aid veterinary clinicians in communication with pet owners about prognoses, recommendations for splenectomy, and subsequent histopathological diagnoses. This study will facilitate further investigations with more detailed comparisons of splenic mass lesions between small- and large-breed dogs.

Primary central nervous system B-cell lymphoma in a young dog.

This report describes a primary central nervous system B-cell lymphoma in a 3-year-old intact female Maltese dog. Canine primary central nervous system lymphomas constitute about 4% of all intracranial primary neoplasms, but comprehensive histopathologic classifications have rarely been carried out. This is the first report of this disease in a young adult dog.

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.

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.

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.

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.

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.

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.

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.

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.

Canine classical seminoma: a specific malignant type with human classifications is highly correlated with tumor angiogenesis.

BACKGROUND: Human seminoma is classified as classical seminoma (SE) and spermatocytic seminoma (SS). Human SE is known to be more malignant and metastasizing more frequently than SS. Tumor angiogenesis is highly related with tumor progression and metastasis, with microvessel density (MVD) being an important parameter of metastatic potential. Canine seminoma is not yet well-established as SE or SS type including correlation with angiogenesis. We classified canine SE and SS, and then compared them to tumor associated vessels. METHODS: Twenty-three cases of canine seminomas (2 intratubular, 9 diffuse, and 12 intratubular/diffuse seminomas showing both intratubular and diffuse patterns) were classified as SE or SS by immunohistochemistry (IHC) using monoclonal antibody against PLAP and by PAS stain. The histopathological data were then compared to see if there was a correlation with SE or SS. Angiogenesis of seminomas were evaluated by immunohistochemical assay using polyclonal antibody against Von Willebrand factor (vWF) and by calculating the means of MVD, vessels area and perimeters using computerized image analysis. Statistical Package for Social Sciences (SPSS) program was used for various statistical analyses. RESULTS: The numbers of PLAP+/PAS+ canine SEs were 8/23 (34.8%) and PLAP-/PAS- SSs were 15/23 (61.2%). All SE cases (8/8, 100%) were intratubular/diffuse types. SS types included 2 intratubular (2/15, 13.3%), 9 diffuse (9/15, 60%), and 4 intratubular/diffuse (4/15, 26.7%) types. MVD and vascular parameters in SEs were significantly higher than in SSs, showing the highest value in the intratubular/diffuse type. Seminomas observed with neoplastic cells invasion of vessels presented higher perimeter and area values than seminomas without conformed neoplastic cells invasion. CONCLUSION: In this study, we demonstrated a positive relationship between canine SE and tumor angiogenesis. Furthermore, we also showed that a tumor cells invasion of vessels were a correlated vascular parameter. Although metastasis of canine seminomas has rarely been reported, our results support that canine SE could have high metastatic potential similar to the human counterpart. Further studies are required to clarify the relationship between canine SE and clinical data with metastatic factors.