Establishment of a Patient-Derived Xenograft of Canine Enteropathy-Associated T-Cell Lymphoma, Large Cell Type.

The pathogenesis of canine T-cell lymphoma remains incompletely understood, partly because there are no well-established in-vivo models to study these malignancies. For this study, we generated a patient-derived tumour xenograft (PDTX) from a 10-year-old neutered male golden retriever dog with enteropathy-associated intestinal T-cell lymphoma, large cell type. One of two female, 15-week-old beige/nude/XID mice developed a visible tumour 7 weeks after sections of tumour material from the spleen were surgically implanted. The histological appearance, immunophenotype and clonal antigen receptor rearrangements of the tumour from the recipient mouse showed that it was derived from the primary canine tumour. Our results indicate that immunodeficient mice are receptive hosts to develop in-vivo PDTX models to study the pathogenesis and management of canine T-cell lymphomas.

Interactions between CXCR4 and CXCL12 promote cell migration and invasion of canine hemangiosarcoma.

The CXCR4/CXCL12 axis plays an important role in cell locomotion and metastasis in many cancers. In this study, we hypothesized that the CXCR4/CXCL12 axis promotes migration and invasion of canine hemangiosarcoma (HSA) cells. Transcriptomic analysis across 12 HSA cell lines and 58 HSA whole tumour tissues identified heterogeneous expression of CXCR4 and CXCL12, which was associated with cell movement. In vitro, CXCL12 promoted calcium mobilization, cell migration and invasion that were directly proportional to surface expression of CXCR4; furthermore, these responses proved sensitive to the CXCR4 antagonist, AMD3100, in HSA cell lines. These results indicate that CXCL12 potentiates migration and invasion of canine HSA cells through CXCR4 signalling. The direct relationship between these responses in HSA cells suggests that the CXCR4/CXCL12 axis contributes to HSA progression.

Ovarian large cell neuroendocrine carcinoma in the youngest woman.

Large cell neuroendocrine carcinoma (LCNC) of the ovary is a rare tumor in gynecologic oncologic field. An 18-year-old woman presented with abdominal distention and a pelvic mass measuring ten cm in diameter, who previously underwent laparoscopic ovarian cystectomy due to large borderline mucinous ovarian neoplasm 18 months prior. A debulking operation was optimally performed, which included total abdominal hysterectomy with bilateral salpingo-oophorectomy, bilateral pelvic lymph node dissection, bilateral paraaortic lymph node dissection, omentectomy, optimal debulking of gastrohepatic mass and subdiaphragmatic mass, and pelvic peritonectomy. Despite adjuvant chemotherapy with paclitaxel and carboplatin, the patient died of progressive disease seven months after surgery. The authors report the youngest case of LCNC of the ovary, that failed chemotherapy and had the previous history of the conservative surgical treatment due to mucinous borderline tumor.

Anti-adhesive film mimicking local recurrence during follow up after surgical treatment of gynecologic malignancy.

A 51-year-old woman received a laparoscopic surgical staging operation due to endometrial carcinoma. Adjuvant pelvic radiotherapy was performed when the endometrial carcinoma was staged at FIGO Stage IIIC1, adnexa metastasis. Three months completing adjuvant pelvic radiotherapy, a 2.5-cm vaginal stump mass was found by abdomino-pelvic computed tomography (AP-CT). To rule out local recurrence, diagnostic laparoscopic exploration was performed. The pathologic report revealed chronic inflammation due to the presence of a foreign body. To avoid unnecessary surgery during the follow-up of patients with gynecologic malignancies, anti-adhesive material should be avoided which can possibly cause a lesion mimicking local recurrence.

CD44+/CD24- Cancer Stem Cells Are Associated With Higher Grade of Canine Mammary Carcinomas.

The CD44+/CD24- phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24- phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)-negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24- phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.

Effects of Obesity and Obesity-Related Molecules on Canine Mammary Gland Tumors.

Obesity can affect the clinical course of a number of diseases, including breast cancer in women and mammary gland tumors in female dogs, via the secretion of various cytokines and hormones. The objective of this study was to examine the expression patterns of obesity-related molecules such as aromatase, leptin, and insulin-like growth factor 1 receptor (IGF-1 R) in canine mammary carcinomas (CMCs) on the basis of the body condition score (BCS). Comparative analyses of the expression of these molecules, together with prognostic factors for CMCs, including hormone receptors (HRs; estrogen and progesterone receptors), lymphatic invasion, central necrosis of the tumor, and histologic grade, were performed on 56 CMCs. The mean age of CMC onset was lower in the overweight or obese group (8.7 ± 1.9 years) than in the lean or ideal body weight group (10.4 ± 2.7 years). The proportion of poorly differentiated (grade III) tumors was significantly higher in the overweight or obese female dogs. Aromatase expression was significantly higher in the overweight or obese group and was correlated with the expression of HRs (P = .025). These findings suggest that overweight or obese status might affect the development and behavior of CMCs by tumor-adipocyte interactions and increased HR-related tumor growth.

Obesity, expression of adipocytokines, and macrophage infiltration in canine mammary tumors.

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (n = 28) and malignant (n = 70) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subject's body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted. The mean age of MC development was lower in overweight or obese dogs (9.0 ± 1.8 years) than in lean dogs or optimal bodyweight (10.2 ± 2.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.

Evaluation of clinicopathological characteristics and oestrogen receptor gene expression in oestrogen receptor-negative, progesterone receptor-positive canine mammary carcinomas.

The existence of the oestrogen receptor-negative (OR(-))/progesterone receptor-positive (PR(+)) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR(-)/PR(+) phenotype and compare their clinicopathological features with OR(+)/PR(+) tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR(-)/PR(+) and showed intermediate characteristics between those of OR(+)/PR(+) and OR(-)/PR(-) cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR(-)/PR(+) tumours (11.4 ± 16.34) was between that of the OR(-)/PR(-) (mean 4.7 ± 6.35) and OR(+)/PR(+) (mean 15.8 ± 11.95) (P = 0.033) tumours. Only three of the 36 OR(-)/PR(+) tumours completely lacked OR mRNA expression. The OR(-)/PR(+) tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR(-)/PR(+) tumours were associated with factors related to poor prognosis compared with OR(+)/PR(+) tumours, but OR(-)/PR(-) tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR(-)/PR(+) phenotype.

Analysis of a new histological and molecular-based classification of canine mammary neoplasia.

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

Infiltrating Foxp3+ regulatory T cells and histopathological features in canine classical and spermatocytic seminomas.

In humans, regulatory T (T reg) cells are known to play a critical role in both the regulation of immune homoeostasis and the progression of cancer. However, there is little information about the identification, characterization and the function of T reg cells in canine tumours. We identified T reg cells in 28 canine seminoma samples using a Forkhead box P3 (Foxp3) antibody and investigated the relationship between T reg cell infiltration and histopathological features of classical and spermatocytic seminomas (SE and SS, respectively). The Foxp3 protein showed nuclear immunostaining in infiltrating lymphocytes, and Foxp3+ cells were diffused or focally distributed in seminoma tissues. Foxp3+ cells were frequently present in the SS histotype, in seminomas that showed no evidence of tumour cell invasion into the vessels and in seminomas showing a diffuse growth pattern with three cell types. Neither the SE/SS histotype nor the histopathological features of the tumour correlated with Foxp3+ cell counts. These results indicate that Foxp3+ T reg cells may be associated with a less malignant histological phenotype or may not play a critical role in the immune response of canine seminomas. Moreover, Foxp3+ T reg cells may be associated with SS seminoma, but further studies, involving a larger number of samples, are required to better understand whether these cells play a critical role in the immune response in canine seminomas. This is the first report to demonstrate the characteristics of T reg cell infiltration in canine seminoma.

Identification of triple-negative and basal-like canine mammary carcinomas using four basal markers.

Molecular-based classification of canine mammary carcinomas (CMCs) has been a recent research focus. In human breast cancer, triple-negative and basal-like phenotypes are distinct molecular subgroups that are known for their poor prognosis, but these tumours are not yet well defined in the dog. The aim of this study was to determine whether CMCs include triple-negative and basal-like phenotypes by immunohistochemical assessment of expression of the oestrogen receptor (OR), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and four basal markers, cytokeratin (CK) 14, CK5/6, p63 and the epidermal growth factor receptor (EGFR). In this study of 241 CMCs, 45 triple-negative tumours (OR(-), PR(-) and HER2(-)) were identified and this phenotype was associated with an unfavourable prognosis. In these tumours, the expression of CK14, CK5/6 and EGFR was related to clinicopathological parameters, while the expression of p63 was not relevant. The majority of the triple-negative tumours were of the basal-like phenotype, given that 75.6% of them expressed more than two basal markers. However, three of the basal markers were not uniformly expressed; therefore, the proportion of the basal-like phenotype was altered on the basis of the selection of the markers. Although both triple-negative and basal-like phenotypes are distinct entities in CMC, further study is needed to differentiate one from the other.

P-glycoprotein expression in canine mammary gland tumours related with myoepithelial cells.

P-glycoprotein is influential in chemotherapy-resistance in numerous cancers and has been widely studied in human breast cancer research, but is less studied in canine mammary gland tumour (MGT). The study was to evaluate P-glycoprotein expression and its localisations related with prognostic factors with monoclonal antibody C219, by immunohistochemistry (IHC) of 68 cases of canine malignant (n=54) and benign (n=14) MGT. Additional immunofluorescence (IF) and reverse transcriptase-polymerase chain reaction (RT-PCR) were also performed. There was a novel finding that P-glycoprotein expression with C219 localised at two different cell types: epithelial and myoepithelial cells. Myoepithelial localised tumours were 5 benign (35.5%) and 21 malignant (63.6%), while epithelial localised tumours were 12 cases, all malignant (36.5%). Unlike conventional belief, semi-quantitative evaluation of IHC intensity scores of C219 expression in malignant MGT was related with favourable histopathological parameters.

Possible role of Snail expression as a prognostic factor in canine mammary neoplasia.

Epithelial cells adhere tightly to each other by cell-to-cell adhesion and through the basement membrane barrier to prohibit movement. In carcinomas, neoplastic epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype during the epithelial-mesenchymal transition (EMT) for invasion and metastasis. The aim of this study was to identify Snail expression and examine the role of Snail protein in canine mammary tumour progression. Immunohistochemical expression of Snail, E-cadherin, oestrogen receptor, human epidermal growth factor receptor-2, cytokeratin 14 and p63 was analyzed in 54 samples of canine mammary epithelial tumours (11 adenomas and 43 carcinomas). Expression of mRNA encoding Snail was evaluated in seven samples (one normal mammary gland, two adenomas and four carcinomas) by reverse transcriptase-polymerase chain reaction. Snail mRNA was detected in all samples. Snail expression correlated significantly with histological type, grade and lymphatic invasion. However, there was no association between Snail expression and molecular subtype and between Snail expression and that of E-cadherin. Snail, a hallmark of EMT, might play an important role in invasion and metastasis of canine mammary carcinomas.

Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma.

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 µm) was also significantly different from normal canine kidney (23.2 ± 3.4 µm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis.

Correlation of Foxp3 positive regulatory T cells with prognostic factors in canine mammary carcinomas.

Regulatory T cells (Treg) cells play a crucial role in tumor progression by suppressing anti-tumor immunity, but are not well-documented in veterinary oncology. To identify the characteristics of Treg cells in tumor microenvironments, the numbers of Treg cells were analyzed and compared with histological prognostic factors and molecular biomarkers in canine mammary carcinoma (MC) tissues (n=37). Abundant Treg cells were associated with high histological grade and lymphatic invasion. The numbers of Treg cells infiltrating intratumoral areas markedly increased in tumors with poor prognostic factors, such as high histological grade, lymphatic invasion, and necrosis. These findings suggest that Treg cells play a role in canine MC progression. Furthermore, Treg cell numbers in intratumoral compartments may provide a potential prognostic factor when assessing canine MCs, which may in turn lead to the development of new immunologic therapeutics.

Histopathological and immunohistochemical comparison of the brain of human patients with Alzheimer's disease and the brain of aged dogs with cognitive dysfunction.

Alzheimer's disease (AD) is the most common progressive form of dementia in aged people. Microscopical changes in the brains of AD patients include the formation of senile plaques (SPs), neurofibrillary tangles (NFTs) and granulovacuolar degeneration and the deposition of amyloid-beta (Aß). Aged dogs are known to suffer from cognitive dysfunction and this state is associated with deposition of Aß in the brain. The aim of the present study was to investigate tau phosphorylation of neurons and astrocytes in the brain of aged dogs with progressive cognitive impairment. Changes in the brain of aged dogs with cognitive dysfunction were compared with those in the brain of patients with AD of Braak stage V. Immunohistochemically, Aß deposition, phosphorylated tau Ser396 (p-tau Ser396) and ubiquitin were observed in the parietal cortex and hippocampus of aged dogs with cognitive dysfunction. Astrocytes with expression of p-tau Ser396 and neurons with co-localization of p-tau Ser396 and ubiquitin were observed. Expression of p-tau Ser396 and accumulation of ubiquitin were significantly increased in the parietal cortex and dorsal part of the hippocampus of the brain of aged dogs when compared with expression of these molecules in human AD.

CDX-2 and HER-3 expression in canine gastric and colorectal adenocarcinomas.

CDX-2 is used as a specific cell marker for human intestinal adenocarcinoma. In human studies, HER-3 overexpression predicts poor survival for patients with various cancers including gastric cancer. Gastrointestinal adenocarcinoma is less common in dogs than in man and the expression of immunological markers by the canine tumours has not yet been extensively studied. CDX-2 and HER-3 expression was determined in 18 canine gastrointestinal adenocarcinomas: 13 were of colorectal origin and five were of gastric origin. CDX-2 expression was predominantly observed in the nuclei of normal colonic epithelium and in neoplastic epithelium and neoplastic gastric epithelial cells that which had metastasized to the gastric lymph node. CDX-2 was expressed in 11 of 13 (84.6%) colorectal adenocarcinomas and in all five (100%) gastric adenocarcinomas. HER-3 was consistently expressed in the cytoplasm of neoplastic epithelial cells. HER-3 expression was detected in 12 of 13 (92.3%) colorectal and in all five (100%) gastric adenocarcinomas. CDX-2 and HER-3 may be useful markers for canine gastrointestinal adenocarcinoma.

Tryptase-positive mast cells correlate with angiogenesis in canine mammary carcinoma.

Angiogenesis plays a crucial role in tumour growth, invasion and metastasis. Mast cells (MCs) release angiogenic factors that promote endothelial proliferation and differentiation. Previous studies have suggested that MCs are involved in tumour angiogenesis due to the release of various pro-angiogenic factors. This study evaluated samples from 40 canine mammary carcinomas and eight healthy non-neoplastic canine mammary glands. Toluidine blue staining was performed to characterize the MCs. Immunohistochemical labelling was performed to detect the number of tryptase-positive MCs and microvessels. MCs accumulated in tumour tissue and were closely associated with blood or lymphatic vessels in the tumour microenvironment. Angiogenesis, as measured by microvessel density, increased in direct proportion to the number of MCs. The correlation coefficient was significantly higher for tryptase-positive MCs than for toluidine blue-stained MCs. These results suggest that MCs are involved in tumour angiogenesis, which in turn influences tumour growth, invasion and metastasis. In particular, MC tryptase may be influential in mediating this function of MCs.

Expression of HER-2 and nuclear localization of HER-3 protein in canine mammary tumors: histopathological and immunohistochemical study.

HER-2 and HER-3 are transmembrane receptor proteins that are considered to be important but poorly understood biomarkers in canine tumors. In this study, the expression and the localization of HER-2 and HER-3 were evaluated immunohistochemically in canine mammary tumors (n=64; 12 benign, 52 malignant). HER-2 overexpression was identified in 2/12 (16.7%) benign and in 18/51 (35.3%) malignant cases. HER-3 was expressed in a non-nuclear localization in 11/12 (91.7%) benign and 18/52 (34.6%) malignant tumors. In contrast, HER-3 was expressed in the nucleus of neoplastic cells in 0/12 (0%) benign and 22/52 (42.3%) malignant tumors. Nuclear HER-3 expression was higher in neoplastic epithelial cells compared to myoepithelial cells, and positively correlated with high histological grade and lymphatic vessel invasion. These results suggest that nuclear HER-3 expression is significantly associated with tumor progression and metastasis and may serve as a useful prognostic biomarker in canine malignant mammary tumors.

Effects of a priming dose of fentanyl during anaesthesia on the incidence and severity of fentanyl-induced cough in current, former and non-smokers.

Fentanyl is commonly used during anaesthesia and can cause fentanyl-induced cough (FIC). This study investigated whether a priming dose of fentanyl reduced FIC, and determined the factors associated with increased risk of FIC. Subjects undergoing elective surgery under general anaesthesia (n = 800) were randomized into four groups: group 1 received 2 µg/kg fentanyl bolus; groups 2, 3 and 4 received a priming dose of fentanyl 0.5 µg/kg followed by 1.5 µg/kg after 1, 2 or 3 min, respectively. The incidence of FIC was 17.0%, 10.0%, 12.5% and 11.5% for groups 1, 2, 3 and 4, respectively, with no significant between-group differences in FIC incidence or severity. The mean FIC onset time was 22 s. Former smokers were 2.91 times more likely than current smokers to experience cough. A fentanyl priming dose did not reduce the incidence and severity of FIC. Former smokers were hyper-reactive to fentanyl compared with current smokers.