Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

Malignant peripheral nerve sheath tumor originating from the adrenal gland in a dog.

A large abdominal mass was found in a dog. Histopathologically, the surface of the mass was covered with compressed adrenal gland tissue. The neoplastic cells showed typical features of malignant peripheral nerve sheath tumor (MPNST), including Antoni type A and type B pattern, and nuclear palisading. Immunohistochemically, these cells were positive for S100 protein, nerve growth factor receptor, nestin and claudin-1. The dog was euthanized because of the developing multiple metastatic lesions. The metastatic cells showed quite similar histopathological and immunohistochemical features as those in the original tumor. Although MPNST can develop at many body sites, this is the first report of MPNST originating from the adrenal gland in a dog.

Coagulase-negative staphylococcal bacteraemia in cancer patients. Time to positive culture can distinguish bacteraemia from contamination.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most common contaminants of blood cultures, however, we sometimes have difficulties in determining their clinical significance. It is still controversial that there is a significant difference between the contamination group and the true bacteraemia group in the time to positivity (TTP) of blood cultures. We validated the relationship between a TTP and the presence of CoNS bacteraemia in cancer patients by using an objective, non-judgmental definition for CoNS contamination. METHODS: We retrospectively reviewed 175 sets of blood cultures drawn from 95 patients that yielded CoNS from October 2011 to March 2013. We considered as contamination if an isolate of CoNS was identified in one out of multiple sets of blood cultures. We investigated the TTP, the threshold values and corresponding likelihood ratios to distinguish CoNS bacteraemia from contamination. RESULTS: The median TTP in CoNS bacteraemia group was significantly shorter than that in contamination group (14 h 45 min and 20 h 31 min, respectively, p = .0157). A TTP of ≤16 h had a specificity of 83% for predicting CoNS bacteraemia, and that of >20 h had a sensitivity of 86% for predicting CoNS contamination. CONCLUSIONS: We validated that the median TTP in CoNS bacteraemia group was significantly shorter than that in their contamination group, and that a TTP of ≤16 h was associated with CoNS bacteraemia, while that of >20 h was associated with CoNS contamination, if evaluated with an objective, non-judgmental definition for CoNS contamination.

Flow cytometric analysis of peripheral blood and tumor-infiltrating regulatory T cells in dogs with oral malignant melanoma.

It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma.

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.

Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors.

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0].