Whole exome and transcriptome analysis revealed the activation of ERK and Akt signaling pathway in canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients.

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.

Lymphoma in Miniature Dachshunds: A retrospective multicenter study of 108 cases (2006-2018) in Japan.

BACKGROUND: Miniature Dachshunds (MD) are predisposed to lymphoma with disease onset of young age and long-term survival. OBJECTIVES: To compare clinical features and survival time of lymphoma in MD and non-MD. ANIMALS: One hundred and eight MDs with lymphoma and 149 non-MD breed dogs with lymphoma were included in the study. METHODS: This was a retrospective multicenter observational study. Lymphoma was classified based on signalment, histopathology/cytology, and anatomical site of the disease. For each type of lymphoma, median survival time was analyzed by Kaplan-Meier estimates and life table analysis. Prognostic factors for large-cell gastrointestinal lymphoma (LGIL) were analyzed using Cox regression. RESULTS: Gastrointestinal lymphomas were more common in MDs (53/108) compared to non-MDs (41/149). The multicentric lymphoma was most common in non-MD breed dogs (74/149) compared to MDs (33/108). The median age that dog developed lymphoma in MD and non-MD were both 10 years old; however, lymphomas were more frequently observed in younger dogs (<4 years) in MDs (20/108) compared to non-MDs (9/149; P = .002). Seventy percent were diagnosed with B-cell with median age of diagnosis was 3 (1-14) years. Mott cell differentiation was observed in 6 dogs. Age <4 years and B-cell phenotype were significant factors for longer survival time in MD with LGIL. CONCLUSIONS AND CLINICAL IMPORTANCE: Lymphomas in MDs involved gastrointestinal lesions at higher frequency compared to other dog breeds examined. B-cell lymphoma was more common in early-onset LGIL in MD and cases that involved Mott cell differentiation were observed. Awareness of this specific presentation of lymphoma in dogs will possibly affect the treatment decision process for the owners of MD with LGIL.

mRNA sequencing analysis and growth inhibitory effects of palbociclib on cell lines from canine histiocytic proliferative disorders.

Canine histiocytic proliferative disorders include aggressive and fatal diseases, such as histiocytic sarcoma (HS) and histiocytosis (SyH). The molecular mechanisms underlying cell proliferation need to be elucidated for the development of effective treatments. In the present study, mRNA expression levels were comprehensively analysed in cell lines derived from localized HS, disseminated HS, SyH and Langerhans cell histiocytosis (LCH) in dogs. Based on the results obtained, the growth inhibitory effects of palbociclib, a CDK4/6 inhibitor, were verified with the cell lines in vitro and in xenograft mouse model. Hierarchical clustering and principal component analysis plots of mRNA expression profiles divided the cell lines into three groups: a localized HS group, disseminated HS/SyH group, and LCH. The results of an ingenuity pathway analysis suggested that the MAPK signalling pathway was activated in the localized HS and LCH cell lines, and the PI3K signalling pathway in the disseminated and localized HS cell lines. In all cell lines, the expression of the tumour suppressor genes TP53, CDKN2A and CDKN1A was down-regulated, whereas that of Rb was preserved. In vitro assessments revealed the growth inhibitory effects of palbociclib in all cell lines examined. In a xenograft mouse model using a cell line from disseminated HS, palbociclib exerted significant growth inhibitory effects. These results suggest the potential of palbociclib as a therapeutic drug candidate for the treatment of malignant histiocytic proliferative disorders of the dog.

Establishment and characterisation of cell lines and xenograft mouse models of canine systemic histiocytosis and disseminated histiocytic sarcoma.

Canine histiocytic proliferative disorders include reactive diseases (histiocytosis) and neoplastic diseases (histiocytic sarcoma [HS]), however discrimination is challenging due to their overlapping pathological features. In the present study, novel cell lines and xenograft mouse models of systemic histiocytosis (SyH) and disseminated HS were established, and examined together with cell lines previously established from localized HS and Langerhans cell histiocytosis (LCH). The chromosomal numbers of the SyH and HS cell lines were abnormal, and their population doubling time and morphological features were comparable. Immunophenotypically, SyH and HS cells were CD204+/E-cadherin+ in vitro and in vivo, like their original tissues. Western blot analysis for E-cadherin revealed an immunopositive band of full-length E-cadherin (120 kDa) in cultured cells of localized HS and LCH but not in disseminated HS and SyH; expression level was weaker in localized HS than in LCH. An immunopositive band of fragmented E-cadherin (45 kDa) was detected in cell lines of disseminated HS and SyH. These results suggest that cultured SyH cells have features that are similar to disseminated HS, including chromosomal aberration, high proliferation activity, weak cell adhesion, and expression of fragmented E-cadherin. Fragmentation of the E-cadherin cell adhesion molecule may be associated with the loss of cell adhesion and increased abilities of invasion and migration of neoplastic cells. The established cell lines and xenograft mouse models will aid in understanding the pathogenesis and developing novel treatments of canine histiocytic proliferative disorders.

Immunophenotyping of Nonneoplastic and Neoplastic Histiocytes in Cats and Characterization of a Novel Cell Line Derived From Feline Progressive Histiocytosis.

Histiocytic proliferative diseases are rare in cats, and their pathogenesis is poorly understood. In the present study, 25 cases of histiocytic sarcoma (HS) and 6 of feline progressive histiocytosis (FPH) were examined, and survival times were recorded in 19 cases. The immunophenotypes of tumor cells in these cases as well as of nonneoplastic feline histiocytes were characterized using formalin-fixed, paraffin-embedded tissues. An FPH cell line (AS-FPH01) and xenotransplant mouse model of FPH were also established. The median survival time of HS (150 days) was significantly shorter than that of FPH (470 days). Immunohistochemically, nonneoplastic histiocytes were immunopositive for various combinations of Iba-1, HLA-DR, E-cadherin, CD204, CD163, CD208, and MAC387. By immunohistochemistry, dermal interstitial dendritic cells (iDCs) and macrophages were CD204+/E-cadherin-, while epidermal Langerhans cells (LCs) were CD204-/E-cadherin+. Neoplastic cells of 4 FPH and 18 HS were CD204+/E-cadherin- (iDC/macrophage immunophenotype), while 2 FPH and 2 HS were CD204-/E-cadherin+ (LC immunophenotype), and 5 HS were CD204+/E-cadherin+ (LC-like cell immunophenotype). Furthermore, immunohistochemical and western blot analyses of AS-FPH01 cells derived from E-cadherin-negative FPH revealed that cultured cells were immunopositive for both CD204 and E-cadherin in vitro and in vivo. These results indicate that the neoplastic cells of feline HS and FPH were variably positive for iDC/macrophage and LC markers, and their immunophenotype changed in different microenvironments. The novel cell line established in the present study may serve as an experimental model of FPH that will enable further molecular and therapeutic studies on this disease.

A retrospective survey on canine intracranial tumors between 2007 and 2017.

To clarify the prevalence of canine intracranial tumors in Japan, a retrospective study was performed using data on 186 canine intracranial tumors. Of 186 cases, 159 cases (85.5%) were primary and 27 cases (14.5%) were secondary intracranial tumors. Among primary intracranial tumors, meningioma (50.9%) was the most common, followed by glial tumors (21.4%) and primary intracranial histiocytic sarcoma (12.6%). These 3 tumors were most frequently found in middle-aged to elderly dogs without any sex predilection. Regarding glial tumors, the incidence of oligodendroglial tumors (79.4%) was higher than that of astrocytic tumors (17.6%). A significant breed predisposition (P<0.05) was observed for meningioma in Rough Collie, Golden Retriever, Miniature Schnauzer, and Scottish Terrier; for glial tumors in Bouvier de Flandres, French Bulldog, Newfoundland, Bulldog, and Boxer; for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi, Siberian Husky, and Miniature Schnauzer. The high incidence of oligodendroglial tumors in dogs and the breed predisposition for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi have not been reported in previous epidemiological studies on canine tumors. Since the incidence of intracranial tumors was vary among dog breeds, the present results demonstrate the uniqueness of the canine breed population in Japan.

Blastic natural killer cell lymphoma/leukaemia in a cat.

CASE SUMMARY: A 7-year-old mixed-breed cat presented with subcutaneous oedema and erythema extending from the right axilla to the abdomen. Fine-needle aspiration of the subcutaneous lesion revealed large, atypical, round cells. A clonality analysis for the T-cell receptor-gamma and immunoglobulin heavy chain genes showed no clonal rearrangement. The presumed diagnosis was lymphoma and the cat was treated with prednisolone and L-asparaginase but died 78 days after initial treatment. At necropsy, an oedematous subcutaneous mass in the right axilla, hepatomegaly, splenomegaly and lymphadenopathy of the mediastinum and left axilla were observed. Histopathological examination revealed diffuse infiltration of large atypical round cells in the subcutaneous mass, liver, spleen, lymph nodes and bone marrow. Immunohistochemically, the tumour cells were strongly positive for CD56, and negative for CD3, CD20, CD79a, CD57, granzyme B and perforin. Based on these findings, the cat was diagnosed with blastic natural killer (NK) cell lymphoma/leukaemia. RELEVANCE AND NOVEL INFORMATION: Here, we report the pathological and clinical findings of NK cell lymphoma/leukaemia in a cat. The antibody for human CD56, a diagnostic marker for human NK cell neoplasms, showed cross-reactivity with feline CD56 by immunohistochemistry and Western blotting analysis. The antibody could be a useful diagnostic marker for feline NK cell neoplasms.

Immunohistochemical studies on meningoencephalitis in feline infectious peritonitis (FIP).

The present study describes the association between inflammatory cell types and feline infectious peritonitis virus (FIPV) antigen in the brain of 4 cats diagnosed as feline infectious peritonitis (FIP). Immunohistochemically, FIPV antigens were detected in the inflammatory foci of the leptomeninges, choroid plexus and ventricles in 3 of the 4 cats. In 3 cases, inflammatory foci mainly consisted of CD204- and Iba1-positive macrophages, and the FIPV antigens were found in the macrophages. In the other case which was negative for FIPV antigen, severe inflammation predominantly consisting of CD20-positive B lymphocytes was observed in the leptomeninges and subventricles, accompanied with diffuse proliferation of gemistocytic astrocytes. The difference in histopathology may reflect the inflammatory process or the strain variation of FIP virus.

Thrombin-antithrombin complex measurement using a point-of-care testing device for diagnosis of disseminated intravascular coagulation in dogs.

Reference interval for thrombin-antithrombin complex (TAT) level was determined using an in-house TAT measurement device, and its validity for diagnosis of disseminated intravascular coagulation (DIC) was evaluated in dogs. One hundred and two clinically healthy dogs and 247 diseased dogs with conditions that potentially caused DIC were recruited in the study. Six diagnostic testing for DIC were evaluated in diseased dogs and the diseased dogs were categorized into five groups depending on abnormal findings. TAT was measured in all study animals and between-group differences were evaluated. TAT level was positively associated with severity of DIC. There were no significant differences in TAT levels among clinically healthy dogs, diseased dogs without any abnormal finding and diseased dogs with one abnormal finding in the DIC diagnostic testing. TAT levels in groups with two or more abnormal findings were significantly higher than clinically healthy dogs. Reference interval of TAT level for clinically healthy dogs was ≤ 0.25 ng/ml. Validity of using TAT for early detection of DIC was evaluated. In-house TAT measurement was suggested to be a clinically relevant and useful tool for early detection of canine DIC.