Mid-to-caudal partial hemipelvectomy with limb preservation for ischial tumor in a dog.

A 10-year-old spayed female Golden Retriever was referred for hindlimb lameness. A firm mass was palpated over the right caudal pelvis. Computed tomography revealed an osteolytic bone region and an associated periosteal reaction in the ischium, including the acetabulum. The histological diagnosis was sarcoma of unknown origin. A mid-to-caudal partial hemipelvectomy was performed to remove the mass. Femoral head and neck osteotomy was performed to allow hindlimb preservation. Following surgery, the dog regained satisfactory hindlimb use over time and was alive for 821 days with no recurrence or metastatic disease. This report indicates that mid-to-caudal partial hemipelvectomy with femoral head and neck osteotomy is technically feasible and allows for tumor control with preservation of the hindlimb and its function.

Prognostic role of ΔNp63 expression in canine transitional cell carcinoma of the urinary bladder.

BACKGROUND: Decreased p63 protein expression in canine transitional cell carcinoma (TCC) of the urinary bladder is associated with vascular invasion of the tumor, metastasis, and shortened survival. ΔNp63, an isoform of p63, is downregulated in high-grade invasive urothelial carcinoma in humans. However, the clinical significance of ΔNp63 expression in canine urinary bladder tumors is unknown. Therefore, it is essential to investigate ΔNp63 expression patterns in TCC, the most common urinary bladder tumor in dogs. AIM: This study aimed to evaluate the expression and role of ΔNp63 in canine TCC of the urinary bladder. METHODS: ΔNp63 expression was compared between the normal canine urinary bladder, polypoid cystitis, and TCC. The correlation of ΔNp63 expression with histopathological and clinical findings were further evaluated, and its usefulness as a prognostic factor was examined. RESULTS: We observed that ΔNp63 was highly expressed in dogs' normal urinary bladder and polypoid cystitis, and its expression levels were low in TCC. Furthermore, low levels of ΔNp63 expression were associated with vascular invasion, metastasis, and shortened survival in dogs with TCC. CONCLUSION: These results indicate that ΔNp63 expression could serve as a valuable biomarker for invasion, metastasis, and prognosis of canine TCC of the urinary bladder.

Author Correction: Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Spindle assembly checkpoint competence in aneuploid canine malignant melanoma cell lines.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents unequal segregation of chromosomes during mitosis. Abnormalities in the SAC are associated with chromosome instability and resultant aneuploidy. This study was performed to evaluate the SAC competence in canine malignant melanoma (CMM) using four aneuploid cell lines (CMeC1, CMeC2, KMeC, and LMeC). After treatment with nocodazole, a microtubule disrupting agent, CMeC1, KMeC, and LMeC cells were arrested in M phase, whereas CMeC2 cells were not arrested, and progressed into the next cell cycle phase without cytokinesis. Chromosome spread analysis revealed a significantly increased rate of premature sister chromatid separation in CMeC2 cells. Expression of the phosphorylated form of the SAC regulator, monopolar spindle 1 (Mps1), was lower in CMeC2 cells than in the other CMM cell lines. These results indicate that the SAC is defective in CMeC2 cells, which may partially explain aneuploidy in CMM. Thus, CMeC2 cells may be useful for further studies of the SAC mechanism in CMM and in determining the relationship between SAC incompetence and aneuploidy.

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

Cancer-promoting inflammation is an important event in cancer development. Canine urothelial carcinoma (cUC) overexpresses prostaglandin E2 (PGE2) and has a unique sensitivity to cyclooxygenase 2 (COX2)-inhibiting therapy. In addition, majority of cUC harbour BRAFV595E mutation. However, mechanisms underlying aberrant PGE2 production in BRAFV595E cUC patients remain unclear. Drug screening revealed that inhibition of RAF/MEK/ERK pathway, p38 and JNK pathway reduced PGE2 production in cUC cells. By pharmacological inhibition of the multiple components in the pathway, activation of the ERK MAPK pathway was shown to mediate overexpression of COX2 and production of PGE2 in BRAFV595E cUC cells. In silico gain-of-function analysis of the BRAF mutation also implicated involvement of mutation in the process. The positive association between ERK activation and COX2 expression was further validated in the clinical patients. Moreover, it was also suggested that p38 and JNK regulates PGE2 production independently of ERK pathway, possibly through COX2-dependent and COX1-/COX2- independent manner, respectively. In conclusion, this study demonstrated that activation of ERK induces production of PGE2 in BRAFV595E cUC cells, which is also independently regulated by p38 and JNK. With its unique vulnerability to COX-targeted therapy, BRAFV595E cUC may serve as a valuable model to study the tumour-promoting inflammation.

DNA aneuploidy and centrosome amplification in canine tumor cell lines.

DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.

Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines.

Transitional cell carcinoma (TCC) accounts for >90% of canine malignant tumours occurring in urinary bladder, and the prognosis is poor. Our previous study, using RNA sequencing, showed that human epidermal growth factor 2 (HER2) was the most activated upstream regulator related to carcinogenesis in canine TCC. The aim of this study was to examine the anti-tumour effect of lapatinib, a tyrosine kinase inhibitor of HER2, on canine TCC cell lines in vitro and in vivo. Five canine TCC cell lines (TCCUB, Love, Sora, LCTCC, and MCTCC) were used. Western blotting showed that HER2 protein expression was observed in all of the canine TCC cell lines. Lapatinib inhibited phosphorylation of HER2 and cell growth in a dose-dependent manner. Cell cycle analyses using flow cytometry showed that lapatinib significantly increased the sub-G1 and G0 /G1 phase fractions and significantly decreased the S and G2 /M phase fractions in the cell lines (Sora and TCCUB). For the in vivo experiments, the canine TCC cells (Sora) were subcutaneously injected into nude mice. Six days after inoculation, lapatinib (100 mg/kg) or vehicle was administered daily via intraperitoneal administration for 14 days. Tumour volume was significantly smaller in the lapatinib group compared with the vehicle control group. Histologically, lapatinib significantly increased necrotic areas in the tumour tissues. These findings suggest that lapatinib exerts anti-tumour effects on canine TCC cells by inhibiting HER2 signalling and inducing cell cycle arrest.

Apoptosis and Ki-67 as predictive factors for response to radiation therapy in feline nasal lymphomas.

Nasal lymphoma is the most common nasal tumor in cats and is generally a solitary and radiosensitive tumor. We retrospectively evaluated the response to radiation and survival time in relation to apoptosis and Ki-67 indices in feline nasal lymphomas treated with radiation therapy. The apoptotic and Ki-67 indices were evaluated with TUNEL and immunohistochemical staining in 30 biopsy tissues that were taken before any treatment. These two indices were compared, and differences between different treatment response groups were analyzed. The correlation between the median survival times (MST) and the indices was estimated using the Kaplan Meier method, and statistical differences between survival curves were analyzed using a log-rank method. With regard to apoptotic index, a statistical difference was observed between the samples taken from cats with complete response and stable disease (1.22% vs. 0.45%; P=0.045). The Ki-67 index in cats with both complete response and partial response was significantly higher than in cats with stable disease (44.4% and 39.6% vs. 16.3%; P<0.001 and P=0.008, respectively). The cats with a high level of apoptosis (>0.9%) nasal lymphoma were not significantly prolonged MSTs (P=0.202), however, high Ki-67-positive (>40%) cats experienced a statistically significant relationship with longer survival time (P=0.015). Our results indicate that spontaneous apoptotic and Ki-67 indices are strong predictors for response to radiation therapy in feline nasal lymphomas.

Identification and preservation of the parathyroid gland during total thyroidectomy in dogs with bilateral thyroid carcinoma: a report of six cases.

Simultaneous removal of bilateral thyroid tumors was performed while preserving the parathyroid gland in six dogs. At least one external parathyroid gland was identified in all dogs. In five cases, the external parathyroid gland and its blood supply were preserved intact. In one dog, the vessels supplying the external parathyroid gland had been invaded by the tumor, and the gland was thus removed and reimplanted into the sternohyoid muscle. That dog required postoperative treatment with oral calcium gluconate and vitamin D3. Local tumor recurrence was not observed in any of the cases. The mean survival time was 920 days. We found that the external parathyroid gland could be identified and preserved in most dogs undergoing total thyroidectomy.

Epidermal growth factor receptor expression in canine transitional cell carcinoma.

Transitional cell carcinoma (TCC), a urinary bladder tumor with high mortality, is encountered commonly in dogs. Whereas overexpression of epidermal growth factor receptor (EGFR) is associated with development of human urinary bladder cancer, information on EGFR expression in canine TCC is lacking. In this study, EGFR protein and mRNA expression in canine normal bladder (n=5), polypoid cystitis (n=5) and TCC (n=25) were examined by immunohistochemistry and real-time polymerase chain reaction. EGFR protein expression was significantly higher in TCC than that in normal healthy bladder (P<0.001) and polypoid cystitis (P<0.005). High EGFR protein expression was significantly (P<0.01) associated with TCC with a sensitivity of 72% and specificity of 100%. Comparative analysis of protein and mRNA expression levels in TCC showed significant positive correlation (r=0.88, P<0.05) between mRNA and protein expression. These findings suggest that intense expression of EGFR protein could be used as a marker to help canine TCC diagnosis.

Prognostic utility of apoptosis index, Ki-67 and survivin expression in dogs with nasal carcinoma treated with orthovoltage radiation therapy.

Apoptosis, Ki-67 and survivin expression have been reported as prognostic values in human cancer treated with radiation therapy. The aim of this study was to evaluate the correlation between the outcome of canine nasal carcinomas treated with radiation therapy and these cancer markers. The apoptotic index (AI) was evaluated with TUNEL assays, and an immunohistochemical evaluation was performed on Ki-67 and survivin in 33 biopsy samples taken before treatment. Median survival times were estimated using Kaplan-Meier curves and the log-rank method. The AI ranged from 0 to 0.7%, and the percentage of Ki-67-positive cells defined as the proliferative index (PI) ranged from 0.8 to 77% in all samples. Neither the AI nor the PI had a significant relationship with survival time (P=0.056 and 0.211). Survivin expression was detected in 84.9% of samples of canine nasal carcinoma. Dogs with high survivin expression were associated with poorer response to treatment and had shorter survival times (P=0.017 and 0.031). Advanced-stage tumors were also significantly associated with a high level of survivin (P=0.026). Overexpression of survivin was shown to be an unfavorable prognostic factor in dogs with nasal carcinomas treated with radiation therapy.

Serum big endothelin-1 as a clinical marker for cardiopulmonary and neoplastic diseases in dogs.

AIMS: Many studies of human subjects have demonstrated the utility of assessing serum levels of endothelin-1 (ET-1) and big ET-1 as clinical biomarkers in cardiopulmonary and neoplastic diseases. In this study we explored the feasibility of using serum big ET-1 as a reliable veterinary marker in dogs with various cardiopulmonary and neoplastic diseases. MAIN METHODS: Serum big ET-1 levels were measured by ELISA in dogs with cardiopulmonary (n=21) and neoplastic diseases (n=57). Dogs exhibiting cardiopulmonary disease were divided into two groups based on the velocity of tricuspid valve regurgitation (3.0>m/s) measured by ultrasound: without and with pulmonary hypertension. Big ET-1 levels for the dogs with the diseases were compared with levels in normal healthy dogs (n=17). KEY FINDINGS: Dogs with cardiopulmonary disease (4.6±4.6 pmol/l) showed a significantly (P<0.01) higher level of big ET-1 than healthy control dogs (1.1±0.53 pmol/l). Serum levels in the dogs with pulmonary hypertension (6.2±5.3 pmol/l) were significantly (P<0.01) higher than those without pulmonary hypertension (2.0±0.6 pmol/l). Dogs with hemangiosarcoma (5.6±2.2 pmol/l), adenocarcinoma (2.0±1.8 pmol/l), histiocytic sarcoma (3.3±1.9 pmol/l), chondrosarcoma or osteosarcoma (3.0±1.6 pmol/l) and hepatocellular carcinoma (2.7±1.8 pmol/l) showed significantly (P<0.05) higher levels than healthy control dogs. SIGNIFICANCE: These findings point to the potential of serum big ET-1 as a clinical marker for cardiopulmonary and neoplastic diseases in dogs.

Ultrasonographic findings related to prognosis in canine transitional cell carcinoma.

In human bladder cancer patients, ultrasonography is extensively used not only to identify tumor masses but also to evaluate tumor size, shape, echogenicity, location, and degree of tumor invasion into the bladder wall. The information revealed by ultrasonography delineates the tumor's biological features and facilitates prediction of prognosis. However, in veterinary medicine the feasibility of using ultrasonography for these purposes has not been fully investigated. In this retrospective study, we reviewed cases of dogs with histologically confirmed bladder mass lesions, including transitional cell carcinoma (n = 22) and polypoid cystitis (n = 5), to determine whether ultrasonography could reliably predict bladder wall involvement. By following patients with transitional cell carcinoma until death, we also determined whether ultrasonographic tumor size, shape, echogenicity, and mass location were related to prognosis. Wall involvement as revealed by ultrasound was significantly (P = 0.00005) associated with histological muscular layer involvement with a sensitivity of 93% (95% Confidence interval, 79-98%) and specificity of 92% (95% Confidence interval, 76-98%). Ultrasonographic wall involvement (P = 0.03, vs. noninvolvement), heterogeneous mass (P = 0.02, vs. homogeneous mass), and trigone location (P = 0.01, vs. other locations) characteristics were significantly associated with shorter survival times in transitional cell carcinoma cases. Findings indicated that ultrasonographic characteristics such as wall involvement, heterogeneous mass, and trigone location could be reliable prognostic indicators in canine transitional cell carcinoma.

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): a pilot study in a canine model.

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

Peripheral lymphocyte subsets as a prognostic indicator of mortality and morbidity in healthy dogs.

To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3(+), CD4(+), CD8(+) and CD21(+) cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4(+) and the CD4(+):CD8(+) ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3(+) lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity.

Pathological classification of canine mammary tumor based on quantifying mRNA levels of hormonal receptors, SATB1, and Snail in tissue and fine needle biopsy samples.

Cytological diagnosis is not generally conclusive enough to identify histopathological malignancy in canine mammary tumors (CMTs). To establish cytological examination using fine needle biopsy (FNB) samples, gene expressions of hormonal receptors, human epidermal growth factor receptor 2 (HER2), and transcription regulators (Special AT-rich binding protein 1: SATB1 and Snail) were investigated in both tissue and FNB samples of CMTs. In tissue samples of malignant CMTs, especially invasive ones, low expressions of hormonal receptors and high expressions of SATB1 and Snail were detected. On discriminant analysis of tissue samples, 73.2% of CMTs were correctly classified according to histopathological examinations. In FNB samples of malignant CMTs, low expressions of hormonal receptors were detected. On discriminant analysis of FNB samples, 74.2% of CMTs were correctly classified according to histopathological examination. In conclusion, FNB gene expressions had a utility for diagnosis of CMTs malignancy in some degree. By researching more sensitive genes for malignant CMTs, the gene examination of FNB samples from CMTs will become a useful diagnostic tool that can be performed easily without anesthesia and could predict tumor malignancy and invasion prior to surgical removal.

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues.

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.

Significance of tumor-infiltrating immune cells in spontaneous canine mammary gland tumor: 140 cases.

The numbers of tumor infiltrating T lymphocytes, B lymphocytes and antigen presenting cells were evaluated in an immunohistochemical manner in 140 canine spontaneous mammary gland tumor (MGT) tissues. As a result, we found a statistically significant increase in the number of intratumoral T lymphocytes (23.2 ± 23.8) in the malignant MGT group (n=51) compared with the benign MGT group (14.0 ± 16.0, n=89; P<0.05). Moreover, the high T lymphocyte infiltration in the malignant group correlated with poor prognosis in multivariate analysis (P<0.05). This study indicated the relationship between increased infiltrating T lymphocytes and canine MGT malignancy.

Effects of NF-κB expression and its inhibition on canine mammary cancer cell lines in an immunodeficient mice model.

Two canine mammary gland tumor (MGT) cell lines, CHMp-5b and -13a, were transplanted into nude mice, and their tumor development and metastatic potential were evaluated. In addition, NF-κB, Ki-67 and cyclin D1 expressions in the tumor masses were evaluated, and the relationship between these expressions and malignancy of the tumors developed in nude mice was investigated. Lower activation of NF-κB was positively correlated with a lower potential of metastasis and better prognosis in nude mice xenografted with CHMp-13a compared with CHMp-5b. Then, CHMp-5b cells were treated with BAY11-7082, an inhibitor of NF-κB, and the expressions of I-κBα, p-I-κBα, cyclin D1 and Bcl-2 in these cells were evaluated by Western blot analysis. In addition, BAY11-7082 at a dose of 6 mg/kg was administered intraperitoneally to nude mice xenografted with CHMp-5b, and relationships between tumor growth and lung and lymph node metastasis in nude mice and NF-κB, I-κBα and p-I-κBα expressions in tissues of these mice were evaluated. BAY11-7082 treatment induced decreased expressions of p-I-κBα, cyclin D1 and Bcl-2 in a dose- and time-dependent manner, suggesting that BAY inhibited NF-κB in this cell line. CHMp-5b-xenografted mice treated with BAY11-7082 showed a reduction in tumor growth and metastasis. Therefore, inhibition of the NF-κB signaling pathway may be a promising novel therapeutic approach for canine MGTs.

Alterations of lymphocyte subpopulations in healthy dogs with aging and in dogs with cancer.

Changes in an individual's immune status are considered major contributing factors towards the morbidity of cancer and mortality of aging. To evaluate age-related changes in the immune status of dogs, the immunophenotypes (CD3, CD4, CD8 and CD21) of peripheral blood lymphocytes were measured in 160 healthy dogs aged from 1 to 17 years, and in 365 dogs with various tumors and at various stages. In healthy dogs, the absolute numbers of white blood cells, lymphocytes, and CD3(+), CD4(+) and CD21(+) lymphocytes decreased significantly with age. The relative percentages of lymphocytes and CD4(+) cells decreased significantly, while CD8(+) cells increased significantly with age. The CD4:CD8 ratio showed a significant age-related decrease. In contrast, dogs with tumors possessed significantly lower absolute numbers and relative percentages of all lymphocyte phenotypes, while the CD4:CD8 ratio was significantly higher than in age-matched controls. The relative percentages of CD3(+) and CD8(+) lymphocytes were significantly lower in dogs with distant metastases compared with dogs without metastases, and the CD4:CD8 ratio increased with advanced stage. These observations illustrate the significant changes in immune status with age and the presence of marked immunological defects in a large-scale study of dogs with advanced tumors.