Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers.

Cancer in dogs has increased in recent years and is a leading cause of death. We have developed a retroviral replicating vector (RRV) that specifically targets cancer cells for infection and replication. RRV carrying a suicide gene induced synchronized killing of cancer cells when administered with a prodrug after infection. In this study, we evaluated two distinct RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) in canine tumor models both in vitro and in vivo. Despite low infection rates in normal canine cells, both RRVs efficiently infected and replicated within all the canine tumor cells tested. The efficient intratumoral spread of the RRVs after their intratumoral injection was also demonstrated in nude mouse models of subcutaneous canine tumor xenografts. When both RRVs encoded a yeast cytosine deaminase suicide gene, which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil, they caused tumor-cell-specific 5-FC-induced killing of the canine tumor cells in vitro. Furthermore, in the AZACF- and AZACH-cell subcutaneous tumor xenograft models, both RRVs exerted significant antitumor effects. These results suggest that RRV-mediated suicide gene therapy is a novel therapeutic approach to canine cancers.

A feline spinal dermoid cyst treated with surgical intervention.

A 5-year-old neutered female mixed cat presented with reduced activity and ataxia of the hind limbs. Computed tomography and magnetic resonance imaging revealed an extradural mass compressing the spinal cord on the dorsal aspects from the 7th to 8th thoracic vertebra. Dorsal laminectomy was performed on the 7-8th thoracic vertebra and the cyst was totally removed, giving full resolution of the clinical signs. The cyst was diagnosed as a dermoid cyst. To our knowledge, this is the first report of feline dermoid cyst compressing the spinal cord that was diagnosed antemortem. The prognosis is favorable when the cyst is completely resected.

microRNA-203 inhibits migration and invasion of canine tonsillar squamous cell carcinoma cells by targeting SLUG.

Objective: Squamous cell carcinoma (SCC) occurring in the tonsils (TSCC) has a poorer prognosis than SCC occurring in other regions of the oral cavity (non-tonsillar SCC [NTSCC]) because it easily metastasizes to distant organs. This study aimed to elucidate the molecular mechanisms underlying the migration and invasion of TSCC cells in vitro. Materials and methods: This study focused on differential microRNA (miRNA) expression using microRNA microarrays and quantitative polymerase chain reaction in canine TSCC and NTSCC tissues and cell lines. A target gene of the miRNA involved in cell migration and invasion was validated by wound healing, transwell, and luciferase assays. Results: miR-203 expression was lower in TSCC tissues than in the normal oral mucosa and NTSCC tissues. Transfection of the miR-203 mimic resulted in the downregulation of mesenchymal marker protein expression and attenuation of cell migration and invasion in TSCC cells, but not in NTSCC cells. A dual-luciferase assay revealed that miR-203 directly targeted the mesenchymal transcription factor SLUG. SLUG overexpression enhances the migration of TSCC cells. Conclusion: Our study indicates that the miR-203/SLUG axis may be involved in the metastatic mechanisms of TSCC.

Podoplanin promotes cell proliferation, survival, and migration of canine non-tonsillar squamous cell carcinoma.

Podoplanin (PDPN) is a prognostic factor and is involved in several mechanisms of tumor progression in human squamous cell carcinoma (SCC). Canine non-tonsillar SCC (NTSCC) is a common oral tumor in dogs and has a highly invasive characteristic. In this study, we investigated the function of PDPN in canine NTSCC. In canine NTSCC clinical samples, PDPN overexpression was observed in 80% of dogs with NTSCC, and PDPN expression was related to ki67 expression. In PDPN knocked-out canine NTSCC cells, cell proliferation, cancer stemness, and migration were suppressed. As the mechanism of PDPN-mediated cell proliferation, PDPN knocked-out induced apoptosis and G2/M cell cycle arrest in canine NTSCC cells. These findings suggest that PDPN promotes tumor malignancies and may be a novel biomarker and therapeutic target for canine NTSCC.

Successful management of nasopharyngitis caused by Schizophyllum commune in a captive cheetah (Acinonyx jubatus).

Herein, we describe the management of nasopharyngitis caused by Schizophyllum commune infection in a captive cheetah. Computed tomography revealed a nodule in the nasal cavity and pharynx, and an endoscopic biopsy was performed. As a result, the nodule was surgically resected because of a suspected carcinoma. However, the surgical specimen was histologically re-evaluated and a fungal granuloma was diagnosed. Sequence analysis of DNA from formalin-fixed, paraffin-embedded samples revealed S. commune infection. The cheetah was administered fluconazole orally for 73 days. However, the drug was ineffective and itraconazole was administered for 14 days. Symptoms such as nasal discharge and sneezing have completely resolved for 4 years.

Potential therapeutic efficiency of pan-ERBB inhibitors for canine glioma.

Canine glioma is one of the most common brain tumors with poor prognosis, making effective chemotherapy highly desirable. Previous studies have suggested that ERBB4, a signaling molecule involving one of the epidermal growth factor receptors (EGFR), may be a promising therapeutic target. In this study, the anti-tumor effects of pan-ERBB inhibitors, which can inhibit the phosphorylation of ERBB4, were evaluated both in vitro and in vivo using a canine glioblastoma cell line. The results demonstrated that both afatinib and dacomitinib effectively reduced the expression of phosphorylated ERBB4, and significantly decreased the number of viable cells, ultimately prolonging the survival time of orthotopically xenografted mice. Further downstream of ERBB4, afatinib was found to suppress the expression of phosphorylated Akt and phosphorylated Extracellular signal-related kinases1 and 2 (ERK1/2) and induced apoptotic cell death. Thus, pan-ERBB inhibition is a promising therapeutic strategy for the treatment of canine gliomas.

Contrast-enhanced CT features of pyloric lesions in 17 dogs: Case series.

Pyloric outflow obstructions can be caused by several types of lesions. When a thickened gastric wall and pyloric mass are detected, malignant neoplasia must be differentiated from chronic hypertrophic pyloric gastropathy. CT can characterize gastric tumors. However, based on the authors' review of the literature, there is limited information about the CT findings of pyloric lesions. The purpose of this retrospective case series study was to assess the CT findings of canine pyloric lesions. The following CT parameters were recorded: anatomical area, involved area, lesion shape, growth patterns of wall thickening lesions, enhancement pattern of the lesion in the early and delayed phases, lymphomegaly, and pulmonary metastasis. Seventeen dogs were included in this study and had the following final diagnoses: hyperplasia (five dogs), adenoma (five dogs), adenocarcinoma (three dogs), gastrointestinal stromal tumor (GIST; two dogs), polyposis (one dog), and pyogenic granuloma (one dog). Hyperplasia, adenoma, and polyposis formed mass lesions that involved the mucosal layer. Lymphomegaly was detected in two Jack Russell terriers with hyperplasia; however, the causes were unknown because we did not perform biopsies. All adenocarcinomas formed wall-thickened lesion that involved the outer layer, with lymphomegaly. All GISTs formed mass lesion that involved the outer layer. The pyogenic granulomas formed symmetric wall-thickened lesion that involved the mucosal and outer layers. CT facilitated the characterization of canine pyloric lesions using contrast enhancement, based on the involved area and lesion shape. However, polyposis may require caution in diagnosis based on CT findings alone.

Functional analysis of the miR-145/Fascin1 cascade in canine oral squamous cell carcinoma.

OBJECTIVES: Canine oral squamous cell carcinoma (SCC) often develops in the gingiva and tonsils. The biological behavior of canine oral SCC is similar to that of human head and neck SCC (HNSCC). Inhibiting invasion and metastasis is major importance for the treatment of canine and human HNSCC. In this study, the significance of microRNA (miR)-145 and Fascin1 (FSCN1) in the invasion of canine oral SCC was explored. MATERIALS AND METHODS: Canine oral SCC tissues and cell lines were used for miR-145 and FSCN1 expression analysis via real-time PCR and immunohistochemistry. Canine oral SCC cell lines were used for in vitro assays. RESULTS: miR-145 was downregulated while FSCN1 mRNA was upregulated in canine oral SCC. Immunohistochemistry revealed that FSCN1 was upregulated in SCC when compared to normal mucosa. Transfection of canine SCC cells with miR-145 or FSCN1 siRNA suppressed cell growth and attenuated cell migration as well as invasion by inhibiting the epithelial-to-mesenchymal transition. Furthermore, the promoter region of miR-145 was highly methylated in SCC cell lines and tissues. CONCLUSION: The expression profile and functions of miR-145 in canine oral SCC are similar to those in human HNSCC. Thus, canine oral SCC may represent a valuable preclinical model for human HNSCC.

Perioperative Management of Robotic-Assisted Gynecological Surgery in a Super Morbidly Obese Patient.

We report the perioperative management of a 32-year-old woman with super-morbid obesity (body mass index (BMI) of 60.9 kilograms per meter squared (kg/m2)) who underwent a robotic-assisted total laparoscopic hysterectomy in a hospital that was not specialized for obese patients. She successfully reduced her BMI by 10% using dietary restrictions in five weeks, and her surgery was performed two weeks later by consultation between gynecologists and anesthesiologists. This case demonstrates that the determination of the optimal time for surgery by consultation between physicians is crucial in the care of such a complex patient in hospitals that are not specialized for morbidly obese patients. Weight reduction in the preoperative period should be considered for super-morbid obesity patients with malignant diseases, even if the duration of preoperative optimization is shorter than four to eight weeks.

Computed tomography and magnetic resonance imaging findings in dogs with vaginal leiomyoma and leiomyosarcoma.

BACKGROUND: In humans, magnetic resonance imaging (MRI) is preferred over computed tomography (CT) for the assessment of pelvic lesions. Although CT findings of several pelvic tumours have been reported in veterinary medicine, MRI findings are limited. OBJECTIVES: The purpose of this study was to retrospectively compare the CT and MRI findings in dogs with vaginal leiomyoma and leiomyosarcoma. METHODS: This retrospective study of five dogs compared the CT and MRI findings of intrapelvic lesions, including vaginal leiomyoma (n = 4) and leiomyosarcoma (n = 1). No invasion of the surrounding tissue was detected on histopathological examination. In this retrospective study, the following parameters of CT and MRI were recorded for each dog: the border between the lesion and the adjacent pelvic organs, including the prostate, rectum or urethra; signal intensity (SI) of the lesion; enhancement pattern; presence of haemorrhage; necrosis or cystic areas and lymphadenopathy. Because SI on MRI is affected by cell density, tumour cell density was analysed using a microscope slide. RESULTS: In vaginal leiomyoma, the border between the lesion and the surrounding pelvic organ tends to be clearer on MRI than on CT. In vaginal leiomyosarcoma, the border was comparable between MRI and CT. Each lesion showed heterogeneous enhancement on CT and MRI scans. In each lesion, the assessment of haemorrhage, necrosis, cystic areas and lymphadenopathy was comparable between MRI and CT. The SI of the lesion on T2WI of the vaginal leiomyoma and leiomyosarcoma were hyperintense in four cases (4/4; 100%) and mixed intense in one case (1/1; 100%), respectively. The cell density of leiomyosarcoma is higher than that of leiomyomas. CONCLUSIONS: The SI on T2WI may be useful for differentiating leiomyoma from leiomyosarcoma. MRI may be useful to differentiate vaginal leiomyomas from leiomyosarcomas and evaluate margins.

Single-modality palliative radiotherapy versus palliative radiotherapy after chemotherapy failure for cats with nasal lymphoma.

Published studies describing outcomes for cats with nasal lymphoma (NLSA) receiving first-line palliative radiation (PRT) versus PRT after chemotherapy failure are currently lacking. The aims of this retrospective observational study were to compare outcomes for cats with NLSA that were treated with these two methods. A total of 48 cats were included in analyses; 32 receiving PRT alone and 16 receiving PRT after chemotherapy failure. The treatment response, progression-free survival (PFS), disease-specific survival (DSS), overall survival (OS), and incidence rate of systemic disease were compared between the two groups. The overall response rate (ORR) was calculated from the same target lesions between pre-RT (within a week before starting PRT) and post-RT (on date of PRT completion) by computed tomography (CT) imaging. The ORR was 94% in cats that received PRT alone, 13 had a complete response (CR) and 17 had a partial response (PR). The ORR was 88% in cats that received PRT after chemotherapy failure, with five having CR and nine with PR. There were no significant differences in the ORR between the two groups. The PFS, DSS, and OS significantly increased in the cats that received PRT alone compared to the cats that received PRT after chemotherapy failure (median PFS: 336 vs 228 days, P = 0.0012, median DSS: 360 vs 242 days, P = 0.0025, median OS: 346 vs 242 days, P = 0.0036, respectively). The incidence rate of systemic disease significantly increased in 75% (12/16) of cats receiving PRT after chemotherapy failure compared to 41% (13/32) of cats receiving PRT alone. The results suggested that clinical outcomes may improve in cats with NLSA with first-line PRT compared to PRT after chemotherapy failure.

SLUG is upregulated and induces epithelial mesenchymal transition in canine oral squamous cell carcinoma.

SLUG, encoded by the Snai2 gene, is known to play a role in epithelial-mesenchymal transition (EMT), which contributes to cell invasion and metastasis in some types of human carcinomas. However, the mechanisms and roles of EMT in canine squamous cell carcinoma (SCC) have not yet been elucidated. We have previously established canine oral SCC cell lines, including tonsillar SCC, and in this study, we evaluated the effects of SLUG on the phenotypes regarding EMT of canine SCC cells. First, immunohistochemical analysis revealed that SLUG is upregulated in canine oral SCC tissues compared to that in non-tumoural oral mucosa. Furthermore, gain-of-function and loss-of-function of SLUG revealed that SLUG partly contributed to migration and invasion of cells, as well as the upregulation of EMT markers such as vimentin and SNAIL. Thus, the current study suggests that SLUG promotes cell migration and invasion through EMT induction in canine oral SCC, as well as human cancers.

Comparing the CT and MRI findings for canine primary hepatocellular lesions.

BACKGROUND: Triple-phase CT and gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) MRI have been used to differentiate hepatocellular carcinomas (HCCs) in dogs. METHODS: This retrospective case series aimed to compare the CT findings with MRI findings of 20 canine hepatocellular lesions, including eight poorly/moderately-differentiated HCCs, eight well-differentiated HCCs and four hyperplasias. CT data were analysed, and the following parameters were noted: vessel enhancement, enhancement pattern in the equilibrium phase, maximal transverse diameter, the lowest enhancement, and the attenuation values of each hepatocellular lesion in the precontrast and triple-phase series, including the arterial phase, portal phase and equilibrium phase. MRI data were analysed, and the following parameters were noted: signal intensities of each hepatocellular lesion on T2-weighted images and T1-weighted images, and signal intensity ratio of the hepatocellular lesions in the hepatobiliary phase. RESULTS: In 62.5% of poorly/moderately-differentiated HCC and 75% of well-differentiated HCC, presumptive necrosis was detected on CT and MRI. In the hepatobiliary phase on MRI, the median signal intensity ratio of poorly/moderately-differentiated HCC (0.54 [range: 0.3-0.71]) was significantly lower than that of well-differentiated HCC (0.75 [range: 0.6-0.96]) and hyperplasia (0.79 [range: 071-0.98]; p = 0.02 and p = 0.02, respectively). CONCLUSION: Gd-EOB-DTPA-enhanced MRI may be a superior modality for differentiating hepatocellular origin lesions.

Preliminary study of CT features of intermediate- and high-grade alimentary lymphoma and adenocarcinoma in cats.

CASE SERIES SUMMARY: The ultrasonographic findings of many feline intestinal tumours are similar. This study evaluated the CT features of intermediate- and high-grade alimentary lymphoma and adenocarcinoma in cats. CT was performed on six cats with adenocarcinoma and 14 cats with lymphoma. Comparisons between tumour types were conducted, focusing on CT features, including obstruction (present or absent), growth patterns of lesions (symmetry or asymmetry), layering enhancement (present or absent), location of the lesion, number of lesions (solitary or multiple), lymphadenopathy (present or absent), location of lymphadenopathy, pulmonary metastasis (present or absent) and maximum thickness (mm) of the lesion. The cats with adenocarcinoma (n = 5/6 [83%]) experienced intestinal obstruction significantly more often than cats with lymphoma (n = 0/14 [0%]; P = 0.0004). Layering enhancement was observed significantly more often in cats with adenocarcinoma (n = 6/6 [100%]) than in cats with lymphoma (n = 1/14 [7%]; P = 0.0002). Lymphadenopathy was detected significantly more often in cats with lymphoma (n = 14/14 [100%]) than in cats with adenocarcinoma (n = 2/6 [33%]) (P = 0.003). In cats with lymphoma, the intestine (12.1 ± 3.9 mm) was significantly thicker than that in cats with adenocarcinoma (6.4 ± 2.3 mm; P = 0.005). RELEVANCE AND NOVEL INFORMATION: To the best of our knowledge, no reports have described the characteristics of feline intestinal tumours using CT. Layering enhancement was observed in cats with intestinal adenocarcinomas. No layering enhancement was observed in alimentary lymphoma in cats, but enlarged regional nodes were noted. Lesions with lymphoma were thicker than those with adenocarcinoma. These findings may help differentiate between adenocarcinomas and lymphomas.

Prognostic Factors for the Efficiency of Radiation Therapy in Dogs with Oral Melanoma: A Pilot Study of Hypoxia in Intraosseous Lesions.

Unresectable oral melanoma is often treated with radiation therapy (RT) and may show a temporary response to therapy. The clinical stage is one of the well-known prognostic factors for canine oral melanoma. However, the factors that directly affect the response to RT have remained unclear. This study aimed to validate the risk factors for recurrence after RT. Sixty-eight dogs with oral melanomas were included in this study. All dogs were treated with palliative RT using a linear accelerator without adjuvant therapies. After RT, the time to local recurrence (TTR) and overall survival (OS) were evaluated using the log-rank test. As a result, clinical stage and response to therapy were the significant independent prognostic factors in the multivariate analysis. The presence of local bone lysis and non-combination with cytoreductive surgery were associated with a worse response to RT. Immunohistochemical analysis for hypoxia-inducible factor-1α indicated that tumor cells invading the bone are under hypoxic conditions, which may explain a poorer efficiency of RT in dogs with bone lysis. In conclusion, clinical stage and combination with debulking surgery were needed to improve the efficiency of RT.

Radiotherapy-induced tumor lysis syndrome in a dog with thymoma.

A 13-year-old, female, mixed-breed dog with a huge cranial mediastinal mass underwent radiotherapy (RT). On the following day, the dog presented with lethargy and anorexia. Hematological examination revealed elevated levels of blood urea nitrogen, creatinine, inorganic phosphorus, potassium, lactate dehydrogenase, creatine phosphokinase and aspartate aminotransferase, decreased calcium level, and metabolic acidosis. Urine output markedly decreased. The patient recovered with fluid therapy and diuretic therapy; however, died suddenly from an unknown cause 11 days after RT completion. Histopathological examination after necropsy showed thymoma in the cranial mediastinum and extensive tubular necrosis of both kidneys which may be due to RT-induced tumor lysis syndrome (TLS). This report suggests that the risk of TLS should be evaluated in dogs with thymoma who undergo RT.

The NRG3/ERBB4 signaling cascade as a novel therapeutic target for canine glioma.

Canine glioma is a common brain tumor with poor prognosis despite surgery and/or radiation therapy. Therefore, newer and more effective treatment modalities are needed. Neuregulin 3 (NRG3) has known to be a ligand of ERBB4. This study aimed to investigate the usefulness of the NRG3/ERBB4 signaling cascade as a novel therapeutic target in canine glioma. We found out that microRNA (miR)-190a was downregulated in canine brain tumor tissues, including glioma and meningioma. miR-190a directly targeted NRG3 and inhibited the growth of canine glioma cells. The level of p-Akt, which is a downstream target of ERBB4 signaling, was decreased by transfection with miR-190a. NRG3 silencing also suppressed cell growth and decreased the levels of p-Akt and p-ERK1/2, and NRG3 overexpression exhibited opposed effects in canine glioma J3T-1 cells. The mRNA level of erbb4 was significantly upregulated in glioma tissues compared with that in normal brain tissues and meningioma tissues. Furthermore, compared with gefitinib and lapatinib, afatinib exerted a greater inhibitory effect on the growth of canine glioma cells. In conclusion, NRG3/ERBB4 signaling is negatively regulated by miR-190a and contributes to the growth of canine glioma cells, indicating that it may be a promising therapeutic target in canine glioma.

Assessment of biomarkers influencing treatment success on small intestinal lymphoma in dogs.

This study aimed to determine a reliable therapeutic biomarker for localized small intestinal lymphoma (SIL) in dogs based on clinical and histopathological features. We retrospectively investigated 84 dogs with localized SIL, including 36 dogs receiving surgery and 48 dogs receiving chemotherapy. The dogs receiving surgery were divided into two subgroups: 18 dogs (group 1) with overall survival (OS) <120 days (median OS) and 18 dogs (group 2) with OS ≥120 days. Correspondingly, the dogs receiving chemotherapy were divided into 24 dogs (group 3) with OS <98 days (median OS) and 24 dogs (group 4) with OS ≥98 days. Clinical, haematological, histopathological and immunohistochemical analyses were comparatively evaluated among the four subgroups. There was no significant difference in OS between the surgery and chemotherapy groups. In dogs receiving surgery, the rate of Ki67-positive cells was significantly increased in group 1 compared to group 2 and showed no significant difference between groups 3 and 4. In dogs receiving chemotherapy, the rate of O6-methylguanine-DNA methyltransferase (MGMT) was significantly higher in group 3 than in group 4 and showed no significant difference between groups 1 and 2. Additionally, our data showed that OS in dogs with higher Ki67 expression might be significantly increased by chemotherapy than by surgery, that of those with higher MGMT expression might be significantly increased by surgery than by chemotherapy, and Ki67 and MGMT were independent of each other. Indices of Ki67 and MGMT are suggested therapeutic biomarkers to determine the optimal first-line treatment for localized SIL in dogs.

A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs.

Inhibition of programmed death 1 (PD-1), expressed on activated T cells, can break through immune resistance and elicit durable responses in human melanoma as well as other types of cancers. Canine oral malignant melanoma is one of the most aggressive tumors bearing poor prognosis due to its high metastatic potency. However, there are few effective treatments for the advanced stages of melanoma in veterinary medicine. Only one previous study indicated the potential of the immune checkpoint inhibitor, anti-canine PD-L1 therapeutic antibody in dogs, and no anti-canine PD-1 therapeutic antibodies are currently available. Here, we developed two therapeutic antibodies, rat-dog chimeric and caninized anti-canine PD-1 monoclonal antibodies and evaluated in vitro functionality for these antibodies. Moreover, we conducted a pilot study to determine their safety profiles and clinical efficacy in spontaneously occurring canine cancers. In conclusion, the anti-canine PD-1 monoclonal antibody was relatively safe and effective in dogs with advanced oral malignant melanoma and other cancers. Thus, our study suggests that PD-1 blockade may be an attractive treatment option in canine cancers.

The inhibitory effect of canine interferon gamma on the growth of canine tumors.

Recombinant canine interferon-γ (rc-IFNγ; InterdogⓇ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.