Kiwamu Hanazono

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.

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.

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 tumors 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.

CONTRAST AGENT Gd-EOB-DTPA (EOB·Primovist®) FOR LOW-FIELD MAGNETIC RESONANCE IMAGING OF CANINE FOCAL LIVER LESIONS

Contrast-enhanced magnetic resonance (MR) imaging with a new liver-specific contrast agent gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA; EOB·Primovist®) was studied in 14 normal beagles and 9 dogs with focal liver lesions. Gd-EOB-DTPA accumulates in normally functioning hepatocytes 20 min after injection. As with Gd-DTPA, it is also possible to perform a dynamic multiphasic examination of the liver with Gd-EOB-DTPA, including an arterial phase and a portal venous phase. First, a reliable protocol was developed and the appropriate timings for the dynamic study and the parenchymal phase in normal dogs using Gd-EOB-DTPA were determined. Second, the patterns of these images were evaluated in patient dogs with hepatic masses. The optimal time of arterial imaging was from 15 s after injection, and the optimal time for portal venous imaging was from 40 s after injection. Meanwhile, the optimal time to observe changes during the hepatobiliary phase was from 20 min after injection. In patient dogs, 11 lesions were diagnosed as malignant tumors; all were hypointense to the surrounding normal liver parenchyma during the hepatobiliary phase. Even with a low-field MR imaging unit, the sequences afforded images adequate to visualize the liver parenchyma and to detect tumors within an appropriate scan time. Contrast-enhanced MR imaging with Gd-EOB-DTPA provides good demarcation on low-field MR imaging for diagnosing canine focal liver lesions.

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.

L-type amino acid transporter 1 (LAT1): A new therapeutic target for canine mammary gland tumour

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. LAT1 has recently received attention because of its preferential and upregulated expression in a variety of human tumours which is in contrast to its limited distribution and low-level expression in normal tissues. In this study, the feasibility of using an LAT1 inhibitor as a new therapeutic agent was explored for mammary gland tumours (MGT). [(3)H]l-leucine uptake by CHM, a cell line established from MGT, and effects on cell growth were analysed in the presence or absence of two LAT1 inhibitors, namely, BCH (2-amino-2-norbornane-carboxylic acids) or melphalan (LPM). [(3)H]l-leucine uptake and cellular growth activities in CHM were inhibited in a dose-dependent manner by both LAT1 inhibitors. The inhibitory growth activities of various conventional anti-cancer drugs used for MGT treatment, including carboplatin, cyclophosphamide, doxorubicin, mitoxantrone, vinblastine and vincristine, were significantly enhanced by combining use with BCH or LPM. The findings suggest that LAT1 could be a new therapeutic target for canine MGT.

Big endothelin-1 as a tumour marker for canine haemangiosarcoma

Haemangiosarcoma (HSA) is an important malignant neoplasm of dogs that originates from vascular endothelial cells. This study explored the suitability of using serum big endothelin-1 (ET-1) as a tumour marker for canine spontaneous HSA. Serum big ET-1 was measured in dogs with splenic HSA (n = 14), splenic malignant tumours other than HSA (n = 10), benign splenic lesions (n = 11) and normal healthy dogs (n = 17) by ELISA. Serum big ET-1 levels in dogs with HSA were significantly (P < 0.01) higher than in other dogs. High sensitivity (100%, 95% confidence interval 86-100%) and specificity (95%, 95% confidence interval 86-95%) for HSA diagnosis were obtained using a cut-off of 17 pg/mL according to receiver operating characteristic (ROC) curves (area under ROC curve 0.93). PPET1, ETA, VEGF and Hif1-α mRNA expression, measured by real-time PCR, were elevated in HSA compared with normal tissues. These findings suggest that elevated serum big ET-1 could be used as a diagnostic marker for canine HSA.

Porencephaly in dogs and cats: relationships between magnetic resonance imaging (MRI) features and hippocampal atrophy

Porencephaly is the congenital cerebral defect and a rare malformation and described few MRI reports in veterinary medicine. MRI features of porencephaly are recognized the coexistence with the unilateral/bilateral hippocampal atrophy, caused by the seizure symptoms in human medicine. We studied 2 dogs and 1 cat with congenital porencephaly to characterize the clinical signs and MRI, and to discuss the associated MRI with hippocampal atrophy. The main clinical sign was the seizure symptoms, and all had hippocampal atrophy at the lesion side or the larger defect side. There is association between hippocampal atrophy or the cyst volume and the severe of clinical signs, and it is suggested that porencephaly coexists with hippocampal atrophy as well as humans in this study.

Immunohistochemical expression of p63, Ki67 and β-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder.

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or β-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and β-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.