Effects of trastuzumab emtansine on canine urothelial carcinoma cells in vitro and in vivo.

Urothelial carcinoma (UC) is the most common malignancy of the urinary tract in dogs and has aggressive behaviour. Although human epidermal growth factor receptor 2 (HER2) is a known therapeutic target with evidence in canine UC, the efficacy of anti-HER2 antibody drugs remains unknown. This study aimed to investigate the effects of anti-HER2 antibody drugs including trastuzumab and trastuzumab emtansine (T-DM1) on canine UC cell lines in vitro and in vivo. Four canine UC cell lines (Nene, TCCUB, Love, and Sora) were used. In western blotting, HER2 protein expression was observed in all the cell lines. Although both trastuzumab and T-DM1 showed dose-dependent growth inhibitory activity in the cell lines, T-DM1 showed much stronger activity than that of trastuzumab. In flow cytometry analyses with the canine UC cell line (Sora), T-DM1 but not trastuzumab significantly increased the percentages of early and late apoptotic cells in annexin V apoptotic assays and the sub-G1 phase fraction in cell cycle analyses. For the in vivo experiment, the canine UC cells (Sora) were subcutaneously injected into nude mice. Four days after inoculation, trastuzumab, T-DM1, or vehicle was administered intraperitoneally once a week for three times. Tumour volumes were significantly smaller in the T-DM1 group compared to the trastuzumab and vehicle control groups. These findings indicate that T-DM1 exerts a stronger antitumour effect than that of trastuzumab on canine UC cells in vitro and in vivo, possibly by inducing apoptosis due to DM1.

Dystrophin-deficient muscular dystrophy in a Toy Poodle with a single base pair insertion in exon 45 of the Duchenne muscular dystrophy gene.

A 10-month-old, intact male Toy Poodle was referred for a postural abnormality. Blood biochemical tests revealed a marked increase in plasma creatine phosphokinase (CPK) concentration. The isoenzyme test showed that 99% of serum CPK consisted of CPK-MM. Histopathological evaluation of muscle biopsy samples confirmed scattered degeneration and necrosis of myofibers. Immunohistochemistry for dystrophin showed an absence of staining in muscle cells. Based on these findings, the dog was diagnosed with dystrophin-deficient muscular dystrophy. Whole genome sequencing using genomic DNA extracted from blood revealed a single base pair insertion in exon 45 of the Duchenne muscular dystrophy (DMD) gene. This is the first report on muscular dystrophy in Toy Poodles and identified a novel mutation in the DMD gene.

Lapatinib as first-line treatment for muscle-invasive urothelial carcinoma in dogs.

Epidermal growth factor receptors 1 and 2 (EGFR and HER2) are frequently overexpressed in various malignancies. Lapatinib is a dual tyrosine kinase inhibitor that inhibits both EGFR and HER2. Although a phase III trial failed to show the survival benefits of lapatinib treatment after first-line chemotherapy in patients with EGFR/HER2-positive metastatic urothelial carcinoma, the efficacy of lapatinib for untreated urothelial carcinoma is not well defined. Here, we describe the therapeutic efficacy of lapatinib as a first-line treatment in a canine model of muscle-invasive urothelial carcinoma. In this non-randomized clinical trial, we compared 44 dogs with naturally occurring urothelial carcinoma who received lapatinib and piroxicam, with 42 age-, sex-, and tumor stage-matched dogs that received piroxicam alone. Compared to the dogs treated with piroxicam alone, those administered the lapatinib/piroxicam treatment had a greater reduction in the size of the primary tumor and improved survival. Exploratory analyses showed that HER2 overexpression was associated with response and survival in dogs treated with lapatinib. Our study suggests that lapatinib showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use for untreated advanced urothelial carcinoma in dogs. The use of lapatinib as a first-line treatment may be investigated further in human patients with urothelial carcinoma.

Retrospective evaluation of nimustine use in the treatment of feline lymphoma.

BACKGROUND: Nimustine, similar to lomustine, is an alkylating agent from the nitrosourea family. There have been some reports regarding lomustine treatment for tumour-bearing cats. However, information regarding nimustine treatment for tumour-bearing cats is limited. OBJECTIVES: To retrospectively evaluate adverse events and clinical outcomes in tumour-bearing cats receiving nimustine. METHODS: Information regarding diagnosis, treatment condition, adverse events, and clinical outcomes was collected in tumour-bearing cats receiving nimustine through reviews of medical records. RESULTS: Nine cats with lymphoma were treated with nimustine in the primary therapy (n = 2) and in the rescue therapy (n = 7). Median starting dose of nimustine was 25 mg/m2 (range: 20-30 mg/m2 ) with dosing interval of three weeks and 1-11 administrations. Adverse events were mild gastrointestinal toxicity (grade 1) including diarrhoea (n = 2) and vomiting (n = 2) and mild myelosuppression (grade 1 or 2) including thrombocytopenia (n = 3) and neutropenia (n = 1). No severe adverse events were observed. Progression-free survival durations among cats receiving nimustine in the primary therapy and in the rescue therapy were 274-688 days (median: 481 days) and 9-671 days (median: 102 days), respectively. Overall survival durations among cats receiving nimustine in the primary therapy and in the rescue therapy were 275-745 days (median: 510 days) and 14-671 days (median: 109 days), respectively. CONCLUSIONS: Nimustine was well tolerated and showed clinical outcomes similar to lomustine in cats with lymphoma. These findings suggest that nimustine might be an alternative to lomustine in the treatment of feline lymphoma.

ErbB2 copy number gain is associated with adverse outcome in canine mammary carcinoma.

Copy number gain (CNG) and/or protein overexpression of ErbB2 have been observed in human breast cancer patients and are associated with poor prognosis. Similarly, ErbB2 overexpression has also been observed in canine mammary carcinoma; however, data on ErbB2 copy number is limited. The purposes of this study were to evaluate ErbB2 copy number in dogs with mammary carcinoma and to investigate associations of ErbB2 CNG with ErbB2 expression, histological and clinical characteristics, and survival. DNA samples were isolated from 59 formalin-fixed paraffin-embedded canine mammary gland tissues (34 carcinoma, 14 adenoma, and 11 normal). Using a digital PCR assay, the ErbB2 copy number in these samples was determined as compared to a reference gene on canine chromosome 8. ErbB2 CNG was detected in 14/34 (41%) carcinomas and 2/14 (14%) adenomas. ErbB2 overexpression was observed in 3/34 (9%) carcinomas but not in adenomas. Neither ErbB2 CNG nor ErbB2 overexpression were detected in the normal controls. There was no significant association of the ErbB2 CNG with histological and clinical characteristics such as age, neutered status, histological grade, tumor size, lymph node involvement, distant metastasis, and clinical stage in the dogs with mammary carcinoma. The presence of ErbB2 CNG, but not ErbB2 overexpression, was significantly related to the shorter overall survival. These findings suggest that ErbB2 CNG is a prognostic factor in dogs with mammary carcinoma.

Protease-Activated Receptor-2 Is Associated With Adverse Outcomes in Canine Mammary Carcinoma.

Protease-activated receptor-2 (PAR2) is a G protein-coupled receptor that is activated by serine proteases. In humans, PAR2 is highly expressed in various cancers, including breast cancer, and is associated with cancer progression and metastasis. However, the expression and roles of PAR2 in canine mammary carcinoma remain unclear. The purpose of this study was to examine the expression of PAR2 in canine mammary carcinoma, the association between PAR2 expression and clinical characteristics, and the role of PAR2 in the metastatic phenotypes of tumor cells. Mammary carcinoma from 31 dogs and 10 normal mammary glands were included in this study, and used for immunohistochemical analysis of PAR2 expression. Normal mammary glands did not express PAR2. In contrast, mammary carcinomas showed PAR2 immunoreactivity in the cytoplasm, and its expression level varied between specimens from negative to strongly positive. The overall survival of dogs with high PAR2 expression was shorter than that of dogs with low PAR2 expression. Moreover, PAR2 expression level was associated with the presence of lymph node involvement, advanced clinical stage, and high histopathological grade. In vitro analyses revealed that a PAR2 agonist accelerated cell migration and invasion in a canine mammary carcinoma cell line. In addition, the PAR2 agonist induced epithelial-mesenchymal transition and actin polymerization. These results suggest that PAR2 expression plays a role in tumor progression and clinical outcomes in canine mammary carcinoma.

ErbB2 Copy Number Aberration in Canine Urothelial Carcinoma Detected by a Digital Polymerase Chain Reaction Assay.

Urothelial carcinoma (UC) is the most common tumor affecting the urinary bladder of dogs. Protein overexpression of ErbB2 (the canine homolog of HER2) has been observed in dogs with UC. However, no study regarding ErbB2 copy number aberration (CNA) is reported in dogs with UC. In this study, a digital PCR assay for detecting CNA of canine ErbB2 was developed. DNA samples were isolated from 83 formalin-fixed, paraffin-embedded urinary bladder tissues (36 UC, 8 polypoid cystitis, and 39 normal) and 94 urinary sediments (54 UC, 30 nonneoplastic, and 10 normal). The copy number of canine chromosome 8 (CFA8) was used as a control. In the urinary bladder tissues, ErbB2 CNA was detected in 12 of 36 (33%) UC, 2 of 8 (25%) polypoid cystitis, and 0 of 39 (0%) normal controls. In the urinary sediments, ErbB2 CNA was also detected in 19 of 54 (35%) UC; however, no ErbB2 CNA was detected in nonneoplastic diseases or normal controls. The sensitivity and specificity of ErbB2 CNA in urinary sediment for the detection of UC were 35% and 100%, respectively. There was a positive correlation between the copy number ratios of ErbB2 to CFA8 in the urinary bladder tissues and urinary sediments. Our findings indicate that the digital PCR assay of urinary sediments may be a useful, noninvasive method for detecting ErbB2 CNA in dogs with UC.

Assessment of HER2 Expression in Canine Urothelial Carcinoma of the Urinary Bladder.

Canine urothelial carcinoma (UC) has a poor prognosis and high metastatic rate. Human epidermal growth factor receptor 2 (HER2), a receptor tyrosine kinase involved in cell proliferation and differentiation regulation, has been attracting interest as a therapeutic target molecule for human breast cancer. This study investigated expression of the canine homolog of HER2 (ERBB2) in canine UC, and its association with clinical factors. Since it has been controversial whether commercial anti-human HER2 antibody (Dako A0485) correctly recognizes the canine homolog of HER2, an application of the antibody using a canine UC cell line was validated first. By Western blot, a single band at the appropriate size for canine HER2 (185 kDa) was recognized. Immunohistochemistry for HER2 was performed on 23 samples of UC, 8 samples of polypoid cystitis, and 8 samples of normal urinary bladder, and the results were scored as either 0, 1+, 2+, or 3+ with reference to the evaluation method for human UC. Intense membranous HER2 immunoreactivity was frequently observed in neoplastic cells, especially in grade 2 UC. Minor HER2 expression was found in the epithelial cells of polypoid cystitis and normal bladder. The incidence of HER2 positivity (scores of 2+ or 3+) was 14 of 23 (60.9%) in UC, 3 of 8 (37.5%) in polypoid cystitis, and 0 of 8 (0%) in normal bladder. There was no significant correlation between HER2 positivity and clinical factors. While increased HER2 expression was observed in a subset of urothelial carcinomas, further mechanistic studies are needed to determine its role in the pathogenesis and targeted therapy of this cancer.

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.

Decreased plasma amino acid concentrations in cats with chronic gastrointestinal diseases and their possible contribution in the inflammatory response.

In humans, plasma amino acids (AAs) levels are used as dynamic nutritional markers. Moreover, some AAs are associated with chronic inflammation. In this study, we analyzed plasma AA profiles in cats with chronic gastrointestinal (GI) diseases. Eight healthy controls (HCs) and 12 client-owned cats with chronic GI diseases including chronic enteritis (n=8) and neoplasms (n=4) were recruited. Plasma albumin, total protein, and 22 AAs (11 essential and 11 non-essential AAs) levels were estimated. There was no significant difference in plasma albumin and total protein concentrations between the cats with chronic GI diseases and HCs. The plasma concentrations of 7 essential AAs (arginine, histidine, lysine, methionine, phenylalanine, taurine, and tryptophan) and 7 non-essential AAs (asparagine, aspartic acid, glutamic acid, glycine, hydroxyproline, proline, and serine) were significantly decreased in the cats with chronic GI diseases (P<0.05). Moreover, plasma histidine and tryptophan levels were inversely correlated with severity of symptoms (histidine: rs=-0.7781, P<0.005; tryptophan: rs=-0.6040, P<0.05). To examine the contribution of altered AAs levels in the inflammatory response, feline macrophages were stimulated by lipopolysaccharides (LPS) with or without histidine, and the expression of interleukin-8 (IL-8) mRNA was quantified. The expression of IL-8 mRNA was significantly increased in the LPS-stimulated feline macrophages (P<0.05). Histidine almost suppressed the LPS-induced IL-8 expression in the feline macrophages (P<0.05). Our findings suggest that plasma AAs levels are more sensitive nutritional markers than albumin and total protein levels in cats with chronic GI diseases. There is a possibility that the decrease of histidine levels in cats with GI diseases is associated with chronic inflammation.

Fecal microbiome in dogs with inflammatory bowel disease and intestinal lymphoma.

Although alteration of commensal microbiota is associated with chronic gastrointestinal (GI) diseases such as inflammatory bowel disease (IBD) in dogs, the microbiota composition in intestinal lymphoma, an important differential diagnosis of canine IBD, has not been investigated. The objective of this study was to compare the fecal microbiota in dogs with IBD, dogs with intestinal lymphoma, and healthy dogs. Eight dogs with IBD, eight dogs with intestinal lymphoma, and fifteen healthy dogs were included in the study. Fecal samples were analyzed by 16S rRNA gene next-generation sequencing. Rarefaction analysis failed to reveal any difference in bacterial diversity among healthy dogs and diseased dogs. Based on PCoA plots of unweighted UniFrac distances, the bacterial composition in dogs with intestinal lymphoma was different from those observed in dogs with IBD and healthy dogs. When compared with healthy dogs, intestinal lymphoma subjects showed significant increases in organisms belonging to the Eubacteriaceae family. The proportion of the family Paraprevotellaceae and the genus Porphyromonas was significantly higher in dogs with IBD compared to healthy dogs. These observations suggest that dysbiosis is associated with intestinal lymphoma as well as IBD in dogs.

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor.

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.