A de novo nonsense variant in the DMD gene associated with X-linked dystrophin-deficient muscular dystrophy in a cat.

BACKGROUND: X-linked dystrophin-deficient muscular dystrophy (MD) is a form of MD caused by variants in the DMD gene. It is a fatal disease characterized by progressive weakness and degeneration of skeletal muscles. HYPOTHESIS/OBJECTIVES: Identify deleterious genetic variants in DMD by whole-genome sequencing (WGS) using a next-generation sequencer. ANIMALS: One MD-affected cat, its parents, and 354 cats from a breeding colony. METHODS: We compared the WGS data of the affected cat with data available in the National Center for Biotechnology Information database and searched for candidate high-impact variants by in silico analyses. Next, we confirmed the candidate variants by Sanger sequencing using samples from the parents and cats from the breeding colony. We used 2 genome assemblies, the standard felCat9 (from an Abyssinian cat) and the novel AnAms1.0 (from an American Shorthair cat), to evaluate genome assembly differences. RESULTS: We found 2 novel high-impact variants: a 1-bp deletion in felCat9 and an identical nonsense variant in felCat9 and AnAms1.0. Whole genome and Sanger sequencing validation showed that the deletion in felCat9 was a false positive because of misassembly. Among the 357 cats, the nonsense variant was only found in the affected cat, which indicated it was a de novo variant. CONCLUSION AND CLINICAL IMPORTANCE: We identified a de novo variant in the affected cat and next-generation sequencing-based genotyping of the whole DMD gene was determined to be necessary for affected cats because the parents of the affected cat did not have the risk variant.

Successful treatment of sclerosing encapsulating peritonitis in a cat using bioresorbable hyaluronate-carboxymethylcellulose membrane after surgical adhesiolysis and long-term prednisolone.

Case summary: An 8-year-old neutered male domestic shorthair indoor cat was presented with an 8-week history of intermittent vomiting, anorexia and weight loss that had been unresponsive to supportive treatment. Abdominal ultrasound revealed plication of the small intestine and fluid accumulation proximal to the lesion, and a linear foreign body was suspected. An exploratory celiotomy showed cocoon-like encapsulation of the entire intestine. Surgical adhesiolysis and full-thickness biopsy were performed, and histopathologic examination revealed mild thickening of the visceral peritoneum with fibrin deposition, as well as mild neutrophil and lymphocyte infiltration. These findings were compatible with sclerosing encapsulating peritonitis (SEP). The cat recovered well postoperatively and was discharged the next day. Prednisolone was administered for 7 weeks to prevent recurrence of SEP. Five months after surgery, the cat was re-presented with anorexia and chronic vomiting. Based on the clinical examination findings, recurrent SEP was suspected. At the second surgery, surgical adhesiolysis was repeated and a bioresorbable hyaluronate-carboxymethylcellulose membrane was used to cover the serosal surface and thus prevent adhesion formation. Histopathologic findings of the peritoneal biopsy specimen confirmed SEP. Long-term prednisolone treatment (1 mg/kg for the first dose and 0.5 mg/kg every 48 h for maintenance) was administered postoperatively. The cat survived for more than 1239 days without recurrence. Relevance and novel information: To our knowledge, this is the first report of SEP in a cat with long-term survival. The use of a bioresorbable hyaluronate-carboxymethylcellulose membrane and long-term prednisolone treatment may have prevented short-term and long-term recurrence, respectively, in this case.

Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.

Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

Diverse genome-wide DNA methylation alterations in canine hepatocellular tumours.

BACKGROUND: Canine hepatocellular tumours (HCTs) are common primary liver tumours. However, the exact mechanisms of tumourigenesis remain unclear. Although some genetic mutations have been reported, DNA methylation alterations in canine HCT have not been well studied. OBJECTIVES: In this study, we aimed to analyse the DNA methylation status of canine HCT. METHODS: Tissues from 33 hepatocellular carcinomas, 3 hepatocellular adenomas, 1 nodular hyperplasia, 21 non-tumour livers from the patients and normal livers from 5 healthy dogs were used. We analysed the DNA methylation levels of 72,367 cytosine-guanine dinucleotides (CpG sites) in all 63 samples. RESULTS AND CONCLUSIONS: Although a large fraction of CpG sites that were highly methylated in the normal liver became hypomethylated in tumours from most patients, we also found some patients with less remarkable change or no change in DNA methylation. Hierarchical clustering analysis revealed that 32 of 37 tumour samples differed from normal livers, although the remaining 5 tumour livers fell into the same cluster as normal livers. In addition, the number of hypermethylated genes in tumour livers varied among tumour cases, suggesting various DNA methylation patterns in different tumour groups. However, patient and clinical parameters, such as age, were not associated with DNA methylation status. In conclusion, we found that HCTs undergo aberrant and diverse patterns of genome-wide DNA methylation compared with normal liver tissue, suggesting a complex epigenetic mechanism in canine HCT.

Enhanced Systemic Antitumour Immunity by Hypofractionated Radiotherapy and Anti-PD-L1 Therapy in Dogs with Pulmonary Metastatic Oral Malignant Melanoma.

Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.

Systemic mucoid degeneration of the arterial tunica intima in a young dog.

A 27-mo-old, spayed female mixed-breed dog was presented with left forelimb pain, which progressed to full thickness necrosis of the soft tissues of multiple limbs. Clinical imaging and postmortem examination suggested multiple large arterial thromboemboli. Histologic examination of vascular lesions revealed markedly thickened tunica intima with polypoid intraluminal projections, which partially to entirely occluded the arterial lumen. The expanded tunica intima was comprised of intimal accumulation of Alcian blue-positive matrix with scattered spindle-to-satellite cells. These cells were positive for von Willebrand factor and vimentin but negative for α-smooth muscle actin, suggesting endothelial origin. Deposition of the intimal mucoid matrix was observed in the elastic and muscular arteries associated with regional ischemic changes. Mucoid emboli, likely from fragmentation of proliferative intimal tissue, were identified in smaller vessels supplied by affected arteries. Based on these findings, we diagnosed systemic mucoid degeneration of the arterial tunica intima. Such systemic arterial degeneration characterized by deposition of mucoid matrix in the tunica intima has not been reported previously in dogs, to our knowledge, and should be distinguished from thromboembolism and other degenerative vascular diseases.

Immunohistochemical evaluation of HER2 expression in canine thyroid carcinoma.

The human epidermal growth factor receptor 2 (HER2) is expressed in various human cancers including thyroid cancers (TC) and is used as a diagnostic marker and therapeutic target. Canine TC (cTC), the most common endocrine malignancy in dogs, shows a high metastasis rate, and HER2-targeted therapy could be a candidate for treatment. Here, we immunohistochemically evaluated HER2 expression in 21 paraffin-embedded cTC tissues and scored the degree of expression based on intensity and positivity (score: 0-3+). Four samples (19%) scored 3+; 6 (29%), 2+; 7 (33%), 1+; and 4 (19%), 0. Therefore, 48% of the cTC tissues were HER2 positive (scored ≥2+). These data may lead to further evaluation of the role of HER2 in cTC as a mechanism of malignancy and a therapeutic target.

Detection of human epidermal growth factor receptor 2 overexpression in canine anal sac gland carcinoma.

Canine anal sac gland carcinoma (ASGC) frequently occurs in the apocrine glands of the canine anal sac and shows aggressive biological behavior. The expression of human epidermal growth factor receptor 2 (HER2) has been reported in various human and canine tumors. HER2 is a promising therapeutic target of these tumors, and HER2-targeted drugs, such as trastuzumab and lapatinib, have improved the outcome of these patients. In this study, HER2 expression in ASGC was evaluated to investigate its potential as a therapeutic target for canine ASGC. HER2 mRNA expression in surgically resected ASGC tissues was significantly higher than that in normal anal sac tissue. To evaluate the expression of HER2 protein, paraffin-embedded ASGC tissues were immunohistochemically evaluated. Strong and broad staining of HER2 was detected in ASGC tissues, while HER2 was weakly to moderately stained in normal anal sac apocrine glands and squamous epithelia. The degree of HER2 expression in ASGC tissues was scored based on its intensity and positivity (score: 0-3+). Scoring of HER2 expression revealed 6 samples (24%) scored 3+, 14 (56%) scored 2+, and 5 (20%) scored 1+, with no samples scoring 0. In all, 80% of canine ASGC tissues were positive for HER2 (scored ≥2+). Furthermore, putative HER2-overexpressed cells in ASGC were detected with trastuzumab by flow cytometry. These preliminary data may lead to further evaluation of the role of HER2 in canine ASGC as a mechanism of malignancy and as a therapeutic target for HER2-targeted therapy.

Histopathologic and Immunohistochemistry Findings in Feline Renal Cell Carcinoma.

The biological behavior and immunohistochemical features of feline renal cell carcinoma (RCC) have not been well characterized. In the present study, immunohistochemical examinations were performed in 12 feline cases of RCC. The RCC consisted of solid ( n = 2), solid-tubular ( n = 2), tubular ( n = 3), papillary ( n = 2), tubulopapillary ( n = 2), and sarcomatoid ( n = 1) type lesions. Of the cases with RCC, 1 developed metastatic disease and 6 cases had no evidence of recurrence at 80 to 2292 days after surgery. One papillary-type tumor had cuboidal cells with scant cytoplasm and monomorphic nuclei, and the other had pseudostratified columnar cells with abundant cytoplasm. Immunohistochemistry revealed that the tumor cells in most cases were positive for cytokeratin (CK)7, CK20, KIT, and CD10, with the exception of cases of the solid type with clear cytoplasm (solid anaplastic), papillary type with columnar cells, and sarcomatoid types. A small number of tumor cells in the solid anaplastic and in the sarcomatoid types were positive for aquaporin-1. Increased expression of N-cadherin and Twist along with nuclear accumulation of ß-catenin were observed in the sarcomatoid type. These results indicated that CK, KIT, and CD10 are relatively strongly expressed in most feline RCC. The solid anaplastic RCC exhibited CD10 expression with the absence of distal tubule marker expression. Although immunohistochemistry profiles were relatively consistent with those described in human RCC, the histopathologic features were different from those seen in humans. Epithelial-mesenchymal transition (EMT) marker expression in the current cases may suggest the involvement of an EMT-like mechanism in the development of sarcomatoid RCC in cats.

Density of tumor-infiltrating granzyme B-positive cells predicts favorable prognosis in dogs with transitional cell carcinoma.

Although tumor-infiltrating lymphocytes (TILs) play a key role in anti-tumor immunity, their involvement in canine transitional cell carcinoma (TCC) is not well-documented. The objective of this study was to investigate the association between TIL number and prognosis in dogs with urinary bladder TCC. Immunohistochemical analysis of CD3 and granzyme B was performed using canine TCC (n=32) and normal bladder (n=10) tissues. The numbers of CD3+ and granzyme B+ cells located in peritumoral stroma of canine TCC were significantly higher than those in normal controls. In TCC cases, the number of CD3+ TILs was not significantly related to prognosis, whereas the abundant granzyme B+ TILs were associated with favorable outcome. Since granzyme B+ TILs were not associated with the tumor stage, the presence of granzyme B+ TILs may be an independent prognostic factor. These results suggest that granzyme B+ TILs play a role in anti-tumor immunity and inhibit tumor progression in canine TCC.

The effect of L-citrulline ingestion on ECG QT interval and autonomic nervous system activity.

The purpose of this study was to investigate the effect of L-citrulline ingestion on ECG QT interval. To accomplish this purpose, nine male subjects (age: 23.4±0.5 years, weight: 57.7±5.6 kg) participated, who had no history of cardiovascular or respiratory function disorders. Each subject performed two tests (L-citrulline and placebo ingestion) on a separate day. ECG was taken before, and 60 min and 90 min after L-citrulline ingestion. The heart-rate-corrected QT interval (QTc) decreased significantly 60 min (p=0.0004) and 90 min (p=0.011) after L-citrulline ingestion. However, there were no significant changes after placebo ingestion. In addition, the interaction between L-citrulline ingestion and placebo was significant (two-way ANOVA: interaction, p=0.010). Our major finding was that L-citrulline ingestion decreases the QT interval in healthy subjects. This result suggests that transient L-citrulline ingestion may shorten the time required to complete myocardial depolarization and repolarization.