Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.

BACKGROUND: Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. RESULTS: In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci. CONCLUSIONS: SG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted.

Epidemiology of mammary tumours in bitches under veterinary care in the UK in 2016.

BACKGROUND: There is limited information on the epidemiology of canine mammary tumours. This study aimed to estimate the incidence and risk factors for mammary tumours in UK bitches. METHODS: A nested case-control study was conducted within VetCompass to estimate the frequency and risk factors for clinically diagnosed mammary tumours during 2016 (VetCompass study). A second case-control study explored further breed associations for cases confirmed histopathologically compared to the VetCompass controls (laboratory study). Multivariable logistic regression was used to evaluate associations between risk factors and mammary tumours. RESULTS: The incidence of mammary tumours was 1340.7/100,000 per year (95% confidence interval: 1198.1-1483.3). A total of 222 clinical cases (VetCompass study) and 915 laboratory cases (laboratory study) were compared to 1515 VetCompass controls in the two analyses. In the VetCompass study, Springer and Cocker Spaniels, Boxers, Staffordshire Bull Terriers and Lhasa Apsos had increased odds of developing mammary tumours. Neutering was associated with reduced odds, while odds increased with increasing age and a history of pseudopregnancy. In the laboratory study, increasing age was associated with greater odds of mammary tumours, and the breeds most at risk were similar to those identified in the VetCompass study. LIMITATIONS: The timing of neutering was not consistently available. Comparing laboratory cases to VetCompass controls provided only exploratory evidence for the breed associations identified. CONCLUSIONS: The study provides an update on the frequency of canine mammary tumours.

High-Grade Cutaneous Mast Cell Tumour with Widespread Intrathoracic Metastasis and Neoplastic Pericardial Effusion in a Dog.

An 8-year-old neutered male French Bulldog was presented with a 2-day history of intermittent vomiting, reduced appetite and recent rapid development of multiple cutaneous masses over the head and neck regions. On presentation, the patient had a moderate volume of pericardial and bilateral pleural effusion. Echocardiography demonstrated irregular, heterogeneous thickening of the walls of the right ventricle and right atrium, consistent with infiltrative intramyocardial disease. Cytological examination of fine needle aspirates from one of the cutaneous masses confirmed a mast cell tumour. Pericardial fluid analysis revealed a haemorrhagic neoplastic effusion due to mast cell neoplasia. Histopathological and immunohistochemical examination of tissues obtained post mortem confirmed a high-grade cutaneous mast cell tumour with metastasis to the heart, pericardium, mediastinum and spleen. No metastatic disease was present in the submandibular lymph nodes or liver. Immunohistochemistry demonstrated KIT staining pattern 2. There was strong nuclear Ki67 labelling in an average of 65.0 cells per grid and an average of three positive AgNORs per nucleus in neoplastic cells. Polymerase chain reaction for the activating duplication mutation in exons 8 and 11 of c-Kit were negative. To the authors' knowledge, this is the first report of a canine cutaneous mast cell tumour associated with neoplastic pericardial effusion and widespread intrathoracic metastasis.

Gene Expression Profiling of B Cell Lymphoma in Dogs Reveals Dichotomous Metabolic Signatures Distinguished by Oxidative Phosphorylation.

Gene expression profiling has revealed molecular heterogeneity of diffuse large B cell lymphoma (DLBCL) in both humans and dogs. Two DLBCL subtypes based on cell of origin are generally recognized, germinal center B (GCB)-like and activated B cell (ABC)-like. A pilot study to characterize the transcriptomic phenotype of 11 dogs with multicentric BCL yielded two molecular subtypes distinguished on the basis of genes important in oxidative phosphorylation. We propose a metabolic classification of canine BCL that transcends cell of origin and shows parallels to a similar molecular phenotype in human DLBCL. We thus confirm the validity of this classification scheme across widely divergent mammalian taxa and add to the growing body of literature suggesting cellular and molecular similarities between human and canine non-Hodgkin lymphoma. Our data support a One Health approach to the study of DLBCL, including the advancement of novel therapies of relevance to both canine and human health.

Evaluation of variants of melanoma-associated antigen genes and mRNA transcripts in melanomas of dogs.

OBJECTIVE-To characterize variability in melanoma-associated antigen (MAA) genes and gene expression in melanomas of dogs. ANIMALS-18 dogs with malignant melanomas and 8 healthy control dogs. PROCEDURES-cDNA was prepared from malignant melanoma biopsy specimens and from pigmented oral mucocutaneous tissues of healthy control dogs. Genomic DNA was extracted from poorly pigmented melanomas. A PCR assay was performed by use of Melan-A, SILV, or tyrosinase-specific primers. RESULTS-Splice variants of Melan-A and SILV were identified in malignant melanomas and also in healthy pigmented tissues, whereas a tyrosinase splice variant was detected in melanoma tissues only. A short interspersed nuclear element (SINE) insertion mutation was identified in the SILV gene in 1 of 10 poorly pigmented melanomas. Six novel exonic single nucleotide polymorphisms (SNPs; 3 synonymous and 3 nonsynonymous) were detected in the tyrosinase gene, and 1 nonsynonymous exonic SNP was detected in the SILV gene. CONCLUSIONS AND CLINICAL RELEVANCE-Variants of MAA mRNA were detected in malignant melanoma tissues of dogs. The importance of MAA alternative transcripts expressed in melanomas and normal pigmented tissues was unclear, but they may have represented a means of regulating melanin synthesis. The tyrosinase splice variant was detected only in melanomas and could potentially be a tumor-specific target for immunotherapy. A SILV SINE insertion mutation was identified in a melanoma from a Great Dane, a breed known to carry this mutation (associated with merle coat color). The nonsynonymous SNPs detected in tyrosinase and SILV transcripts did not appear to affect tumor pigmentation.

Phenotypic characterisation of regulatory T cells in dogs reveals signature transcripts conserved in humans and mice.

Regulatory T cells (Tregs) are a double-edged regulator of the immune system. Aberrations of Tregs correlate with pathogenesis of inflammatory, autoimmune and neoplastic disorders. Phenotypically and functionally distinct subsets of Tregs have been identified in humans and mice on the basis of their extensive portfolios of monoclonal antibodies (mAb) against Treg surface antigens. As an important veterinary species, dogs are increasingly recognised as an excellent model for many human diseases. However, insightful study of canine Tregs has been restrained by the limited availability of mAb. We therefore set out to characterise CD4+CD25high T cells isolated ex vivo from healthy dogs and showed that they possess a regulatory phenotype, function, and transcriptomic signature that resembles those of human and murine Tregs. By launching a cross-species comparison, we unveiled a conserved transcriptomic signature of Tregs and identified that transcript hip1 may have implications in Treg function.

Phenotypic and transcriptomic characterization of canine myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are key players in immune evasion, tumor progression and metastasis. MDSCs accumulate under various pathological states and fall into two functionally and phenotypically distinct subsets that have been identified in humans and mice: polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. As dogs are an excellent model for human tumor development and progression, we set out to identify PMN-MDSCs and M-MDSCs in clinical canine oncology patients. Canine hypodense MHC class II-CD5-CD21-CD11b+ cells can be subdivided into polymorphonuclear (CADO48A+CD14-) and monocytic (CADO48A-CD14+) MDSC subsets. The transcriptomic signatures of PMN-MDSCs and M-MDSCs are distinct, and moreover reveal a statistically significant similarity between canine and previously published human PMN-MDSC gene expression patterns. As in humans, peripheral blood frequencies of canine PMN-MDSCs and M-MDSCs are significantly higher in dogs with cancer compared to healthy control dogs (PMN-MDSCs: p < 0.001; M-MDSCs: p < 0.01). By leveraging the power of evolution, we also identified additional conserved genes in PMN-MDSCs of multiple species that may play a role in MDSC function. Our findings therefore validate the dog as a model for studying MDSCs in the context of cancer.

Owner perceptions of their cat's quality of life when treated with a modified University of Wisconsin-Madison protocol for lymphoma.

Objectives The objectives of this study were to assess owner perceptions of their cat's quality of life during treatment for lymphoma with a doxorubicin-containing multi-agent chemotherapy protocol, whether various health-related parameters correlated with quality of life scores, and to assess owner satisfaction with the protocol. Methods A postal questionnaire was sent to the owners of 33 treated cats. Owners retrospectively assessed their cat's quality of life using a Likert scale (1-10) before lymphoma was diagnosed, at diagnosis and during chemotherapy. Owners assigned scores to various health-related parameters previously reported to affect quality of life at the three time points, and correlations with quality of life scores were sought. Owners were asked to rate the importance of these health-related parameters. Satisfaction with the protocol was investigated. Results Twenty questionnaires were completed (61% response rate). The median quality of life score before diagnosis (10, range 5-10) was higher than at diagnosis (3, range 1-9) ( P <0.05). The median quality of life score during chemotherapy (7, range 3-9) was lower than before diagnosis ( P <0.05) and higher than at diagnosis, but this was not statistically significant. Quality of life scores did not correlate with individual health-related parameter scores consistently; however, quality of life scores did correlate with appetite scores during chemotherapy. Appetite, vomiting and diarrhoea were parameters perceived as important in affecting quality of life. Most owners (75%) were happy they had treated their cat. Conclusions and relevance The quality of life scores observed were comparable to a previous study using cyclophosphamide, vincristine and prednisolone, employing the same scoring system. Although quality of life scores during chemotherapy were not significantly improved at diagnosis, owner satisfaction with the protocol was high. The factors perceived by owners to determine quality of life in their pets may be different to those previously conjectured, but appetite during chemotherapy remains important.

Hemothorax in Three Dogs with Intrathoracic Extracardiac Hemangiosarcoma.

Intrathoracic extracardiac hemangiosarcoma (HSA) is rare in dogs. This report describes three dogs with acute onset dyspnea due to hemorrhagic pleural effusion resulting from intrathoracic extracardiac masses, which were confirmed as HSA by histopathology. The dogs were stabilized with thoracocentesis and intravascular fluid resuscitation. Computed tomography identified intrathoracic masses, which were not originating from the heart or pulmonary parenchyma. Surgical exploration was performed in all cases. Case 1 was euthanized intraoperatively as the tumor could not be dissected from the aorta. In cases 2 and 3, hemostasis and resection of the tumors was successful. Case 2 was euthanized 1 mo after surgery and case 3 was alive at the time of writing, 5 mo postoperatively. Intrathoracic extracardiac HSA should be considered as a differential for nontraumatic hemothorax and surgical treatment can be palliative.

Prevalence and risk factors for mast cell tumours in dogs in England.

BACKGROUND: Mast cell tumour (MCT) appears to be a frequent tumour type in dogs, though there is little published in relation to its frequency in dogs in the UK. The current study aimed to investigate prevalence and risk factors for MCTs in dogs attending English primary-care veterinary practices. METHODS: Electronic patient records from practices participating in the VetCompass animal surveillance project between July 2007 and June 2013 were searched for MCT diagnosis. Various search terms and standard diagnostic terms (VeNom codes) identified records containing MCT diagnoses, which were evaluated against clinical criteria for inclusion to the study. MCT prevalence for the entire dataset and specific breed types were calculated. Descriptive statistics characterised MCT cases and multivariable logistic regression methods evaluated risk factors for association with MCT (P < 0.05). RESULTS: Within a population of 168,636 dogs, 453 had MCT, yielding a prevalence of 0.27% (95% confidence interval (CI) 0.24% - 0.29%). The highest breed type specific prevalences were for the Boxer at 1.95% (95% CI 1.40% - 2.51%), Golden Retriever at 1.39% (0.98% - 1.81%) and Weimaraner at 0.85% (95% CI 0.17% to 1.53%). Age, insurance status, neuter status, weight and breed type were associated with MCT diagnosis. Of dogs of specific breed type, the Boxer, Pug and Staffordshire Bull Terrier showed greater odds of MCT diagnosis compared with crossbred dogs. Conversely, the German Shepherd Dog, Border Collie, West Highland White Terrier, Springer Spaniel and Cocker Spaniel had reduced odds of MCT diagnosis compared with crossbred dogs. No association was found between MCT diagnosis and sex. CLINICAL SIGNIFICANCE: This study highlights a clinically significant prevalence of MCT and identifies specific breed types with predisposition to MCT, potentially aiding veterinarian awareness and facilitating diagnosis.

Appendicular arterial tumor embolization in two cats with pulmonary carcinoma.

A 13-year-old neutered male Persian cat and an 11-year-old neutered female Persian cat were examined because of an acute onset of lameness. In both cats, conscious proprioception and reflexes were diminished in the affected limb. In 1 cat, no blood flow was detected in the left brachial artery with a Doppler ultrasonic flow detector, whereas blood flow in the right brachial artery was easily documented. In the other cat, the right femoral pulse was not palpable. Neither cat had any echocardiographic evidence of cardiac disease. In both cats, treatment was primarily supportive. One cat died, and the other was euthanatized. At necropsy, lung lobe consolidation was seen. Microscopically, there was multifocal infiltration of the lung parenchyma with cuboidal to columnar neoplastic epithelial cells. Neoplastic epithelial cells of similar morphology were identified in nodular masses in sections of muscle, and intravascular tumor emboli were identified obliterating small and large arterioles. Immunohistochemical staining of pulmonary and muscular tissue for pan-cytokeratin antigen revealed intense cytoplasmic staining of neoplastic cells. Staining for factor VIII-related antigen confirmed that clusters of neoplastic cells represented intravascular emboli. Clinical signs in the cats were attributed to arterial occlusion by tumor emboli.

Evaluation of low-dose metronomic (LDM) cyclophosphamide toxicity in cats with malignant neoplasia.

Oral administration of low-dose cyclophosphamide in pets with spontaneously occurring malignant neoplasms has become a common practice in veterinary medicine. The purpose of this retrospective study was to investigate toxicity events in cats with spontaneous malignancies receiving cyclophosphamide as a metronomic therapy for at least 1 month. The number and severity of clinical, haematological and biochemical adverse events were recorded according to the Veterinary Cooperative Oncology Group's Common Terminology Criteria for Adverse Events v1.1 classification scheme. Twenty-four cats were enrolled in the study with a total number of 27 neoplasms: 13 sarcomas, 12 carcinomas, one melanoma and one neuroendocrine tumour. Seventeen cats presented with macroscopic disease, while seven had microscopic disease. Seven cats (29%) had metastasis either to the regional lymph nodes and/or distant sites at the time of study enrolment. Additional medications, administered concurrently, included non-steroidal anti-inflammatory drugs (17), toceranib (4) and thalidomide (7). Four cats showed grade I gastrointestinal toxicity during the first month of treatment, which was controlled with antiemetics. Overall, 2/24 cats (8%) showed grade I haematological toxicities and 1/24 (4%) showed grade I renal toxicity in the first 4 weeks. Median follow-up for all cats was 30 days (range 30-360 days). For the 15 cats with follow-up longer than 1 month the only additional toxicities observed were two grade III and one grade II azotaemia that occurred after 2 months of therapy. Low-dose cyclophosphamide seems to be a well-tolerated option for cats bearing primary or metastatic tumours. Evaluation of toxicity after long-term administration is still needed.

Feline mediastinal lymphoma: a retrospective study of signalment, retroviral status, response to chemotherapy and prognostic indicators.

Historically, feline mediastinal lymphoma has been associated with young age, positive feline leukaemia virus (FeLV) status, Siamese breed and short survival times. Recent studies following widespread FeLV vaccination in the UK are lacking. The aim of this retrospective multi-institutional study was to re-evaluate the signalment, retroviral status, response to chemotherapy, survival and prognostic indicators in feline mediastinal lymphoma cases in the post-vaccination era. Records of cats with clinical signs associated with a mediastinal mass and cytologically/histologically confirmed lymphoma were reviewed from five UK referral centres (1998-2010). Treatment response, survival and prognostic indicators were assessed in treated cats with follow-up data. Fifty-five cases were reviewed. The median age was 3 years (range, 0.5-12 years); 12 cats (21.8%) were Siamese; and the male to female ratio was 3.2:1.0. Five cats were FeLV-positive and two were feline immunodeficiency-positive. Chemotherapy response and survival was evaluated in 38 cats. Overall response was 94.7%; complete (CR) and partial response (PR) rates did not differ significantly between protocols: COP (cyclophosphamide, vincristine, prednisone) (n = 26, CR 61.5%, PR 34.0%); Madison-Wisconsin (MW) (n = 12, CR 66.7%, PR 25.0%). Overall median survival was 373 days (range, 20-2015 days) (COP 484 days [range, 20-980 days]; MW 211 days [range, 24-2015 days] [P = 0.892]). Cats achieving CR survived longer (980 days vs 42 days for PR; P = 0.032). Age, breed, sex, location (mediastinal vs mediastinal plus other sites), retroviral status and glucocorticoid pretreatment did not affect response or survival. Feline mediastinal lymphoma cases frequently responded to chemotherapy with durable survival times, particularly in cats achieving CR. The prevalence of FeLV-antigenaemic cats was low; males and young Siamese cats appeared to be over-represented.

Dissecting the regulatory microenvironment of a large animal model of non-Hodgkin lymphoma: evidence of a negative prognostic impact of FOXP3+ T cells in canine B cell lymphoma.

The cancer microenvironment plays a pivotal role in oncogenesis, containing a number of regulatory cells that attenuate the anti-neoplastic immune response. While the negative prognostic impact of regulatory T cells (Tregs) in the context of most solid tissue tumors is well established, their role in lymphoid malignancies remains unclear. T cells expressing FOXP3 and Helios were documented in the fine needle aspirates of affected lymph nodes of dogs with spontaneous multicentric B cell lymphoma (BCL), proposed to be a model for human non-Hodgkin lymphoma. Multivariable analysis revealed that the frequency of lymph node FOXP3(+) T cells was an independent negative prognostic factor, impacting both progression-free survival (hazard ratio 1.10; p = 0.01) and overall survival (hazard ratio 1.61; p = 0.01) when comparing dogs showing higher than the median FOXP3 expression with those showing the median value of FOXP3 expression or less. Taken together, these data suggest the existence of a population of Tregs operational in canine multicentric BCL that resembles thymic Tregs, which we speculate are co-opted by the tumor from the periphery. We suggest that canine multicentric BCL represents a robust large animal model of human diffuse large BCL, showing clinical, cytological and immunophenotypic similarities with the disease in man, allowing comparative studies of immunoregulatory mechanisms.