Evaluation of a Manual Cytocentrifuge versus the Standard Automated Cytocentrifuge in the Analysis of Canine Cerebrospinal Fluid: A Case Series of 55 Dogs.

Cytospins are important for evaluating fluids with very low cellularity such as cerebrospinal fluid (CSF). The aim of this study was to compare the CSF cytospin preparations obtained from automated and manual cytocentrifugation methods. A prospective case series was performed to analyze canine CSF samples using both centrifugation methods. The cytospins were processed within 30-60 min and prepared simultaneously in a conventional automated cytocentrifuge and in an in-house manual cytocentrifuge, using a fixed volume of CSF fluid. The cellularity, differential cell count and the proportion of cell artifacts (pseudopods and vacuolization) were blindly assessed in the cytospin preparations obtained using the two methods. The agreement and correlation between both methods were analyzed. There were 55 dogs enrolled (48 prospectively and 7 retrospectively) in the study. 38 dogs had normal total nucleated cell counts, while 17 had pleocytosis. Automated and manual cytocentrifugation had similar cell yields, and no significant differences in differential cell counts or the presence of artifacts existed between both methods. In cases with pleocytosis, the cytologic diagnosis obtained using each method was similar. Manual cytocentrifugation of CSF is a reliable and economic method designed for routine clinical practice. Its use reduces the specimen deterioration related to processing and analysis delays when samples are transported to external laboratories for evaluation.

Detection of Lymphoid Markers (CD3 and PAX5) for Immunophenotyping in Dogs and Cats: Comparison of Stained Cytology Slides and Matched Cell Blocks.

Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

Prognostic factors associated with survival and hospitalization time in pediatric canine patients diagnosed with presumptive acute viral gastroenteritis.

Background and Aim: Acute viral gastroenteritis is one of the main causes of hospitalization in dogs during the 1st year of life. This retrospective study aimed to describe a pediatric canine population presumptively diagnosed with acute viral gastroenteritis and to identify potential prognostic factors that influence hospitalization time (HT) and mortality. Materials and Methods: Canine patients up to 12 months of age diagnosed with presumptive acute viral gastroenteritis were searched retrospectively from two veterinary hospitals during a 5-year period (2015-2020). Information regarding patient signalment, prophylactic care, clinical signs, blood test results, presence of systemic inflammatory response syndrome, and additional treatments were recorded to analyze their association with HT and mortality. Only dogs with a complete medical record until death or discharge were included in the study. Results: Ninety-four dogs were identified: 76 dogs (80.9%) survived with a median HT of 5 days (range: 2-16 days) and 18 dogs (19.1%) died with a median HT of 3½ days (range: 1-8 days) after admission. The presence of fever and fresh frozen plasma (FFP) administration was significantly associated with a lower survival rate (p = 0.021 and p = 0.037) in the multivariate analysis. Among survivors, incomplete primo-vaccination, the presence of hematochezia, and FFP administration were considered independent predictors of time to clinical recovery (p = 0.026, p = 0.047, and p = 0.026, respectively), being associated with higher HT. Conclusion: The presence of fever and FFP administration was significantly associated with a lower survival rate. An inadequate primo-vaccination status prior to admission, hematochezia, and FFP administration was associated with longer HT in surviving patients. Further studies are needed to confirm the present results.

Edaravone for acute ischemic stroke - Systematic review with meta-analysis.

INTRODUCTION: Ischemic stroke is a major cause of death and disability. Despite major advances in reperfusion therapies, most patients don´t benefit from these treatments as the time window for such interventions is limited. Therefore, other treatment options are desirable. Edaravone has been demonstrated in previous studies to reduce neurologic deficits in stroke patients. OBJECTIVE: To test the hypothesis that edaravone reduces functional dependence in ischemic stroke patients. MATERIAL AND METHODS: Systematic review and meta-analysis of randomized controlled trials and observational studies comparing edaravone to placebo in adult patients with ischemic stroke. The efficacy outcomes of interest were good and excellent functional outcomes at 90 days, defined as modified Rankin Scale (mRS) scores of 0-2 and 0-1 respectively. The safety outcomes of interest were intracranial hemorrhage and mortality. RESULTS: 19 studies were included. Edaravone treatment was associated with improved chances of 90-day good (OR=1.31, 95% CI 1.06-1.67) and excellent (OR=1.26, 95% CI 1.04-1.54) functional outcomes. Mortality was also lower in edaravone treated patients (OR=0.50, 95% CI 0.45-0.56). There were no differences in terms of intracranial hemorrhage. Most studies were observational and performed in Asian populations, especially Japan. Heterogeneity was high for all outcomes but reduced when analysis was restricted to randomized trials. CONCLUSION: Edaravone is a promising treatment for ischemic stroke patients, with a more favorable time window. However, more randomized studies including patient populations outside Asia are required to confirm this hypothesis.

The Dog as a Model to Study the Tumor Microenvironment.

Cancer is a complex and dynamic disease with an outcome that depends on a strict crosstalk between tumor cells and other components in tumor microenvironment, namely, tumor-infiltrating immune cells, fibroblasts, cancer stem cells, adipocytes, and endothelial cells. Within the tumor microenvironment, macrophages and T-lymphocytes appear to be key effectors during the several steps of tumor initiation and progression. Tumor cells, through the release of a plethora of signaling molecules, can induce immune tolerance, by avoiding immune surveillance, and inhibit immune cells cytotoxic functions. Furthermore, as the tumor grows, tumor microenvironment reveals a series of dysfunctional conditions that potentiate a polarization of harmful humoral Th2 and Th17, an upregulation of Treg cells, and a differentiation of macrophages into the M2 subtype, which contribute to the activation of several signaling pathways involving important tissue biomarkers (COX-2, EGFR, VEGF) implicated in cancer aggressiveness and poor clinical outcomes. In order to maintain the tumor growth, cancer cells acquire several adaptations such as neovascularization and metabolic reprogramming. An extensive intracellular production of lactate and protons is observed in tumor cells as a result of their high glycolytic metabolism. This contributes not only for the microenvironment pH alteration but also to shape the immune response that ultimately impairs immune cells capabilities and effector functions.In this chapter, the complexity of tumor microenvironment, with special focus on macrophages, T-lymphocytes, and the impact of lactate efflux, was reviewed, always trying to demonstrate the strong similarities between data from studies of humans and dogs, a widely proposed model for comparative oncology studies.

Therapeutic Effect of EPA/DHA Supplementation in Neoplastic and Non-neoplastic Companion Animal Diseases: A Systematic Review.

BACKGROUND/AIM: The aim of this study is to identify and describe randomized controlled studies evaluating the therapeutic effect of EPA and DHA supplementation in companion animal diseases. MATERIALS AND METHODS: A systematic search was conducted in PubMed database and the information collected was summarized and evaluated according to the risk of bias, using the revised Cochrane tool (RoB2). RESULTS: Twenty-three studies were eligible for inclusion: twenty performed in dogs and three in cats. A therapeutic benefit was found in canine allergic dermatitis, haircoat disorder, keratoconjunctivitis sicca, valvular disease, and canine and feline osteoarthritis. Dogs diagnosed with chronic heart failure and lymphoma and cats with allergic dermatitis also seem to benefit from supplementation with omega-3 fatty acids, but studies with improved methodological quality are needed to strengthen this evidence. CONCLUSION: EPA and DHA supplementation has proven benefits in the adjuvant treatment of various neoplastic and non-neoplastic diseases in dogs and cats.

The role of COX expression in the prognostication of overall survival of canine and feline cancer: A systematic review.

Cyclooxygenase (COX) isoforms-1 and -2 have been extensively investigated in cancer. Although COX-2 is the isoform most studied and has been described in several malignancies associated with histologic criteria of malignancy and worse prognosis, COX-1 has also been linked to some forms of cancer. With the present review our aim was to summarize the current state of knowledge and clarify if and in which type of tumours COX-1 and/or COX-2 expression have real prognostic implications. We searched PubMed database for prognostic studies using predefined inclusion criteria in order to ascertain the prognostic value of COX-1 and COX-2 in malignant neoplasia in dogs and cats. Eighteen studies were analysed. COX-2 was shown to be a negative prognostic factor in canine and feline mammary tumours, canine mast cell tumour, canine melanoma, canine osteosarcoma and canine renal cell carcinoma. COX-1 showed a negative prognostic value in feline oral squamous cell carcinoma (SCC). We found high heterogeneity among studies regarding COX immunohistochemical evaluation methodology even in the same type of neoplasia pointing out the need for its standardization at least by tumour type. The available data support the use of COX-2 as a prognostic factor in canine (mammary carcinoma, mast cell tumour, melanoma, osteosarcoma and renal carcinoma) and feline (mammary carcinoma) cancers. For COX-1, its use is advised in feline oral SCC.

Metastatic feline mammary cancer: prognostic factors, outcome and comparison of different treatment modalities - a retrospective multicentre study.

OBJECTIVES: Although feline mammary carcinomas (FMCs) are highly metastatic, the literature and treatment options pertaining to advanced tumours are scarce. This study aimed to investigate the clinical outcome of metastatic FMC with or without adjuvant treatment. METHODS: The medical records of 73 cats with metastatic FMC (stage IV) were reviewed and included in this study. Metastatic disease was detected by distinct imaging techniques (radiography, ultrasound and CT) and confirmed by cytology and/or histopathology. Cats with adjuvant chemotherapy treatment (n = 34) were divided into three groups: group 1 (n = 9) cats receiving maximum tolerated dose chemotherapy; group 2 (n = 15) cats receiving metronomic chemotherapy; and group 3 (n = 10) cats treated with toceranib phosphate. The study endpoints were time to progression (TTP) and tumour-specific survival (TSS). Treatment-related toxicity was evaluated according to the Veterinary Co-operative Oncology Group's Common Terminology Criteria for Adverse Events version 1.1 (VCOG-CTCAE). RESULTS: Overall mean TTP and TSS were 23 and 44 days, respectively. Cats with clinical signs at the time of diagnosis had a lower TSS (14 days) than asymptomatic cats (128 days; P <0.001). Cats with pleural effusion had a lower TSS (16 days) than cats without (P <0.001). Median TSS was 58, 75 and 63 days in groups 1, 2 and 3, respectively (P = 0.197). Toxicity was observed in 66.7%, 20% and 30% of cats in groups 1, 2 and 3, respectively. CONCLUSIONS AND RELEVANCE: To the best of our knowledge, this study includes the highest number of patients with metastatic FMC assessed. Despite the overall poor prognosis, some cats survived >6 months, indicating that adjuvant treatment may be an option to consider in metastatic disease. More studies are warranted for better understanding and management of stage IV patients.

Determination of urokinase-type plasminogen activator serum levels in healthy and oncologic cats.

The urokinase plasminogen activator system (uPAS) has been poorly investigated in veterinary oncology. The aim of this study was to determine uPA serum concentrations in healthy and oncologic cats to understand the potential value of uPA as a cancer biomarker. Serum samples were collected from 19 healthy cats and 18 cats with spontaneous malignant neoplasms and uPA was measured through a specific enzyme-linked immunosorbent assay kit. The differences between uPA values and their relation with intrinsic factors and clinicopathological parameters were analyzed using an analysis of variance (ANOVA) and independent t-test. The average serum concentration of uPA in cancerous cats (0.54 ± 0.22 ng/mL) differed from that of healthy cats (1.10 ± 1.16 ng/mL) but was not significantly influenced by cats' clinicopathological parameters or by the presence of metastases. This study describes, for the first time, the serum concentrations of uPA in cats and proposes directions for future studies to uncover the relevance of uPAS in feline carcinogenesis.

Le système activateur de plasminogène de type urokinase (uPAS) a été peu étudié en oncologie vétérinaire. L'objectif de la présente étude était de déterminer les concentrations sériques d'uPA chez des chats en santé et oncologiques afin de comprendre la valeur potentielle d'uPA comme marqueur de cancer. Des échantillons de sérum furent prélevés de 19 chats en santé et de 18 chats avec des néoplasmes malins spontanés et l'uPA fut mesuré à l'aide d'une trousse immuno-enzymatique. Les différences entre les valeurs d'uPA et leur relation avec des facteurs intrinsèques et des paramètres clinico-pathologiques furent analysées par analyse de variance (ANOVA) et test de t indépendant. La concentration moyenne d'uPA chez les chats avec cancer (0,54 ± 0,22 ng/mL) différait de celle des chats en santé (1,10 ± 1,16 ng/mL) mais n'était pas influencée de manière significative par les paramètres clinico-pathologiques des chats ou la présence de métastases. Cette étude décrit, pour la première fois, les concentrations sériques d'uPA chez les chats et propose des orientations pour des études ultérieures afin de révéler la pertinence d'uPAS dans la carcinogénèse chez les chats.(Traduit par Docteur Serge Messier).

Assessing the Interleukin 35 Immunoexpression in Malignant Canine Mammary Tumors: Association With Clinicopathological Parameters and Prognosis.

BACKGROUND/AIM: IL-35 has a prominent immunosuppressive role and its overexpression has been reported in human breast cancer. However, the impact of IL-35 in canine mammary carcinogenesis has not been addressed yet. The present study determined the clinicopathological significance of IL-35 immunoexpression and its correlation with overall survival (OS) in 72 malignant canine mammary tumor (CMT) patients. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded malignant CMT samples (n=72) were submitted to immunohistochemical staining to detect IL-35 expression. Survival curves were obtained by the Kaplan-Meier method and the log-rank test was used for the survival estimates. Cox proportional hazard model for multivariate analysis was also performed. RESULTS: IL-35 overexpression was associated with: skin ulceration, tumor necrosis, mitotic index, nuclear pleomorphism, tumor differentiation, histological grade of malignancy (HGM), neoplastic intravascular emboli and lymph node metastasis. Additionally, IL-35 was also correlated with a worse overall survival in multivariate analysis, arising as an independent predictor of poor prognosis. CONCLUSION: IL-35 is associated with carcinogenesis and worse prognosis of CMT.

Comparison of veterinary health services expectations and perceptions between oncologic pet owners, non-oncologic pet owners and veterinary staff using the SERVQUAL methodology.

AIM: Client satisfaction gained great importance in health care as a measurement of service quality. One of the most popular methods to evaluate client satisfaction is the SERVQUAL inquiry which measures service quality by evaluating client expectations and services towards a service in five dimensions: Tangibles, Empathy, Assurance, Reliability and Responsiveness. MATERIALS AND METHODS: In order to evaluate if owners of pets with cancer constitute a distinctive group from the general pet owner population and if these differences were perceived by the hospital staff we applied a SERVQUAL questionnaire to 51 owners of pet with cancer, 68 owners from the general pet population and 14 staff members. RESULTS: Owners of oncologic pets had different expectations of an ideal service granting importance to Assurance questions (6.75 vs 6.5, p= 0.045) while showing unmet needs in Reliability and Empathy dimensions. Veterinarians failed to understand these specificities and over evaluated characteristics of Tangible dimension (6.75 vs 6.25, p=0.027). CONCLUSION: Owners of pet with cancer seem to constitute a specific subpopulation with special needs and veterinary staff should invest resources towards Assurance instead of privileging tangible aspects of veterinary services. By aligning professionals expectations with those of pet owners veterinarians can achieve better client satisfaction, improved compliance and stronger doctor-owner relationships.

Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors.

The activity of regulatory T cells (Tregs) is closely associated with the expression of FoxP3 transcription factor. FoxP3 regulatory T cells (FoxP3Treg) have immunosuppressive properties and can work for prevention of harmful autoimmune responses, however can also interfere with beneficial anti-tumor immunity. In human breast cancer these cells play a crucial role in tumor progression. In canine mammary tumors (CMT) this topic is not well-documented. This study included 80 malignant CMT and studied, by immunohistochemistry, the intratumoral FoxP3 expression together with microvessel density (MVD), vascular endothelial growth factor (VEGF) and several clinicopathological characteristics. Abundant FoxP3Treg cells were associated with tumor necrosis (p=0.001), high mitotic grade (p<0.001), more marked nuclear polymorphism (p=0.001), poor differentiation of tumors (p<0.001), high histological grade of malignancy (HGM) (p<0.001), presence of neoplastic intravascular emboli (p<0.001) and presence of lymph node metastasis (p<0.001). Intratumoral FoxP3 was correlated with MVD (r=0.827; p<0.001) and associated with VEGF (p=0.001). Additionally tumors with abundant FoxP3Treg cells were associated with shorter overall survival (OS) time in univariate and multivariate analysis (p<0.001 Kaplan-Meier curves and 7.97 hazard ratio, p<0.001 Cox proportional hazard model). Results suggest that Treg cells play a role in CMT progression and may contribute to increased angiogenesis and aggression in these tumors. The association of intratumoral FoxP3 expression with shorter OS in multivariate analysis suggests the usefulness of Treg cells as an independent prognostic marker.

Investigating associations of cyclooxygenase-2 expression with angiogenesis, proliferation, macrophage and T-lymphocyte infiltration in canine melanocytic tumours.

Cyclooxygenase-2 (COX-2) is known to be involved in tumour progression and has been suggested as a therapeutic target in many human and animal malignancies. A number of different pathways subjacent to cancer hallmarks are considered to be involved in COX-2-mediated tumour progression, although these are still largely undefined. Our aim is to investigate associations between COX-2 expression and angiogenesis, proliferation and the inflammatory microenvironment in canine melanocytic tumours. Understanding the involvement of COX-2 with cancer hallmarks might enable us to adapt therapeutic strategies for canine melanomas, an aggressive and often lethal malignancy with value in comparative oncology. Immunohistochemical staining of COX-2, Ki-67 (proliferation index), vascular endothelial growth factor (VEGF), factor VIII (microvessel density), CD3 (lymphocytes) and MAC387 (macrophages) was performed in 51 melanocytic tumours (31 malignant melanomas, 20 melanocytomas). Statistical associations between COX-2 and the other parameters detected were analysed. In melanocytic tumours (n=51), both COX-2 labelling extension and intensity showed a statistically significant association with angiogenesis by factor VIII, VEGF, Ki-67, CD3+ T lymphocytes and MAC387. Within malignant melanomas, COX-2 expression has shown significant associations with microvessel density (factor VIII), lymphocyte and macrophage infiltration and, considering all melanocytic tumours, COX-2 was also associated with VEGF intensity and Ki-67 cell proliferation. Our results point to a role for COX-2 in angiogenesis and in the establishment of an inflammatory microenvironment, favourable to melanoma tumour progression. Further mechanistic studies are warranted to dissect molecular pathways in which COX-2 is involved. Present evidence suggests that COX-2 inhibitors might be useful as an adjuvant treatment to hinder canine melanoma progression.

Intratumoral CD3+ T-lymphocytes immunoexpression and its association with c-Kit, angiogenesis, and overall survival in malignant canine mammary tumors.

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor.

Mammary invasive micropapillary carcinoma in a male cat: Immunohistochemical description and clinical follow-up.

We describe the case of a 14-year-old Domestic Short Hair male cat that presented with a single mammary tumour one centimetre in diameter, classified as invasive micropapillary carcinoma. Histology and immunohistochemistry revealed a high mitotic index, a KI-67 index of 10%, a lack of reactivity to myoepithelial markers, and a dense infiltration with T and B lymphocytes at the periphery of the tumour. Micrometastases were detected in the regional lymph node at the time of surgery. Overall survival time was 10 months with a disease-free interval of 7.5 months. Distant metastases in the sublumbar lymph nodes confirmed the aggressiveness of this tumour, which has recently been subtyped in female cats. This is the first case reported in male cats with a complete follow-up, highlighting the importance of prompt and aggressive treatment in the presence of mammary tumours in male cats.

Evaluation of cyclooxygenase-2 expression in canine mast cell tumours.

Mast cell tumours (MCTs) are among the most common cutaneous neoplasms in dogs and have a highly variable clinical behaviour. Cyclooxygenase (Cox) catalyzes the rate-limiting step in prostanoid biosynthesis and has recently gained attention as a prognostic factor and therapeutic target in human and animal oncology. In order to evaluate the potential value of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of canine MCTs, expression of Cox-2 was determined in 49 such tumours (14 of grade I, nine of grade II and 22 of grade III). Cox-2 was expressed by 86% of the tumours studied. The percentage of labelled cells ranged from isolated positive cells throughout the tumour (n=8) to localized foci of labelled cells (n=3) or diffuse labelling of >50% of the cells (n=31). The intensity of Cox-2 labelling ranged from weak (n=4) to moderate (n=16) and strong (n=22) and was greatest at the advancing margin of the tumour. The intensity of Cox-2 labelling was significantly different between the three histological groups (P=0.018). However, no significant differences were noted for the percentage of Cox-2 positive cells (P=0.122) and for the immunoreactivity score (P=0.348) between the histological grades. The results of this study suggest that NSAIDs, particularly Cox-2 inhibitors, may be of value in the treatment of canine MCTs.

COX-2 over-expression correlates with VEGF and tumour angiogenesis in canine mammary cancer.

This study was designed to investigate the possible roles of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in canine mammary cancer angiogenesis. Immunohistochemistry was performed on 70 tumours (28 benign and 42 malignant) in order to detect COX-2 and VEGF expression. Microvessel density (MVD) was determined by CD31 immunolabelling to assess tumour angiogenesis. There was a significantly higher expression of COX-2 (P<0.001), VEGF (P<0.001) and MVD (P<0.001) in malignant compared to benign tumours. In the malignant group, the MVD of COX-2 positive tumours was significantly higher than that of COX-2 negative tumours (P=0.026). A similar association was observed for VEGF (P<0.001) positive tumours. The results from this study suggested that over-expression of COX-2 and VEGF may contribute to increased angiogenesis and aggression in malignant tumours.