Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

Chemotherapy-induced diarrhoea in dogs and its management with smectite: Results of a monocentric open-label randomized clinical trial.

Chemotherapy-induced diarrhoea (CID) is a frequent chemotherapy adverse event in dogs. Yet, there is currently no consensus regarding its management. Smectite is a natural medical clay, widely used in the treatment of acute diarrhoea in humans. The objectives of this study were to assess the efficacy of smectite in the management of CID in dogs, and to collect epidemiological data on CID. For each episode of diarrhoea, dogs were randomized into two management groups: Smectite group, receiving smectite at 0.5 g/kg PO per day divided in two to three doses initiated at the start of CID; control group, without initial medication. In both groups, rescue metronidazole was prescribed if CID progressed or was not improved within 48 hours. Sixty dogs were recruited and received 426 chemotherapy administrations between June 2017 and March 2019. The incidence rate of CID was 110/426 (25.8%, 95% CI: 21.7%-30.2%), and significantly differed between the chemotherapeutic drugs administered (P < .001). Metronidazole was administered in 5/54 events (9.3%, 95% CI: 3.1%-20.3%) in the smectite group and in 40/56 events (71.4%, 95% CI: 57.5%-82.3%) in the control group (P < .001). The time to resolution of diarrhoea was shorter (P < .001) in the smectite group (median: 19.5 hours, interquartile range [IQR]: 13.5-32 hours) compared with the control group (median: 53 hours, IQR: 31.5-113.5 hours). The results of this study support the administration of smectite in the first-line management of CID in dogs.

Contrast-enhanced ultrasound for sentinel lymph node mapping in the routine staging of canine mast cell tumours: A feasibility study.

Canine mast cell tumours (MCTs) typically spread to lymph nodes (LNs) before reaching distant sites, and LN assessment is an important part of MCT staging. Sentinel LN (SLN) mapping techniques to identify draining LNs are being developed and could improve the accuracy of MCT staging. The primary objective of this feasibility study was to determine the safety and effectiveness of contrast-enhanced ultrasound (CEUS) to identify SLNs. Secondary objectives were to determine if the SLNs identified by CEUS coincided with the regional LN predicted by the anatomical lymphosomes, if previous MCT excision altered CEUS SLN findings, and if CEUS could identify MCT nodal metastases. Between June 2017 and March 2019, 59 dogs with 62 MCTs were enrolled. No adverse events related to CEUS were reported. CEUS detected at least 1 SLN in 59/62 MCTs (95.2%, 95% CI: 86.5-99.0%). In only 32/59 (54.2%) MCTs, clinicians would have correctly predicted the SLN(s) identified by CEUS. Among the 35 MCTs that had histological examination of the SLN(s), the prevalence of metastasis was 60% (95% CI: 42.1-76.1%). Additional staging procedures did not reveal any metastases in dogs with histologically non-metastatic SLNs. Integration of CEUS SLN mapping into the routine staging of MCTs is promising, but future studies are required to refine this procedure and to investigate if it would translate into a clinical benefit.

Evaluation of a 0.75 × 109 /L absolute neutrophil count cut-off for antimicrobial prophylaxis in canine cancer chemotherapy patients.

Absolute neutrophil count (ANC) cut-offs for antimicrobial prophylaxis in veterinary cancer chemotherapy patients are empirical and vary between institutions. Evidence based cut-offs are vital for antimicrobial stewardship, particularly as global antimicrobial resistance rises. The primary objectives of this study were to evaluate the tolerability of a <0.75 × 109 /l ANC cut-off for antimicrobial prophylaxis in dogs after receiving chemotherapy and its impact on antimicrobial prescription. Predicted nadir ANCs (pnANCs) were stratified into six groups (<0.75 × 109 /l [receiving antimicrobial prophylaxis], 0.75-0.99 × 109 /l, 1-1.49 × 109 /l, 1.5-1.99 × 109 /l, 2.0-3.59 × 109 /l and 3.6-12 × 109 /l [reference interval]). The incidences of post-nadir febrile neutropenia (FN) and non-haematological toxicity (NHT) were compared between groups. Five hundred and eighty-six pnANCs were recorded for 181 dogs. There were four episodes of post-nadir FN and 90 episodes of post-nadir NHT. There was no significant difference in incidence of post-nadir FN (P = .063) or post-nadir NHT (P = .084) between pnANC groups. Antimicrobial prophylaxis was prescribed following 8.8% of the chemotherapy administrations; had cut-off values of <1.0 × 109 /l or <1.5 × 109 /l been used it would have been prescribed in 15.3% and 25.8% of cases respectively. An ANC cut-off of <0.75 × 109 /l for antimicrobial prophylaxis appears to be well tolerated and minimizes the prescription of antimicrobials.

A previously undescribed cutaneous paraneoplastic syndrome in a cat with thymoma.

BACKGROUND: Exfoliative dermatitis is a well-recognized cutaneous paraneoplastic syndrome (PNS) associated with thymoma in cats, of which the clinical and histopathological presentation has been well-characterized. OBJECTIVES: To describe a novel clinical skin manifestation associated with thymoma in a cat. ANIMAL: A 14-year-old neutered female domestic short hair cat. METHODS AND MATERIALS: Physical, abdominal ultrasonographic, thoracic radiographic, ultrasonographic and computed tomographic examinations, histopathological assessment of the skin and mediastinal mass. RESULTS: The cat was presented with noninflammatory alopecia, with a dorsal multifocal distribution. Examination of the alopecic areas using a dermascope indicated an apparent lack of follicular ostia. Histopathological assessment of alopecic areas confirmed follicular and epidermal atrophy, trichilemmal keratinization and mild orthokeratotic hyperkeratosis. Diagnostic imaging revealed a mediastinal mass, which was surgically removed. Histopathological and immunohistopathological examination of the mass was consistent with a thymoma, associated with multiloculated cyst formation and multifocal cholesterol granulomas. Following surgery, hair re-growth was noted in the previously alopecic areas. The cat was euthanized 3.5 months later because of recurrent chylothorax suspected to be a postoperative complication. The alopecic lesions had improved markedly. CONCLUSIONS AND CLINICAL IMPORTANCE: Thymoma-associated PNS might not always manifest as an exfoliative dermatitis and should be considered in the differential diagnosis of multifocal noninflammatory alopecia.

Investigation of the utility of lymph node fine-needle aspiration cytology for the staging of malignant solid tumors in dogs.

BACKGROUND: Fine-needle aspiration cytology (FNAC) of lymph nodes (LNs) is routinely used for staging canine malignant solid tumors, but studies evaluating its efficacy are limited. OBJECTIVES: The primary objectives of this study were to evaluate the sensitivity/specificity of FNAC and the significance of nondiagnostic FNAC when staging canine malignant tumors. A secondary objective was to determine the prevalence of multiple nodal metastases. METHODS: Lymph nodes draining malignant solid tumors assessed with FNAC and histopathology were included. The sensitivity/specificity of FNAC was determined for LNs with diagnostic FNAC, using histopathology as the gold standard. The proportion of nondiagnostic FNAC and associated histopathologic prevalence of metastasis were determined. Among the tumors with multiple LNs assessed, the prevalence of multiple nodal metastases was determined. RESULTS: The sensitivity of FNAC (194 LNs) was 67% for sarcomas, 100% for carcinomas, 63% for melanomas, 75% for mast cell tumors, and 100% for other round cell tumors. The specificity varied between 83% and 96%. Nondiagnostic FNAC was reported in 25% of LNs sampled (65/259), most of which were nonenlarged and/or difficult to access, and 20% of which were metastatic on histopathology. When several LNs were assessed (88/189 tumors), the prevalence of multiple nodal metastases was 24%. CONCLUSIONS: Histopathologic LN evaluation cannot be robustly substituted with FNAC when staging selected canine solid tumors. When a diagnostic FNAC is elusive, the histopathologic assessment remains ideal. Finally, staging should not always be limited to the assessment of a single LN.

Real-time elastosonography of lipomatous vs. malignant subcutaneous neoplasms in dogs: Preliminary results.

Real-time elastography is a recently introduced ultrasound technique allowing the investigation of elastic properties of tissues. A diagnostic accuracy study was conducted to test the performance of this technique in the assessment of subcutaneous lesions in dogs. Fifty-two dogs were prospectively included in the preliminary study (34 malignant and 18 benign lesions). B-mode ultrasound was performed assessing the shape, margins, heterogeneity, and echotexture of the lesions. On elastosonography, assessment of the percentage of softness/hardness was recorded. A qualitative assessment was performed according to the Tsukuba elasticity score with a 1-5 score, representing the increased percentage of high stiffness areas. Results were compared with cytology/histopathology of the lesions. Receiver Operating Curves of the overall diagnostic sensitivity and specificity were obtained. Fisher's exact test and Pearsons's Chi-squared test estimated the relationship between the B-mode appearance of the lesions and final diagnosis. A hardness cutoff of 50.25% was identified between lesions, with malignant neoplasms having higher percentages. A 100% specificity and 89% sensitivity for correctly detecting the nature of the lesion on elastosonography was established. Qualitative assessment of the Tsukuba elasticity score established 1.5 as the cutoff between elastograms of lipomatous and malignant lesions, with 100% sensitivity and 61% specificity in differentiating them. Real-time elastosonography is a novel, noninvasive, and accurate technique for differentiating malignant from benign lipomatous lesions in dogs. This method could be considered as a complementary tool with additional diagnostic value for routine invasive procedures, such as fine needle aspirates.