Retrospective evaluation of the effect of acid suppressant drugs on leukocyte ratios in dogs with mast cell tumors.

BACKGROUND: Acid suppressant drugs (ASDs) are commonly used to decrease gastric acid production, but some evidence exists that ASDs exert immunomodulatory effects. Such an effect has not been investigated in dogs for which ASDs are routinely prescribed. HYPOTHESIS: Compared to naïve subjects, dogs treated with ASDs will exhibit differences in leukocyte ratios after treatment. ANIMALS: Fifty-one dogs with mast cell tumors (MCTs). MATERIALS AND METHODS: Dogs with MCT that were either AS naïve or treated with ASDs (i.e., histamine-2-receptor antagonists [H2RA] or proton pump inhibitors [PPI]) were included in this retrospective study. Subjects were categorized into 3 treatment groups (AS naïve, H2RA treated, and PPI treated), and leukocyte ratios (neutrophil:eosinophil, lymphocyte:monocyte, and neutrophil:lymphocyte [NLR]) were calculated before and after treatment. A mixed effects analysis of variance on ranks was used to assess differences in ratios between treatments, between pre- and post-treatment time points, and between pre- and post-time points for each treatment. Concurrent administration of antihistamines, corticosteroids, and chemotherapeutic drugs was assessed as a confounding factor. RESULTS: Famotidine (n = 14/14) and omeprazole (n = 12/12) were the only H2RA and PPI used, respectively. Dogs receiving famotidine had a significant increase in median NLR from pre- to post-treatment (3.429; range, 1.417-15 to 5.631; range, 2.654-92; P < 0.01) compared to PPI treated or AS naïve dogs. No differences existed in chemotherapeutic drug or corticosteroid use between groups. CONCLUSIONS: A significant difference in NLR was identified in famotidine treated dogs compared with omeprazole treated or AS naïve dogs.

Weishaar's classification system for nodal metastasis in sentinel lymph nodes: Clinical outcome in 94 dogs with mast cell tumor.

BACKGROUND: The therapeutic role and prognostic relevance of lymphadenectomy in mast cell tumor (MCT) has historically been evaluated on regional rather than sentinel lymph nodes. HYPOTHESIS/OBJECTIVES: To update information about the association of histological nodal (HN) classes with clinical outcome in dogs with MCT after tumor excision and extirpation of normal-sized sentinel nodes (SLN) guided by radiopharmaceutical. ANIMALS: Ninety-four dogs with histologically-confirmed treatment-naïve MCT (71 cutaneous, 22 subcutaneous and 1 conjunctival MCT) were included if without: distant metastases, lymphadenomegaly, concurrent mixed cutaneous, and subcutaneous MCT. METHODS: This was a monoistitutional cohort study. Tumors characteristics were retrieved and SLNs were classified according to Weishaar's system. Incidence of MCT-related events (local, nodal, distant relapse), de novo MCT or other tumors and death (MCT-related and non-MCT-related), were recorded. Incidence curves were compared among the HN classes. RESULTS: Twenty-seven dogs had HN0, 19 HN1, 37 HN2, and 11 HN3 SLN. Thirteen (2 HN0, 4 HN2, and 7 HN3) received adjuvant chemotherapies. Kiupel high grade, increasing number of SLN and lymphocentrums were associated with higher HN classes. Five dogs died for MCT-related causes: 1 low-grade (HN0) and 1 subcutaneous (HN3) had a local relapse, 2 high-grade had distant relapse (HN3-HN0) and 1 dog developed disease progression from a de novo subcutaneous MCT. No nodal relapse was registered. Fourteen dogs developed de novo MCTs. CONCLUSION/DISCUSSION: Low grade/low-risk MCT with nonpalpable and normal sized SLN have a favorable outcome independently from the HN. Result should be considered strictly related to the successful SLN detection guided pre- and intraoperative by radiopharmaceutical markers.

A retrospective study on prophylactic regional lymphadenectomy versus nodal observation only in the management of dogs with stage I, completely resected, low-grade cutaneous mast cell tumors.

BACKGROUND: While lymphadenectomy of metastatic lymph nodes (LNs) has been associated with improved outcome, the clinical utility of prophylactic lymphadenectomy in dogs with stage I cutaneous mast cell tumors (cMCTs) remains a controversial topic. To assess the therapeutic role of lymphadenectomy of uninvolved regional LNs, the long-term outcome of cMCT-bearing dogs with cytologically negative and surgically unresected regional LNs (observation only, OO) was compared with that of dogs with surgically resected and histologically negative regional LNs (prophylactic regional lymphadenectomy, PRL). RESULTS: A retrospective analysis of 64 dogs with a low-grade, completely resected stage I cMCT was performed: 35 (54.7%) dogs were subjected to OO and 29 (45.3%) underwent PRL. Dogs were monitored for a median of 813 and 763 days in the OO group and PRL group, respectively. The number of dogs undergoing MCT progression was significantly higher in the OO group (P = 0.028) and curve comparison revealed a tendency to a better time to progression in the PRL group (P = 0.058). No significant difference in survival time (P = 0.294) was observed between dogs in the OO and PRL groups. CONCLUSIONS: Our results showed that lack of immediate lymphadenectomy was associated with a higher risk for tumor progression. This preliminary judgement, reinforced by the findings that lymphadenectomy was well tolerated in all cases, and that histopathology provides the definitive assessment of the nodal pathological status, may suggest that prophylactic lymphadenectomy is indicated in the management of stage I MCTs. Larger prospective studies are warranted for generating clinical evidence of this latter hypothesis.

Long-term in-vitro glucocorticoid treatment induces glucocorticoid resistance in canine mast cell tumors.

Although glucocorticoid administration has produced impressive results in treating canine mast cell tumors (MCTs), in some cases, glucocorticoids fail to reduce the tumor volume, leading to tumor relapse even after treatment. To date, mechanisms involved in glucocorticoid resistance in canine MCTs remain poorly defined. The objective of this study was to establish glucocorticoid-resistant canine MCT cell lines derived from glucocorticoid-sensitive cell lines after prolonged treatment with dexamethasone (Dex). Real-time polymerase chain reaction (RT-PCR) revealed that elevation of glucocorticoid receptor (GR)-regulated gene expression was suppressed in Dex-resistant cell lines after Dex stimulation compared with parent Dex-sensitive cell lines. This indicated that GR-regulated transcription was suppressed in Dex-resistant cell lines. Insufficient expression of GRs was not detected in Dex-resistant cell lines. Possible inhibitors of GR-regulated transcription were increased in mRNA expression in Dex-resistant cell lines. In addition, it was determined that mRNA expression of drug efflux pumps and anti-apoptosis factors was higher in Dex-resistant cell lines. In conclusion, glucocorticoid-resistant canine MCT cell lines have been established that are derived from glucocorticoid-sensitive cell lines. These cell lines suggest that multiple mechanisms contribute to glucocorticoid resistance in canine MCT cells. The mechanisms of glucocorticoid resistance after long-term treatment can be further investigated using these cell lines and a novel therapeutic strategy for glucocorticoid-resistant canine MCT cells can be developed.

Bien que l'administration de glucocorticoïdes ait produit des résultats impressionnants dans le traitement des mastocytomes (MCT) canins, dans certains cas, les glucocorticoïdes ne parviennent pas à réduire le volume tumoral, entraînant une rechute de la tumeur même après le traitement. À ce jour, les mécanismes impliqués dans la résistance aux glucocorticoïdes dans les MCT canins restent mal définis. L'objectif de cette étude était d'établir des lignées cellulaires MCT canines résistantes aux glucocorticoïdes dérivées de lignées cellulaires sensibles aux glucocorticoïdes après un traitement prolongé avec de la dexaméthasone (Dex). La réaction d'amplification en chaîne par polymérase en temps réel (RT-PCR) a révélé que l'élévation de l'expression génique régulée par le récepteur des glucocorticoïdes (GR) était supprimée dans les lignées cellulaires résistantes à Dex après stimulation par Dex par rapport aux lignées cellulaires parentales sensibles à Dex. Cela indiquait que la transcription régulée par GR était supprimée dans les lignées cellulaires résistantes à Dex. Une expression insuffisante des GR n'a pas été détectée dans les lignées cellulaires résistantes à Dex. Les inhibiteurs possibles de la transcription régulée par GR étaient augmentés dans l'expression de l'ARNm dans les lignées cellulaires résistantes à Dex. De plus, il a été déterminé que l'expression de l'ARNm des pompes d'efflux de médicaments et des facteurs anti-apoptose était plus élevée dans les lignées cellulaires résistantes au Dex. En conclusion, des lignées cellulaires canines MCT résistantes aux glucocorticoïdes ont été établies qui sont dérivées de lignées cellulaires sensibles aux glucocorticoïdes. Ces lignées cellulaires suggèrent que de multiples mécanismes contribuent à la résistance aux glucocorticoïdes dans les cellules MCT canines. Les mécanismes de résistance aux glucocorticoïdes après un traitement à long terme peuvent être étudiés plus en détail à l'aide de ces lignées cellulaires et une nouvelle stratégie thérapeutique pour les cellules MCT canines résistantes aux glucocorticoïdes peut être développée.(Traduit par Docteur Serge Messier).

MAST CELL TUMORS IN CHEETAH (ACINONYX JUBATUS): A CASE SERIES.

Mast cell tumors in nondomestic felids are rarely reported and their biological characteristics are not well described. A retrospective review of the pathology records of 52 zoo-housed cheetahs (Acinonyx jubatus) identified five cases of mast cell tumor, involving four closely related individuals. The age at initial presentation varied from 14 mo to 6 yr. Four cases presented as solitary or multiple cutaneous masses that were mostly slow growing, up to 20 mm diameter, and predominantly nonulcerated. The diagnosis was made by fine needle aspiration cytology of a lesion in one case and by excisional biopsy in the others. Histopathologically, the lesions resembled low- to intermediate-grade canine mast cell tumors, with variations in the degree of anisocytosis and anisokaryosis. Surgical excision was incomplete for 80% of the cutaneous lesions, but local recurrence was not observed in any case. One animal with cutaneous lesions subsequently developed fatal visceral mastocytosis involving the spleen, liver, and adrenal gland. There was no evidence of lymph node invasion or paraneoplastic gastrointestinal signs in any of the cases.

In situ c-KIT mRNA quantification of canine cutaneous mast cell tumours and its relationship to prognostic factors.

Cutaneous mast cell tumours (MCTs) are the most frequent malignant skin tumours in dogs. Mutations in the c-KIT proto-oncogene are correlated with the pathogenesis and aggressiveness of MCTs. To date, studies have focused on c-KIT mutations and KIT protein localization, with a general lack of mRNA-level analyses. In this study, c-KIT mRNA expression was investigated in canine MCTs by RNA in situ hybridization (RNA-ISH). Furthermore, we evaluated associations between c-KIT mRNA expression and the histological grade, KIT immunohistochemical staining pattern and other clinicopathological parameters. c-KIT mRNA expression was observed in all MCT samples, appearing as clusters of dots in the cytoplasm of neoplastic cells. A significant correlation was detected between c-KIT mRNA expression (quantified according to the H-score and the percentage of positive cells) and the histological grade (determined using two-and three-tier grading systems; P < .05). We also found a significant positive correlation (all P < .05) between c-KIT mRNA expression and the proliferation indices (mitotic index, Ki-67, and Ag67). However, no significant associations with c-KIT expression from RNA-ISH were found with respect to different KIT staining patterns. Overall, these results demonstrate that c-KIT mRNA expression might be an additional tool for measuring the c-KIT status in canine cutaneous MCTs and could serve as a potential prognostic factor. Further studies should evaluate the prognostic significance of c-KIT mRNA expression in a large and uniform cohort of canine MCTs.

The feasibility and utility of optical coherence tomography directed histopathology for surgical margin assessment of canine mast cell tumours.

Histopathologic surgical margin assessment in veterinary patients is an imprecise science with assessment limited to a small proportion of the surgical margin due to time and finances. Incomplete excision of canine mast cell tumours (MCTs) alters treatment recommendations and prognosis. Optical coherence tomography (OCT) is a novel imaging modality that has been reported in a single veterinary study for surgical margin assessment. Twenty-five dogs with 34 MCTs were enrolled in a prospective pilot-study to assess the imaging characteristics of canine MCTs with OCT and to evaluate the feasibility and utility of OCT-guided histopathology. All dogs underwent routine surgical excision of MCTs. OCT imaging was used to assess the entire surgical margin prior to placement in formalin. Either normal areas or areas suspected of incomplete MCT excision were inked. Standard histopathologic sectioning and tangential sectioning of inked areas were performed and compared to OCT results. OCT identified MCT near the surgical margin in 10 of 26 specimens (38.4%). Four specimens suspicious for incomplete margins on OCT had incomplete MCT excision that was missed on standard histopathologic sectioning. Six specimens had OCT-guided sections taken as suspicious, which did not show MCT on histopathology. OCT-guided pathology sections were able to detect incompletely excised MCT near the surgical margin with a sensitivity of 90% and specificity of 56.2% in this preliminary study. OCT imaging shows promise for guiding pathologists to areas of interest to improve the diagnostic accuracy of surgical margin assessment in excised canine MCTs.

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.

miRNA profiles of canine cutaneous mast cell tumours with early nodal metastasis and evaluation as potential biomarkers.

Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and they can function as tumour promoters or suppressors. However, the role of miRNAs in canine MCTs has not yet been elucidated. Thus, the current study aimed to characterize miRNA profiles and to assess their value as biomarkers for MCTs. miRNA expression profiles were assessed in formalin-fixed, paraffin-embedded samples by next-generation sequencing. Ten samples were MCT tissues, and 7 were healthy adjacent tissues. Nine dysregulated miRNAs (DE-miRNAs) were then validated using RT-qPCR in a larger group of MCT samples, allowing the calculation of ROC curves and performance of multiple factor analysis (MFA). Pathway enrichment analysis was performed to investigate miRNA biological functions. The results showed that the expression of 63 miRNAs (18 up- and 45 downregulated) was significantly affected in MCTs. Five DE-miRNAs, namely, miR-21-5p, miR-92a-3p, miR-338, miR-379 and miR-885, were validated by RT-qPCR. The diagnostic accuracy of a panel of 3 DE-miRNAs-miR-21, miR-379 and miR-885-exhibited increased efficiency in discriminating animals with MCTs (AUC = 0.9854) and animals with lymph node metastasis (AUC = 0.8923). Multiple factor analysis revealed clusters based on nodal metastasis. Gene Ontology and KEGG analyses confirmed that the DE-miRNAs were involved in cell proliferation, survival and metastasis pathways. In conclusion, the present study demonstrated that the miRNA expression profile is changed in the MCT microenvironment, suggesting the involvement of the altered miRNAs in the epigenetic regulation of MCTs and identifying miR-21, miR-379 and miR-885 as promising biomarkers.

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior. METHODS/RESULTS: WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs. CONCLUSIONS: These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

Canine and Feline Cutaneous Mast Cell Tumor: A Comprehensive Review of Treatments and Outcomes.

Mast cell tumor (MCT) or mastocytoma is one of the most frequent malignant cutaneous tumors in the dog, and the second most frequent in the cat. Several mast cell tumor therapeutic approaches have been proposed in the past years for dogs and cats, resulting in very distinct outcomes. The current comprehensive literature review presents a critical approach to the scientific information published about the MCTs treatments and the subsequent prognosis and survival times, in dogs and in cats diagnosed with MCTs. A systematic review of the literature following the Cochrane principles and methodology was performed. The authors resorted to MEDLINE, Scopus, Google Scholar and Web of Science databases to select the 133 publications with evidence-based treatments for MCTs in companion animals. Results of the review suggest that the recommended treatment, prognosis and survival times for dogs and cats with MCTs depends at all times on the clinical staging, histological grade and location of the tumor.

Canine cutaneous neoplasms in the metropolitan region of Goiânia, Goiás state, Brazil / Neoplasias cutâneas caninas na região metropolitana de Goiânia, Goiás, Brasil

ABSTRACT: The present study aimed to describe the occurrence and epidemiological features of skin neoplasms diagnosed in dogs in the metropolitan region of Goiânia, Goiás state, Brazil. Diagnoses from dog biopsies from 2011 to 2016 provided by a private veterinary pathology laboratory were analyzed. The main diagnoses were mast cell tumor, hemangiosarcoma, squamous cell carcinoma, malignant melanoma, and hemangioma. Highest frequency of neoplasms was found in female dogs, dogs aged > 8 years, and purebred dogs, particularly the American Pit Bull Terriers and the Poodles. Most common sites affected by the neoplasms were the limb and the head. Using multiple correspondence analysis, groups of neoplasms were found to be associated with different epidemiological features and the size of the neoplasms was associated with the biological behavior. The results of this study described predispositions and verified the importance of different types of skin neoplasms in dogs in the region being studied.(AU)

O objetivo deste estudo foi determinar a prevalência e as características epidemiológicas das neoplasias cutâneas em cães na região metropolitana de Goiânia, Goiás. Foram analisados os diagnósticos de um laboratório do setor privado de 2011 a 2016. Mastocitoma, hemangiossarcoma, carcinoma de células escamosas, melanoma maligno e hemangioma representaram os principais diagnósticos. A maioria dos casos ocorreram em cães de raças definidas, fêmeas e com idade >8 anos. American Pit Bull Terrier e Poodle foram as raças mais encontradas. As neoplasias acometeram principalmente regiões de membros e cabeça. Pela análise de correspondência múltipla, associou-se os grupos de neoplasias com diferentes características epidemiológicas e o tamanho da neoplasia com o comportamento biológico. A comparação dos resultados com pesquisas prévias possibilitou confirmar predisposições previamente descritas e verificar a importância dos diferentes tipos de neoplasias cutâneas em cães na região estudada.(AU)

Prognostic Indicators and Clinical Outcome in Dogs with Subcutaneous Mast Cell Tumors Treated with Surgery Alone: 43 Cases.

The purpose of this study was to determine if clinical findings, histologic grade, or other histologic features were associated with clinical outcome in dogs with subcutaneous mast cell tumors (MCTs). Medical records of 43 client-owned dogs were retrospectively reviewed, and follow-up information was gathered via phone or follow-up examination. Progression-free survival (PFS), disease-free interval (DFI), and overall survival were calculated. Forty-two and twenty-two dogs, respectively, had grade 2 (Patnaik grading system) or low-grade tumors (two-tier grading system). Median PFS was 1474 days. Median DFI was not reached at >1968 days. Overall median survival time was not reached at >1968 days. In univariate analysis, argyrophilic nucleolar organizer regions (AgNORs), proliferating cell nuclear antigen, and mitotic index were negatively prognostic for PFS whereas Ki-67, proliferating cell nuclear antigen, and microvessel density were negatively prognostic for DFI. In multivariate analysis, AgNORs remained negatively prognostic for PFS. Results suggest that proliferation indices, especially AgNORs, may be useful in predicting the rare poor outcomes in dogs with subcutaneous MCTs. The vast majority of subcutaneous MCTs appear to be low or intermediate grade with excellent outcomes from good local tumor control.

Long-term outcomes of dogs undergoing surgical resection of mast cell tumors and soft tissue sarcomas: A prospective 2-year-long study.

OBJECTIVE: Report clinical outcomes of dogs with surgically excised mast cell tumors (MCT) and soft tissue sarcomas (STS). STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Fifty-three dogs with 52 MCT (50 low grade, 2 high grade) and 19 STS (12 grade I, 6 grade II, 1 grade III). METHODS: All dogs were examined at 3, 6, 12, 18, and 24 months postoperatively, with cytologic or histopathologic evaluation of suspected local recurrences. Dogs euthanized because of study tumor-related causes underwent necropsy. RESULTS: Median intraoperative margins were 20 mm and 30 mm wide for MCT and STS, respectively, with 1 fascial plane resected en bloc. The narrowest histologic tumor-free margins measured <1 mm in 21 of 52 (40%) MCT and 7 of 19 (37%) STS. All dogs were followed for 24 months. Two of 50 (4%) low-grade MCT were diagnosed, with local recurrence 181 and 265 days postoperatively. Two of 36 (6%) dogs with low-grade MCT developed visceral metastasis 181 and 730 days postoperatively. One of 2 dogs with high-grade MCT developed local recurrence 115 days postoperatively. No local recurrence or metastasis was diagnosed after excision of 19 STS. CONCLUSION: Local recurrence rates among predominantly low- to intermediate-grade MCT and STS were low, despite a high prevalence of histologic tumor-free margins <1 mm. Surgical recommendations for high-grade tumors cannot be extrapolated from this population. CLINICAL SIGNIFICANCE: Surgeons should seek to achieve microscopically complete excision for MCT and STS while minimizing patient morbidity and considering limitations of histopathology in predicting outcomes.

Grey eosinophils in a Miniature Schnauzer with a poorly differentiated mast cell tumor.

A 10-year-old female spayed Miniature Schnauzer was presented for investigation of an intra-nasal mass. The mass was diagnosed by histopathologic examination as an undifferentiated round cell neoplasm with an infiltrate of segmented leukocytes, interpreted as neutrophilic inflammation. The mass was treated with palliative radiation and systemic chemotherapy due to the presence of regional lymph node metastasis. During subsequent monitoring over several months, the peripheral leukocyte concentration was repeatedly within reference intervals to slightly increased with low numbers of toxic neutrophils. Four months after the initial diagnosis, there was a significant leukocytosis of 66 100 cells/µL, and 39 700 cells/µL of the leukocytes had variably mature, lobulated, and hypolobulated nuclei, and grey cytoplasm with clear vacuoles, resembling grey eosinophils. To further characterize these cells, peripheral blood smears from the patient and a canine control with eosinophilia were stained for alkaline phosphatase (AP), peroxidase, and esterase activities, and with Luxol fast blue (LFB). Histopathologic sections of the nasal mass were stained with LFB and immunohistochemically for tryptase. On blood smears, the cytoplasm of the suspected grey eosinophils stained for AP and granules stained with LFB confirmed that there was an eosinophilic lineage. Peroxidase staining was weak, and esterase staining was absent. On histopathologic sections from the nasal mass, the segmented leukocytes contained LFB-staining granules, indicating an eosinophilic infiltrate was present. Neoplastic cells expressed tryptase, which confirms a mast cell lineage. Our findings suggest that grey eosinophils might be under-recognized and interpreted incorrectly as toxic neutrophils. This report expands the canine breeds in which these eosinophils have been identified.

DNA purification increases PCR-amplifiable DNA extracted from formalin-fixed, paraffin-embedded canine mast cell tumors for routine KIT mutation detection.

DNA amplification by PCR detects KIT exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes (HPRT and KIT) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8-60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A260/A280 and A260/A230) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both HPRT and KIT primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using KIT gene primers.

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE-dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI-1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC50 values ranging between 0.1 and 1 µM in primary MCT cells and between 1 and 3 µM in C2 and NI-1 cells. In C2 cells, the combination "ibrutinib + midostaurin" produced synergistic growth-inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE-dependent histamine release in primary MCT cells, with IC50 values ranging from 0.05 to 0.1 µM in NI-1 cells, and from 0.05 to 1 µM in primary MCT cells. In summary, ibrutinib exerts anti-proliferative effects in canine neoplastic MCs and counteracts IgE-dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.

Prognostic and predictive significance of KIT protein expression and c-kit gene mutation in canine cutaneous mast cell tumours: A consensus of the Oncology-Pathology Working Group.

One of the primary objectives of the Oncology-Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer-reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.

Identification of two molecular subtypes in canine mast cell tumours through gene expression profiling.

Mast cell tumours (MCTs) are common neoplasms in dogs and are usually regarded as potentially malignant. Several studies have attempted to identify biomarkers to better predict biological behaviours for this tumour. The aim of this study was to identify pathways connected to clinical and histopathological malignancies, shorter survival times, and poor prognoses associated with MCTs. We performed genome-wide gene expression analyses on tissues obtained from 15 dogs with single MCTs, and identified two distinct tumour subtypes-high-risk and low-risk-associated with differences in histological grades, survival times, Ki67 indices, and occurrence of death due the disease. Comparative analyses of RNA sequence profiles revealed 71 genes that were differentially expressed between high- and low-risk MCTs. In addition to these analyses, we also examined gene co-expression networks to explore the biological functions of the identified genes. The network construction revealed 63 gene modules, of which 4 were significantly associated with the more aggressive tumour group. Two of the gene modules positively correlated with high-risk MCTs were also associated with cell proliferation and extracellular matrix-related terms. At the top of the extracellular matrix module category, genes with functions directly related to those of cancer-associated fibroblasts (CAFs) were identified. Immunohistochemical analyses also revealed a greater number of CAFs in high-risk MCTs. This study provides a method for the molecular characterisation of canine MCTs into two distinct subtypes. Our data indicate that proliferation pathways are significantly involved in malignant tumour behaviours, which are known to be relevant for the induction and maintenance of MCTs. Finally, animals presenting high-risk MCTs overexpress genes associated with the extracellular matrix that can be robustly linked to CAF functions. We suggest that CAFs in the MCT stroma contribute to cancer progression.

Concentration of extracellular vesicles isolated from blood relative to the clinical pathological status of dogs with mast cell tumours.

Extracellular vesicles (EVs) are membrane-enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki-67 proliferative index, KIT-staining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT-staining pattern and Ki-67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT-staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations.