Cytological grading of canine cutaneous mast cell tumours

A cytological grading for mast cell tumours (MCTs) would be highly desirable, allowing to select the most appropriate therapeutic intervention prior to surgery. This study evaluates the applicability on fine‐needle aspirations (FNAs) of the novel Kiupel grading system, based on number of mitoses, multinucleated cells, bizarre nuclei and presence of karyomegaly. Fifty consecutive cases with pre‐operative cytological diagnosis were included. In cytological specimens, approximately 1000 cells were evaluated, and the histological grade was assessed on the corresponding resected specimens. On cytology, the above parameters were significantly different between histologically low‐grade and high‐grade tumours (P < 0.001). The cytograding correctly predicted the histological grade in 47 cases (accuracy, 94%; sensitivity, 84.6%; specificity, 97.3%). Two high‐grade MCTs (4%) were not detected on cytology. The cytograding can provide helpful insights to assist clinical decisions in most cases. However, the risk of underestimation in a minority of patients represents a limit to the overall utility of the technique.     

Treatment of canine mast cell tumours with prednisolone and radiotherapy

This retrospective study describes 35 dogs with non‐resectable, grade I–III mast cell tumours on the head or limb treated with prednisolone (40 mg m^?2 daily) for 10–14 days prior to radiotherapy (4 × 800 cGy fractions at 7‐day intervals) from a 4 MV linear accelerator. Prednisolone was continued at a reduced dose rate (20 mg m^?2) during radiotherapy and for 2 months or longer afterwards. Eighteen of 24 tumours (75%) decreased in size in response to prednisolone treatment. By 6–8 weeks following radiotherapy, 12 dogs had achieved a complete remission and 19 a partial response. Two tumours remained static and two progressed during the course of treatment. The overall response rate was 88.5%. With long‐term follow‐up, 11 dogs experienced local recurrence (n = 4), metastasis (n = 5) or both (n = 2). The median progression‐free interval was 1031 days (95% CI 277.44–1784.56, Kaplan–Meier), with 1‐ and 2‐year progression‐free rates of 60 and 52%, respectively. Tumour grade did not predict the prognosis for this group of dogs, but tumour location did affect the outcome. Dogs with tumours located on the limb survived longer than those with tumours on the head. The combination of prednisolone with radiotherapy appears to have a useful role in the management of measurable mast cell tumours sited on the head and distal extremities.     

Combination CCNU and vinblastine chemotherapy for canine mast cell tumours: 57 cases

The purpose of this study was to evaluate the efficacy and toxicity of a CCNU and vinblastine chemotherapy protocol for canine mast cell tumours. Fifty-seven tumours in 56 dogs were evaluated, 37 had macroscopic disease and 20 had microscopic disease. A 57% response rate was seen in dogs with macroscopic disease for a median duration of 52 weeks. Dogs with macroscopic disease had a median progression free survival time (PFST) of 30 weeks and a median overall survival time (OST) of 35 weeks. Dogs with microscopic disease had a median PFST of 35 weeks and a median OST of 48 weeks. Toxicity was recorded in 54% of the dogs treated, with the majority of events being mild. This chemotherapy protocol appears to be well tolerated and should be considered for canine mast cell tumours.     

Association of Ki67 index with prognosis for intermediate‐grade canine cutaneous mast cell tumours*

Intermediate‐grade mast cell tumours (MCT) represent a heterogeneous population of tumours. The prognosis for the majority of dogs is excellent following surgical excision, but a minority die because of their disease. A previous study identified Ki67 expression as a predictor of prognosis in all three grades of MCT. The purpose of this study was to validate those results in a new group of dogs, with intermediate‐grade MCT only. Ki67 immunohistochemistry was performed on intermediate‐grade MCT from 163 dogs with known outcome. Digital microscopy images were taken from each tumour, and an index calculated of Ki67‐positive cells. Ki67 index as a binary variable with a cut‐off value of 1.8% was confirmed to be associated with prognosis (hazard ratio = 19.1, P < 0.0001) for this cohort of dogs. The 1‐year, 2‐year and 3‐year survival probabilities (with standard errors) of 127 dogs with a Ki67 index ≤1.8% were [0.95 (0.024), similar for all] and for 36 dogs with a Ki67 index >1.8% were 0.54 (0.100), 0.45 (0.101) and 0.33 (0.104), respectively.     

Retrospective analysis of outcome and prognostic factors of subcutaneous mast cell tumours in dogs undergoing surgery with or without adjuvant treatment

Subcutaneous mast cell tumours (SC MCTs) can display a different biological behaviour in dogs when compared to their cutaneous counterpart. There is a paucity of information with regards to the outcome of dogs with SC MCTs treated with surgery and/or receiving adjuvant chemotherapy. The aim of this study was to retrospectively review the outcome of dogs with surgically excised SC MCTs undergoing adjuvant treatment or not. A secondary aim was to assess prognostic factors in the same group. Fifty-two cases were included. Recurrence rate was 15% and 63% of evaluated lymph nodes were consistent with early or overt metastasis. Median survival time (range 83–1357 days) and median time to progression (range 14–1357 days) were not reached. Factors predictive of shorter overall survival time included increasing age (HR 1.29, 95% CI 1.06–1.55, p = .0092), presence of clinical signs at presentation (HR 10.44, 95% CI 2.69–40.52, p = .0007), mitotic count >4 (HR 8.69, 95% CI 2.55–29.55, p = 0.0005), presence of multinucleation (HR 4.21, 95% CI 1.35–13.18, p = .0135), use of neoadjuvant and adjuvant chemotherapy (HR 7.16, 95% CI 1.26–40.73, p = .0266). The same factors, together with increasing tumour dimensions, were predictive for shorter progression-free survival (PFS), including increasing age (p = .0012), presence of clinical signs at presentation (p = .0045), increasing tumour dimensions (p = .0004), MC > 4 (p = .0004), presence of multinucleation (p = .0282), use of neoadjuvant and adjuvant chemotherapy (p = .0485). No variables remained significant for overall survival using multivariate analysis. There was a longer survival in cases where chemotherapy was not required (HR 0.14, 95% CI 0.03–0.68, p = .0148), and this variable remained significant for PFS on multivariate analysis (HR 0.13, 95% CI 0.02–0.76, p = .02). In conclusion, our study suggests that dogs with SC MCTs, in the absence of negative prognostic factors, may have a prolonged survival when treated with surgery alone. Further studies are needed to clarify the role of adjuvant treatment for biologically aggressive SC MCTs in dogs.     

Evaluation of prognostic indicators in dogs with multiple,simultaneously occurring cutaneous mast cell tumours: 63 cases

Sixty‐three dogs with multiple contemporaneous cutaneous mast cell tumours (MCTs) were identified. The aim of this study was to determine the significance of breed, concurrent dermatological condition; number of cutaneous MCTs, size, location, histological grade and mitotic index; completeness of excision (complete, close or incomplete); local recurrence, metastasis and adjuvant therapy for the prognostic evaluation of dogs with a unique disease presentation of multiple, simultaneously occurring cutaneous MCTs. On the basis of multivariable survival analysis, dogs with one recorded grade 3 MCT had shorter progression‐free survival (PFS) times (18.7 versus 2.2 months) and median survival times (MSTs) (24 versus 3 months). Dogs treated with adjuvant vinblastine/lomustine had a 16 times increased risk of dying. MSTs were found to be significantly longer in dogs with one recorded MCT on an extremity. For all dogs, the PFS (range 14–1835 days) and MSTs (range 28–1835 days) were not reached.     

Evaluation of bone marrow aspirates from multiple sites for staging of canine lymphoma and mast cell tumours

This prospective study evaluated the utility of bone marrow aspirates (BMAs) obtained from multiple sites for staging of canine lymphoma (LSA) and mast cell tumours (MCTs). Forty dogs (LSA, n = 24; MCTs, n = 16) were enrolled, but only 33 (82.5%) had diagnostic bone marrow (BM) aspirates obtained from two sites for inclusion in the study. Nineteen dogs with LSA were included, and 6 (31.6%) had BM involvement. Neoplastic lymphocytes were present in BM from both sites in all of these dogs. Fourteen dogs with MCTs were included, and 3 (21.4%) had BM involvement. Neoplastic mast cells were present at both sites in two dogs and at only one site in the third. These results indicate that BMAs from multiple sites may not be needed for accurate staging of canine LSA patients, but more studies evaluating the pattern of BM infiltration in dogs with high‐grade MCTs are warranted.     

Vinblastine and prednisolone chemotherapy for surgically excised grade III canine cutaneous mast cell tumours

The effect of treatment with vinblastine and prednisolone chemotherapy in dogs undergoing only surgical excision of Patnaik grade III cutaneous mast cell tumours is reported. Potential explanatory variables were explored using Kaplan–Meier survival analysis with log‐rank tests. During a median follow‐up period of 429 days, the overall median survival time (MST) was not reached (lower 95% CI = 322 days). The 1‐year survival probability was 0.71 (standard error 0.1), remaining unchanged at 2 years. Secondary disease at presentation was an independent risk factor for survival (P= 0.045). The MST of dogs presenting with secondary disease was 322 days, with a lower 95% confidence interval of 142 days and a 1‐year survival of probability of 0.47 (standard error 0.19). Adverse effects were recorded in 6 of the 108 (5.6%) vinblastine doses given. This chemotherapy regimen is a well‐tolerated adjunct to surgery for grade III mast cell tumours and appears to prolong survival compared with that expected with surgery alone.     

Diagnostic accuracy of pre‐treatment biopsy for grading cutaneous mast cell tumours in dogs

Mast cell tumours (MCTs) are common tumours of the canine skin, and are estimated to represent up to 20% of all skin tumours in dogs. Tumour grade has a major impact on the incidence of local recurrence and metastatic potential. In addition to helping the clinician with surgical planning, knowledge of the tumour grade also assists in proper prognostication and client education. For pre‐treatment biopsies to be useful, there must exist a high level of correlation between the histopathological grade obtained from the pre‐treatment biopsy and the actual histopathological grade from the excisional biopsy. The aim of this study was to determine concordance of tumour grade between various biopsy techniques (wedge, punch, needle core) and the “gold standard” excisional biopsy method. We found an overall concordance rate of 96% based on the Patnaik grading system, and an overall concordance rate of 92% based on the Kiupel grading system. The accuracy of the various biopsy techniques (wedge, punch and needle core) when compared with excisional biopsy was 92%, 100% and 100%, respectively, based on the Patnaik grading system, and 90%, 95% and 100%, respectively, based on the Kiupel grading system. Of the cases with discordant results, the pre‐treatment biopsies tended to underestimate the grade of the tumour. Based on these results, we conclude that pre‐treatment biopsies are sufficiently accurate for differentiating low‐grade from high‐grade MCTs, regardless of biopsy technique or tumour location.     

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c‐KIT mutations) in cats affected with splenic mast cell tumours. Twenty‐two cats were included. Median survival time was 780 days (range: 1–1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c‐Kit mutations and tumour differentiation, mitotic activity or survival.     

High COX‐2 expression in canine mast cell tumours is associated with proliferation,angiogenesis and decreased overall survival

COX‐2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX‐2, CD31, Ki‐67, MAC‐387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX‐2 intensity (P = 0.016), but not COX‐2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox‐2 intensity was also associated with higher Ki‐67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX‐2 intensity and CD3‐T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC‐387 immunollabelling, suggesting an active role of COX‐2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.     

Morphometrical approach for predicting regional lymph node micrometastatic load in canine mast cell tumours: preliminary results

Canine cutaneous mast cell tumours (MCTs) have a variable biologic behaviour, and accurate staging is necessary to dictate therapy and predict outcome. Regional lymph node (RLN) involvement is a relevant prognostic factor. While obvious lymph node (LN) metastases are relatively easy to be diagnosed, micrometastatic disease recognition is challenging. The main aim of the study was to evaluate the number of mast cells (MCs) in the LNs of clinically healthy dogs ( n = 4, group 1), dogs with inflammatory diseases ( n = 31, group 2) and dogs with cutaneous MCT ( n = 27, group 3), including animals with no RLN metastases (subgroup 3.1), those with occasional MCs in RLNs (3.2) and those with obvious RLN metastasis (3.3). MCs also were morphometrically evaluated for the following nuclear parameters: mean nuclear area (MNA), mean nuclear perimeter (MNP), largest to smallest diameter length (LS ratio), mean nuclear form factor and coefficient of variation of nuclear area. The average percentages of MCs were 0.0 and 0.01 in groups 1 and 2, respectively, and 0.07, 2.4 and 47.1 in subgroup 3.1, 3.2 and 3.3. MNA and MNP were significantly higher in subgroup 3.3 than in group 2 ( P < 0.05). MNA and MNP in subgroup 3.2 suggested the presence of neoplastic MCs; this prediction of micrometastatic load correlated with outcome. Analysis of preliminary results shows that nuclear morphometry is useful to detect micrometastatic disease in RLN of dogs bearing cutaneous MCTs.     

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 radiosensitizing effect of the aurora kinase inhibitors,ENMD‐2076, on canine mast cell tumours in vitro

ENMD‐2076 is an aurora kinase inhibitor that also has multi‐target tyrosine kinase inhibitor properties. In this study, the mRNA and the protein expression of aurora‐A and aurora‐B were evaluated in three canine mast cell tumour cell lines. Dose‐dependent cytotoxicity was seen in the cells treated, and it affected the cell cycle with cells in the G2/M phase being selectively killed. The cells were also evaluated for radiosensitivity with/without ENMD‐2076, and radiosensitization was seen after 3 Gy and 6 Gy exposures with ENMD‐2076 for 48 h. Protein expression of caspase‐3 was gradually increased, and the expression intensity was highest at 24 h post irradiation in cells without ENMD‐2076 treatment, which indicates that radiation exposure with ENMD‐2076‐induced cell death faster than radiation treatment alone. Our study results suggest the potential usefulness of treating canine mast cell tumours with aurora kinase inhibitors alone or in conjunction with radiation therapy.     

Magnetic resonance imaging of canine mast cell tumors

Mast cell tumors (MCT) are the most common cutaneous tumors in dogs. Our purpose was to describe the magnetic resonance (MR) imaging characteristics of cutaneous MCT and to identify imaging characteristics that allow differentiation of metastatic from normal lymph nodes. Eight dogs with a total of nineMCT were imaged as were their presumed draining and associated contralateral lymph nodes. The signal intensity of tumors and lymph nodes was compared to adjacent musculature. On T2-W images, 7/9 MCT were hyperintense to muscle and 2/9 were isointense. On T1-W images, 8/9 MCT were isointense and 1/9 were mildly hypointense. All tumors were strongly contrast enhancing; 5/9 were homogeneous and 4/9 heterogeneous in their enhancement patterns. Six lymph node pairs were included in the evaluation (five sentinel lymph nodes with metastases, one without, and six contralateral lymph nodes). Metastatic lymph nodes were significantly larger than their contralateral lymph nodes (P = 0.039). All lymph nodes were isointense on T1-W images and hyperintense on T2-W images. 5/5 metastatic and 2/7 normal lymph nodes were heterogeneously T2-hyperintense. All lymph nodes were moderately to strongly contrast enhancing. 4/5 metastatic and 2/7 normal lymph nodes had heterogeneous enhancement patterns. While heterogeneity was more common in metastatic than in normal lymph nodes, this difference was not significant (P = 0.058 for T2-W images; P = 0.234 for postcontrast images). MR imaging may be useful in the presurgical evaluation and clinical staging of cutaneous MCT.     

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.     

Immunohistochemical evaluation of prostaglandin E2 and vascular endothelial growth factor in canine cutaneous mast cell tumours

Vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE-2) appear to play a critical role in tumour neovascularization. In this study, we have investigated the expression of VEGF and PGE-2 in 53 canine cutaneous mast cell tumours (MCTs). Immunohistochemistry of tissue sections revealed that VEGF and PGE-2 were expressed in all mast cell tumours studied. When the expression patterns of VEGF and PGE-2 were compared with tumour grade according to Patnaik criteria, the only significant correlation observed was between PGE-2 staining intensity and tumour pathological grade, with grade II and III tumours having higher PGE-2 staining, both in intensity and percentage of cells stained, than grade I tumours ( P < 0.05).     

Retrospective outcome evaluation for dogs with surgically excised,solitary Kiupel high‐grade,cutaneous mast cell tumours

Published outcomes for dogs with specifically high‐grade mast cell tumours (MCTs), controlled for clinical stage, are few. Clinical outcomes for 49 dogs with Kiupel high‐grade, clinical stage I, cutaneous MCTs were evaluated. Median survival time (MST) was 1046 days; 1 and 2‐year survival rates were 79.3% and 72.9%, respectively. At study end 24 dogs had died, 23 dogs were alive (median follow‐up 980 days) and 2 dogs were lost to follow‐up. Death was considered MCT‐related in 14 of 20 dogs with a known cause of death. Local tumour recurrence developed in nine dogs (18.4%); regional lymph node metastasis occurred in six dogs (12.2%); and a new MCT developed in 15 dogs (30.1%). Tumour location, histologic margin size and use of chemotherapy did not affect MST; increasing mitotic count (P = .001) and increasing tumour diameter (P = .024) were independently negatively prognostic. Six dogs that developed lymph node metastasis after surgery had worse MST (451 days) than 42 dogs that did not develop metastasis (1645 days); (P < .001). Our study suggests that dogs with local surgical control of clinical stage I histologically high Kiupel grade cutaneous MCT may have a long survival time; especially those with smaller tumours and a lower mitotic count. Our results suggest that evaluation of staging information and mitotic count may be equally helpful as histologic grading when making a prognosis; and highlight the importance of not relying on histologic grade alone when predicting survival for dogs with MCT.     

Unilateral intraocular mastocytosis and anterior uveitis in a dog with subcutaneous mast cell tumors

A 9‐year‐old male castrated Scottish terrier was referred to the Radiation Oncology Service at the William R. Pritchard Veterinary Medical Teaching Hospital for palliative radiation therapy of an incompletely excised, recurrent subcutaneous mast cell tumor (MCT) located over the right scapula, and surgical removal of a perianal MCT. Three weeks after initial presentation and prior to the fifth radiation treatment, the patient was presented with cloudiness of the left eye of 3–7 days duration. Ophthalmic consultation revealed 3+ aqueous flare with a dependent, swirling component filling approximately one‐third of the anterior chamber. Aqueocentesis was performed under general anesthesia. Cytology revealed mast cells with highly atypical morphology and considered most consistent with neoplasia. The patient died 7 months after pathologic diagnosis of MCT on the right shoulder and 2 months after the cytologic diagnosis of malignant mast cells in the left anterior chamber. To the authors' knowledge, this is the first report of intraocular involvement in a mammal with MCTs, described here as intraocular mastocytosis.