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.     

Metastatic mast cell tumour in a dog presenting with spinal pain

The clinical, advanced imaging and surgical features of a case of canine extradural mast cell tumour are presented. This rare location has only been reported once before as a primary lesion, although the present case may be a metastatic lesion. Given the frequent occurrence and treatment of cutaneous mast cell tumours in dogs, the finding of spinal metastasis is an important reminder to consider metastatic neoplasia in cases of previously treated cancers presenting with spinal pain.     

Aggressive local therapy combined with systemic chemotherapy provides long‐term control in grade II stage 2 canine mast cell tumour: 21 cases (1999–2012)

This retrospective case series evaluates the outcome of 21 dogs with grade II stage 2 mast cell tumour (MCT) treated with adequate local therapy and adjuvant systemic chemotherapy (prednisone, vinblastine and CCNU). The median survival for all dogs was 1359 days (range, 188–2340). Median disease‐free interval was 2120 days (149–2325 days). Dogs treated with surgery and chemotherapy had shorter survival (median, 1103 days; 188–2010 days) than those that underwent surgery, radiation therapy and chemotherapy as part of their treatment (median, 2056 days; 300–2340 days). Two patients had local recurrence in the radiation field and four patients had de novo MCT. Distant metastasis was not observed in any dogs. The results of this study suggest that, in the presence of loco‐regional lymph node metastasis in grade II MCT, the use of prednisone, vinblastine and CCNU after adequate local‐regional therapy can provide a median survival in excess of 40 months.     

TSLC1 tumour-suppressor gene expression in canine mast cell tumours

Tumour suppressor in lung cancer-1 (TSLC1) is a tumour-suppressor gene coding for an adhesion molecule that is expressed by mast cells. Reduced TSLC1 expression is associated with a poor prognosis in several human tumours, and this study sought to investigate if TSLC1 expression could be used to predict outcome in dogs with mast cell tumours (MCTs). Sections of MCTs of different tumour grades from 45 dogs (Group 1) were immunohistochemically assessed for TSLC1 and Ki67 expression. In addition, 35 intermediate-grade MCTs (Group 2) from dogs with known clinical follow-up were immunohistochemically stained for TSLC1 and Ki67. The TSLC1 staining intensity was found to strongly inversely correlate with tumour grade for Group 1 (P = 0.002857). For Group 2 there was a trend towards dogs with lower TSLC1 scores being more likely to die from MCT-related disease (P = 0.058). The intensity of TSLC1 staining inversely correlated with Ki67 expression for both groups.     

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.     

Evaluation of histological grade and histologically tumour‐free margins as predictors of local recurrence in completely excised canine mast cell tumours

Completeness of mast cell tumour (MCT) excision is determined by assessment of histologically tumour‐free margins (HTFM). The HTFM width necessary to prevent local recurrence (LR), recognized as histologic safety margin (HSM) in human oncology, has not been defined. We hypothesized that HTFM width would correlate with risk for LR and high‐grade tumours would require wider HTFM than low‐grade tumours. Records of dogs with completely excised MCTs were included. Signalment, two‐tier tumour grade, tumour size, HTFM width, recurrence and therapy data was collected. High‐grade (n = 39) tumours were more likely to recur than low‐grade (n = 51) tumours (35.9% versus 3.9%), P < 0.0001, with no association between HTFM width and LR. Twenty‐nine percent of low‐grade tumours had HTFM less than 3 mm; none recurred. Narrow (≤3 mm) histologic margins are likely adequate to prevent LR of low‐grade tumours. High‐grade tumours have significant risk of LR regardless of HTFM width.     

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.     

Evaluation of minichromosome maintenance protein 7 as a prognostic marker in canine cutaneous mast cell tumours

Minichromosome maintenance proteins (MCMs) are sensitive markers of cellular proliferation and have been shown to be significant predictors of survival in several human malignancies. MCM7 was evaluated as a prognostic marker in canine cutaneous mast cell tumours (MCTs). MCM7 immunohistochemistry was performed and an index of MCM7-positive cells calculated in dogs with known outcome. The Receiver Operating Characteristics method was used to individuate the best cut-off value of MCM7 score as predictor of survival. Survival analysis and prognostic variables were analysed with statistical methods. Ninety-five dogs were included with 31 dying of MCTs. A value of 0.18 was used as cut-off value of MCM7 score as a binary variable. The median survival time for MCM7 score ≤0.18 was not reached at 3668 days, whereas for MCM7 score >0.18 was 187 days (log-rank test; P < 0.0001). In the multivariable analysis, MCM7 was significantly associated with survival after controlling for age, surgical margins and histological grade (hazard ratio 9.2; P = 0.001).     

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.     

Pre-operative neoadjuvant vinblastine-prednisolone in canine mast cell tumours: A single-centre retrospective cohort study

Neoadjuvant chemotherapy can be used in canine mast cell tumours (MCTs) to optimise surgical margins or to enable marginal excision in challenging locations. The objective of this study was to describe the outcome of dogs with cutaneous and subcutaneous MCTs treated with neoadjuvant vinblastine-prednisolone (NA-VP). Records of treatment-naïve dogs with cutaneous/subcutaneous MCT that received NA-VP were reviewed including signalment, indication for NA-VP, staging results, clinical response, surgical data and histopathology reports. For dogs with post-operative follow-up ≥365 days, predictive factors for local recurrence (LR) were evaluated. Forty-four dogs were included. NA-VP was indicated to optimise surgical margins (group MARG) in 19 dogs (43.2%) and to enable surgery (group MORB) in 25 dogs (56.8%). Complete and partial response were documented in 40.9% of dogs and 30 dogs (68.2%) underwent surgery. The indication for NA-VP was significantly associated with undergoing surgery (p < .001) on multivariable analysis. Twelve (48%) and 18 dogs (94.7%) underwent surgery in the group MORB and MARG, respectively. Five dogs (16.7%) experienced wound dehiscence. Complete excision was achieved in 14 dogs (46.7%). In dogs undergoing surgery with ≥365 days of follow-up, LR was documented in five cases (20.8%). None of the factors analysed including mitotic count, completeness of excision and response to NA-VP were associated with LR; notably, LR occurred in 3/11 (27.2%) completely excised MCTs. In a pre-operative setting, NA-VP appears safe and could be beneficial in selected cases. Prognostic factors such as clinical response, mitotic count and completeness of excision should be interpreted with caution following NA-VP.     

Efficacy and safety of electrochemotherapy combined with peritumoral IL‐12 gene electrotransfer of canine mast cell tumours

Electrochemotherapy combined with peritumoral interleukin‐12 (IL‐12) gene electrotransfer was used for treatment of mast cell tumours in 18 client‐owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL‐12 gene electrotransfer resulted in 78% of patients with detectable serum IFN‐γ and/or IL‐12 levels. In the treated tumours vascular changes as well as minimal T‐lymphocytes infiltration was observed. After 1 week, the plasmid DNA was not detected intra‐ or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL‐12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours.     

Masitinib mesylate for metastatic and non‐resectable canine cutaneous mast cell tumours

Masitinib mesylate is a tyrosine kinase inhibitor approved for the treatment of gross, non‐metastatic grade II and III canine mast cell tumours (MCTs). This study evaluated the use of masitinib as a frontline and rescue agent for metastatic and non‐metastatic canine MCTs. Identification of toxicities and prognostic factors in these dogs was of secondary interest. Twenty‐six dogs were included in this study. The overall response rate to masitinib was 50%. The median survival time for dogs that responded to masitinib was 630 days versus 137 days for dogs that did not respond (P = 0.0033). Toxicity was recorded in 61.5% of treated dogs, but the majority of adverse events were mild and self‐limiting. Response to masitinib, not tumour grade, stage or location, was the most significant prognostic factor for survival in dogs with MCTs.     

Treatment of an invasive equine mast cell tumour in the extensor carpi radialis by extensive tenomyectomy and local corticosteroid injections

Equine mast cell tumours (MCT) have been reported to occur in the skin, respiratory tract, oral cavity, synovial structures and eye. These tumours are typically locally invasive and nonaggressive. In this case, a MCT was located in the extensor carpi radialis (ECR) and was highly infiltrative. Excision of the ECR and local corticosteroid injection was curative and resulted in a good functional outcome. A 12-year-old Thoroughbred gelding presented for lameness, swelling and pruritus associated with the right antebrachium. Sonographic changes were suggestive of severe tenosynovitis and purulent material in the ECR. Surgical exploration revealed maceration at the ECR musculotendinous junction. The extensor sheath contained a large quantity of caseous material and serosanguinous fluid. The ECR tendon and 75% of the muscle was excised. Cytology and histopathology were consistent with a MCT that infiltrated the surgical margins. Post-operatively the horse was treated with multiple local corticosteroid injections along the incision. Right forelimb movement was initially limited, however, there was steady improvement over 3 months with increasing exercise. At 18 months post-operatively, no lameness was detectable in the walk, trot or canter and no recurrence of MCT was apparent. The horse appeared to compensate for loss of the ECR by using the biceps during the swing phase. Partial excision and local corticosteroid treatment can be curative for cases of invasive MCT. Extensive excision of the ECR is a viable treatment option for neoplasia or other conditions affecting the ECR in the horse.     

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.     

Treatment of canine mast cell tumours with vinblastine, cyclophosphamide and prednisone: 35 cases (1997–2004)

The purpose of this retrospective study was to determine the efficacy and toxicity of a combined protocol of vinblastine, cyclophosphamide and prednisone (VCP) in 35 dogs with mast cell tumours (MCTs). Eleven dogs had measurable disease (group 1) and 24 dogs had incompletely excised MCT or were at high risk for metastasis (group 2). Five patients in group 1 achieved complete response, two partial responses, two stable diseases and two progressive diseases. The median progression‐free survival time (PFST) for group 1 and 2 dogs was 74 and 865 days, respectively. The median overall survival time (OST) for group 1 and 2 dogs was 145 and >2092 days, respectively. Significant negative multivariate prognostic factors included macroscopic disease and reduced vinblastine (VBL) treatments for PFST, and presence of MCT in bone marrow analysis, Patnaik grade III MCT and reduced VBL treatments for OST. Toxicity was infrequent and self‐limiting. This study suggests that the VCP protocol should be considered as an option in the treatment of MCT in dogs.     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

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.