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 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).     

Recurrence rates and clinical outcome for dogs with grade II mast cell tumours with a low AgNOR count and Ki67 index treated with surgery alone

Grade II mast cell tumours (MCT) are tumours with variable biologic behaviour. Multiple factors have been associated with outcome, including proliferation markers. The purpose of this study was to determine if extent of surgical excision affects recurrence rate in dogs with grade II MCT with low proliferation activity, determined by Ki67 and argyrophilic nucleolar organising regions (AgNOR). Eighty‐six dogs with cutaneous MCT were evaluated. All dogs had surgical excision of their MCT with a low Ki67 index and combined AgNORxKi67 (Ag67) values. Twenty‐three (27%) dogs developed local or distant recurrence during the median follow‐up time. Of these dogs, six (7%) had local recurrence, one had complete and five had incomplete histologic margins. This difference in recurrence rates between dogs with complete and incomplete histologic margins was not significant. On the basis of this study, ancillary therapy may not be necessary for patients with incompletely excised grade II MCT with low proliferation activity.     

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.     

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.     

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.     

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).     

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.     

Epidemiology of canine cutaneous round cell tumours on the canary archipelago in Spain

In this study we undertook a comprehensive analysis of a Pet Tumour Registry of the Canary Archipelago (PTR-CA) in Spain to investigate the epidemiology of canine cutaneous round cell tumours. From a database of 2526 tumours collected from 2003 to 2020, we conducted a longitudinal analysis of the main trends in diagnosis, age, multiplicity and anatomical distribution as well as a case–control study comparing these cases with the contemporaneous canine population of the Canary Archipelago to analyse breed distribution. In line with former studies, we found histiocytomas mostly affect young dogs (2, IQR 1–5) and mast cell tumours affect middle-to-old dogs (8, IQR 6–10) with grade 1 affecting at younger ages (6.5, IQR 6–8) than both grade 2 (8, IQR 6–10 years) and grade 3 (9, IQR 7–11). Histiocytomas and plasmacytomas showed a similar anatomical distribution appearing mainly on the face, head and neck regions while mast cell tumours occur mainly on limbs and trunk. Higher risk for mast cell tumours and histiocytomas were found for Bulldog-related breeds such as Boxer (OR_MCT = 23.61, CI95%: 19.12–29.15, OR_HCT = 10.17, CI95%: 6.60–15.67), Boston Terrier (OR_MCT 19.47, CI95%: 7.73–49.05, OR_HCT 32.61, CI95%: 11.81–90.07) and Pug (OR_MCT 8.10, CI95%: 5.92–11.07, OR_HCT 7.87, CI95%: 4.66–13.28) while Chihuahua dogs showed significantly less risk (OR_MCT 0.18, CI95%: 0.09–0.33, OR_HCT 0.41, CI95%: 0.21–0.78). Notably, the Canarian Mastiff, a local breed, had a low risk of suffering from a mast cell tumour which raises the question of whether this relates to a genetic peculiarity of this breed or some husbandry and environmental factor.     

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.     

The treatment of canine mast cell tumours with electrochemotherapy with or without surgical excision

To describe the results of electrochemotherapy (ECT) in dogs with mast cell tumours (MCTs) either as first line therapy or as an adjuvant to surgery. The treatment combines administration of low dose chemotherapeutic drugs with the application of microsecond electric pulses, which cause the temporary permeabilization and increased porosity of the tumour cell membranes. The design of this study is a retrospective case series. A total of 51 dogs with MCTs were included and classified according to ECT procedure into 4 groups (ECT only, 15 cases, intra‐surgery ECT, 11, ECT Adjuvant to surgery, 14, Surgery followed by ECT, 11). The four groups (staged with location, size and grade) were evaluated to assess complete or partial remission, disease free interval, overall survival time and local toxicity. In this case series, Boxers, mixed breed and Labrador Retrievers, male dogs, between 4 and 9 years old were more represented. MCTs were predominantly grade 2 (Patnaik) and T stage 0–1, I‐1 (World Health Organization). Treated lesions were most commonly identified on the hindlimb and head where curative surgery would involve cosmetic or functional compromise. The intra‐surgery group of dogs showed the best disease free interval with Kaplan–Meyer analysis. Local toxicity induced by ECT ranged mostly from 1 to 4 in a 5‐point arbitrary scale with 0 – no toxicity to 5 – highest toxicity. In this study, ECT can be applied successfully as an exclusive therapy in smaller MCTs as an alternative to surgery. ECT can be combined with surgery either intra‐operatively or post operatively for larger lesions without significant toxicity.     

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.     

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.     

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.     

Expression of epidermal growth factor receptor (EGFR) and Ki67 in feline oral squamous cell carcinomas (FOSCC)

The aims of this study were to establish expression of epidermal growth factor receptor (EGFR) and Ki67 in 67 archived biopsy samples of feline oral squamous cell carcinomas (FOSCCs) and to establish if the expression of either markers was predictive of survival. Samples were immunohistochemically labelled for the two proteins and scored. Statistical analyses of data, including Kaplan-Meier survival curves, were performed. All samples expressed both markers although levels differed between samples. Median overall survival was 46 days and 1-year survival was 5%. There was no correlation between Ki67 and EGFR scores (Pearson's correlation coefficient, P = 0.861). Low cellular proliferation (low Ki67 score) was positively correlated with an overall longer survival (Log Rank, P = 0.02) and a trend towards better survival for the high EGFR group was observed (Log Rank, P = 0.076). Ki67 and EGFR immunostaining in FOSCC may be of value as biochemical markers for screening of biopsies from cases of FOSCC.     

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.     

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.     

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.