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.     

Patient and tumour factors influencing canine mast cell tumour histological grade and mitotic index

The aim of this study was to identify patient and tumour factors most frequently associated with high histological grades of canine mast cell tumours (MCTs). Search criteria in a shared database of multiple Animal Referral Hospital locations within Australia generated 400 canine MCTs in 286 patients. Patient and tumour data were extrapolated and the association between a tumour being histologically high grade and patient and tumour factors, including: patient breed, patient gender and neuter status, patient age at MCT excision, tumour location and tumour size was assessed using univariate analysis. The study consisted of 90 (21.9%) tumours meeting histological high‐grade criteria. Shar peis were the most likely breed to have high grade MCTs, whereas the Pug and the Golden Retriever were the least likely breeds to develop high‐grade MCTs. No significant difference in risks could be established between the age at which the tumour was excised, or the gender and neuter status of patients. MCTs of the inguinal region were the most likely single location to be high grade. Tumour size did not influence the likelihood of a tumour being high grade or low grade. The results of this study suggest that patient and tumour factors may play a role in the histological grades of canine MCTs.     

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 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.     

Cytological lymph node evaluation in dogs with mast cell tumours: association with grade and survival*

The purpose of this retrospective cohort study is to describe the association of cytological assessment of lymph node metastasis with survival and tumour grade in dogs with mast cell tumours. Regional lymph node aspirates of 152 dogs diagnosed with a mast cell tumour were reviewed and classified according to specific cytological criteria for staging. 97 dogs (63.8%) had stage I tumours, and 55 (36.2%) had stage II tumours. Stage II dogs had a significantly shorter survival time than dogs with stage I disease (0.8 and 6.2 years, respectively; P < 0.0001). Dogs with grade III mast cell tumours were more likely to have stage II disease (P = 0.004). These results suggest that cytological evaluation of lymph nodes in dogs with mast cell tumours provides useful and valuable clinical information, and the results correlate with tumour grade and outcome thus providing a practical and non‐invasive method for staging.     

Evaluation of risk and clinical outcome of mast cell tumours in pug dogs

Mast cell tumours (MCT) are common in dogs and characterized by diverse biologic behaviour. Our objective was to evaluate the risk of MCT in pugs and to describe the clinical behaviour of MCT in this breed. Data obtained from the Veterinary Medicine Database demonstrate significantly increased frequency of MCT in pugs compared with other dogs (OR = 2.28, 95% CI = 1.81–2.86). The medical records for 25 purebred pugs with a histologic diagnosis of MCT were reviewed. Multiple cutaneous tumours were documented in 14 (56 %) of the dogs. Histologic review of 64 tumours from these dogs confirmed that most tumours (94%) were low to intermediate grade. Sixty‐four per cent of these dogs are still living, while only three dogs (12%) have died due to mast cell disease. A median survival time has not been reached. The median follow‐up time is 660 days from the diagnosis of the first MCT. We conclude that MCT in pugs are relatively benign, despite the presence of multiple cutaneous tumours in most cases. Multiple tumours in breeds with predisposition to MCT may indicate separate primaries rather than advanced stage disease.     

A retrospective review of treatment and response of high‐risk mast cell tumours in dogs

This retrospective case series evaluates survival outcome of 94 dogs with high metastatic risk mast cell tumours (MCT). Patients were treated with a cytotoxic chemotherapy protocol or the tyrosine kinase inhibitor masitinib, in the presence of gross disease or as an adjunct to surgical resection of the primary tumour. In patients presenting with metastatic disease, surgical resection of the primary tumour with adjunctive therapy with any chemotherapy incurred a significant survival advantage [median survival time (MST): 278 days] compared to patients receiving chemotherapy without surgical excision of the primary tumour (MST: 91 days, P < 0.0001). Patients with a surgically excised Patnaik grade II tumour and high Ki‐67 in the absence of metastatic disease treated with vinblastine and prednisolone showed a significantly longer survival (MST: 1946 days) than those treated with masitinib (MST: 369 days, P = 0.0037). Further prospective case‐controlled clinical trials of high‐risk MCTs are required to make precise evidence‐based treatment decisions for individual patients.     

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.     

Chlorambucil and prednisolone chemotherapy for dogs with inoperable mast cell tumours: 21 cases

O bjectives : To evaluate the response of measurable canine mast cell tumours unsuitable for other treatment modalities to a chemotherapy protocol comprising chlorambucil and prednisolone.
M ethods : Dogs bearing measurable mast cell tumours, unsuitable for treatment by surgery or radiotherapy, were treated with orally administered prednisolone and chlorambucil, and their responses assessed.
R esults : Twenty-one dogs were enrolled in the study; 13 had intermediate-grade mast cell tumour, six were high grade and two were diagnosed by cytology alone. Eight dogs had multiple tumours and 13 dogs had single tumours, and six dogs had lymph node metastases and no dogs had visceral metastases detected. Three dogs achieved complete remission, five achieved partial remission (overall response rate 38 per cent), nine had static disease and four dogs had progressive disease. Median progression-free interval for the eight responders was 533 days, and median survival time for all dogs in the study was 140 days. Progression-free interval and median survival time were not influenced by the age, sex, weight or neutering status of the patient, by the grade or stage of the tumour or whether the patient had single or multiple tumours. No toxicity was detected.
C linical S ignificance : Response and survival rates of inoperable canine MCT to chlorambucil and prednisolone are comparable to previously described protocols, with no apparent toxicity.     

Association of tumour‐infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours

Regulatory T cells (Tregs) infiltrate into a variety of tumour tissues and associate with poor prognosis in humans. However, data on association of Treg infiltration with prognosis is limited in canine tumours. The purpose of this study was to examine the number of tumour‐infiltrating Tregs and its association with overall survival (OS) in dogs with malignant tumours. The following 168 canine tumours were included: 37 oral malignant melanomas (OMMs); 14 oral squamous cell carcinomas (OSCCs); 16 pulmonary adenocarcinomas (PAs); 37 mammary carcinomas (MCs); 36 mast cell tumours (MCTs) and 28 hepatocellular carcinomas (HCCs). Normal tissues were obtained from 8 healthy dogs as controls. The number of forkhead box P3 (Foxp3)‐positive Tregs in intratumoral and peritumoral areas was investigated by immunohistochemistry. OS was compared between high and low Treg groups. The number of intratumoral and peritumoral Foxp3‐positive Tregs was significantly higher in OMM, OSCC, PA and MC compared with each normal tissue. There were few Foxp3‐positive Tregs in MCT and HCC. With intratumoral Tregs, the OS in the high Treg group was significantly shorter than that in the low Treg group in OMM, OSCC and PA. With peritumoral Tregs, there was no significant difference for OS between the 2 groups in each tumour type. These results suggest that Tregs infiltrate into a variety of canine tumours and the abundance of Tregs are associated with poor prognosis in some solid tumour types.     

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.     

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.     

Increased expression of tissue inhibitor of metalloproteinase‐1 correlates with improved outcome in canine cutaneous mast cell tumours

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP‐2 and ‐9 and tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP‐2, MMP‐9, TIMP‐2 and TIMP‐1 was performed in 46 canine cases of MCTs. TIMP‐1 expression showed an independent prognostic value for post‐surgical survival and disease‐related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP‐1 positivity were more prone to die because of the disease and had a shorter post‐surgical survival. This article suggests the involvement of TIMP‐1 in MCT progression, by contributing to a good outcome in patients with 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.     

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.     

Cutaneous MCTs: associations with spay/neuter status, breed, body size, and phylogenetic cluster

Certain breeds are known to be overrepresented among mast cell tumor (MCT) patients, but other risk factors have not been evaluated. This study presents results from a case-control study of 252 dogs with grade 2 or grade 3 cutaneous MCT. Increased risk for MCT development was found in spayed females (adjusted odds ratio [OR], 4.11), boxers (adjusted OR, 6.09), Labrador retrievers (adjusted OR, 3.95), pugs (adjusted OR, 3.17), golden retrievers (adjusted OR, 2.12), the mastiff and terrier phylogenetic cluster (adjusted OR, 3.19), and breeds classified as large (adjusted OR, 2.10) or giant (adjusted OR, 5.44). Additional studies are needed to evaluate the role of these and other potential risk factors in MCT development.     

The value of molecular expression of KIT and KIT ligand analysed using real‐time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real‐time polymerase chain reaction (RT‐PCR) and the rate of tumour recurrence and tumour‐related deaths in dogs affected with mast cell tumour (MCT). Kaplan–Meier curves were constructed to compare tumour recurrence and tumour‐related death between patients. The log‐rank test was used to check for significant differences between curves. KIT‐I, KIT‐II and KIT‐III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour‐related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT‐PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis.     

Genomic hypomethylation in neoplastic cells from dogs with malignant lymphoproliferative disorders

DNA methylation is an epigenetic modification in which a methyl group is added usually to the fifth carbon position of a cytosine residue. Dysregulation of this process is an important molecular event which has been shown to be associated with neoplastic transformation and tumour progression in humans and mice. Features of methylation dysregulation in many different types of neoplasms include general genomic hypomethylation, focal hypermethylation, and altered expression of genes which encode a series of DNA (cytosine-5) methyltransferases. Interestingly, many types of neoplasia that are recognised in humans also develop spontaneously in the dog. By comparing the restriction patterns of MspI and HpaII, this study demonstrates that as in human, genomic hypomethylation is a feature of neoplastic cells in the majority of canine lymphoma cases and approximately one-third of canine leukemia cases confirming that dysregulation of the DNA methylating machinery is implicated in malignant transformation of lymphoid cells in some dogs.     

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).     

Dose-escalating vinblastine for the treatment of canine mast cell tumour

The purpose of this study was to evaluate the short‐term adverse events (AEs) in dogs with mast cell tumours (MCT) receiving prednisone and dose‐escalating vinblastine (VBL). Twenty‐four dogs were treated with intravenous VBL starting at 2 mg m^?2 and then escalating in weekly increments to 2.33, 2.67 and 3 mg m^?2. AEs were graded using a standardized scoring system. No dogs receiving 2 or 2.33 mg m^?2 experienced grade 3 or 4 AEs. Among the dogs, 9.5 and 5.9% had grade 3 or 4 AEs at dosages of 2.67 and 3 mg m^?2, respectively. Serious AEs included neutropaenia (n = 3) and vomiting (n = 1), only one of which required hospitalization. These data indicate that VBL chemotherapy may be safe to administer at higher than the traditional 2 mg m^?2 dosage for dogs with MCT. Randomized prospective trials are necessary to establish whether dose escalation will translate into improved response rates when compared with the standard 2 mg m^?2 dosage.