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.     

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.     

Comparison of mitotic index and Ki67 index in the prognostication of canine cutaneous mast cell tumours

Proliferation markers are commonly used for prognostication of mast cell tumours. The aim of the study is to compare the relative abilities of Ki67 and mitotic index to predict survival in the same cohort of dogs with cutaneous MCTs. Histological grade, mitotic index and Ki67 index were performed in all samples and clinical information was obtained by a follow‐up questionnaire. Ninety‐five dogs were included in the study with a median follow‐up of 1145 days. Survival times varied significantly between categories of histological grade, mitotic index and Ki67 index. Multivariable analyses showed that the risk of dying due to MCT was similar in dogs with increased Ki67 index [hazard ratio, HR: 3.0 (95% CI 1.3–6.8)] or increased mitotic index [HR: 2.7 (95% CI 1.1–6.5)]. In conclusion, both mitotic index and Ki67 index were able to independently differentiate MCTs with worse prognosis. This distinction is particularly meaningful in selecting intermediate grade MCTs that may benefit from more aggressive local or systemic treatment.     

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.     

Cellular proliferation in canine cutaneous mast cell tumors: associations with c-KIT and its role in prognostication

Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease in dogs. Due to the prevalence of canine MCTs and the variable biologic behavior of this disease, accurate prognostication and a thorough understanding of MCT biology are critical for the treatment of this disease. The goals of this study were to evaluate and compare the utility of the proliferation markers Ki67, proliferating cell nuclear antigen (PCNA), and argyrophilic nucleolar organizing region (AgNOR) as independent prognostic markers for canine MCTs and to evaluate the use of these markers in combination, as each marker assesses different aspects of cellular proliferation. An additional goal of this study was to evaluate the associations between cellular proliferation and c-KIT mutations and between cellular proliferation and aberrant KIT protein localization in canine MCTs. Fifty-six MCTs treated with surgical excision alone were included in this study. Each MCT was evaluated for Ki67 expression, PCNA expression, and KIT protein localization using immunohistochemistry; for AgNOR counts using histochemical staining; and for the presence of internal tandem duplication c-KIT mutations using polymerase chain reaction amplification. In this study, increased Ki67 and AgNOR counts were both associated with significantly decreased survival. On the basis of these results, we recommend that the evaluation of cellular proliferation, including evaluations of both Ki67 expression and AgNORs, should be routinely used in the prognostication of canine MCTs. Additionally, the results of this study show that MCTs with aberrant KIT protein localization or internal tandem duplication c-KIT mutations are associated with increased cellular proliferation, further suggesting a role for c-KIT in the progression of canine MCTs.     

The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors

Cutaneous mast cell tumors (MCTs) are one of the most common tumors in dogs. Currently, prognostic and therapeutic determinations for MCTs are primarily based on the histologic grade of the tumor, but a vast majority of MCTs are of an intermediate grade, and the prognostic relevance is highly questioned. A more detailed prognostic evaluation, especially of grade 2 canine MCTs, is greatly needed. To evaluate the prognostic significance of KIT and tryptase expression patterns in canine cutaneous MCTs, we studied 100 cutaneous MCTs from 100 dogs that had been treated with surgery only. The total survival and disease-free survival time and the time to local or distant recurrence of MCTs were recorded for all dogs. Using immunohistochemistry, 98 of these MCTs were stained with anti-KIT and antitryptase antibodies. Three KIT- and three tryptase-staining patterns were identified. The KIT-staining patterns were identified as 1) membrane-associated staining, 2) focal to stippled cytoplasmic staining with decreased membrane-associated staining, and 3) diffuse cytoplasmic staining. The tryptase-staining patterns were identified as 1) diffuse cytoplasmic staining, 2) stippled cytoplasmic staining, and 3) little to no cytoplasmic staining. Based on univariate and multivariate survival analysis, increased cytoplasmic KIT staining was significantly associated with an increased rate of local recurrence and a decreased survival rate. The tryptase-staining patterns were not significantly associated with any survival parameter. On the basis of these results, we propose a new prognostic classification of canine cutaneous MCTs, according to their KIT-staining pattern, that can be used for the routine prognostic evaluation of canine cutaneous 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.     

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.     

Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis

The Patnaik histologic grading system is commonly used to predict the behavior of cutaneous mast cell tumors (MCTs) in dogs, but it is less useful for grade 2 MCTs because they exhibit considerable variation in biological behavior. In this retrospective study, immunohistochemical staining for Ki-67, proliferating cell nuclear antigen (PCNA), and survivin and a standardized argyrophilic staining of nucleolar organizer regions (AgNOR) protocol were performed on 121 archived paraffin-embedded specimens of canine cutaneous MCTs, for which clinical follow-up data were available. Cox regression models indicated that the Ki-67 score (hazard ratio, 1.92; P < .001) and mean AgNOR score (hazard ratio, 2.57; P < .001) were significantly associated with Patnaik grade and survival time. A binary Ki-67 variable (cutoff point Ki-67 score = 1.8) was a significant predictor of survival for dogs with grade 2 MCTs. The estimated 1-, 2-, and 3-year survival probabilities for dogs with grade 2 MCTs and Ki-67 scores less than 1.8 were 0.92, 0.86, and 0.77, respectively (SEs, 0.08, 0.14, and 0.23, respectively; median not estimable). The corresponding survival probabilities for dogs with grade 2 MCTs and Ki-67 scores higher than 1.8 were 0.43, 0.21, and 0.21, respectively (SEs, 0.19, 0.18, and 0.18, respectively; median survival time, 395 days). No significant association was identified between survival and survivin score or PCNA score. This study shows that both mean AgNOR score and Ki-67 score are prognostic markers for canine MCTs. The Ki-67 score can be used to divide Patnaik grade 2 MCTs into 2 groups with markedly different expected survival times.     

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.     

Incorporation of sentinel lymph node mapping in dogs with mast cell tumours: 20 consecutive procedures

The study hypothesis is that incorporation of sentinel lymph node (SLN) mapping in dogs presenting for mast cell tumour (MCT) removal would impact the recommended adjuvant therapy offered. Nineteen dogs were enrolled having either spontaneously occurring or incompletely excised MCTs. Staging included regional lymph node aspiration. SLN mapping was done with regional lymphoscintigraphy combined with intra‐operative lymphoscintigraphy and blue dye. Twenty MCTs in 19 dogs were excised with SLN mapping. Eight dogs had SLNs different from the closest node. Twelve dogs had metastasis in extirpated SLNs, seven occurred in MCTs with a MI ≤ 5. No correlation was noted between patient stage and the c‐KIT proto‐oncogene. Because of SLN staging, 8 of 19 dogs were offered additional therapy that would have otherwise been excluded. Anatomic sampling of lymph nodes in dogs with MCTs does not accurately reflect which lymph nodes are most likely to be receiving the draining tumour lymph.     

Correlation of histologic grading of canine mast cell tumors with Ki67/PCNA/AgNOR/c‐Kit scores: 38 cases (2002–2003)

Introduction: The Patnaik grading system for canine cutaneous MCT is currently one of the best determinants of prognosis; however, clinical outcome does not always correlate with histologic grade. The development of molecular markers offers a potential advantage and may complement subjective grading. The primary purpose of this study was to correlate histologic grading to Ki67/PCNA/AgNOR/c‐Kit scores. Methods: Thirty‐eight dogs with cutaneous MCT underwent surgical resection. Tumors were graded, with expansion of grade II MCT to low, medium (or II only) and high. For statistical purposes, MCT grade I, II (low, medium, high) and III were assigned a score of 1, 2, 3, 4, or 5, respectively. Sections were processed for AgNOR staining and expression of PCNA, Ki67 and c‐Kit as previously described (modified biotin‐strepavidin with DAB substrate). Paraffin‐embedded canine tissue arrays were used as positive and negative controls (primary antibody replaced with pre‐immune sera). Parametric statistical testing was performed using Statview statistical software with P ≤ .05 as significant. Results: There were 12, 20, 5 and 1 grade II low, grade II medium, grade II high and grade III MCT, respectively. The mean Ki67 score was 9.114 (median 8.0, range 1–28), mean PCNA score was 26.25 (median 24.0, range 5–65), mean AgNOR score was 1.499 (median 1.35, range 1.02–2.76) and c‐Kit scores were +1 (9/37), +2 (19/37) and +3 (9/37). With parametric statistical testing, significantly positive correlations were found for Ki67/Grade, PCNA/Grade, AgNOR/Grade, Ki67/PCNA, Ki67/AgNOR and PCNA/AgNOR (all P < .0001). No significant correlation was found for c‐Kit and grade; however, +3 c‐Kit scores had statistically significantly higher grades than +2 c‐Kit scores (P = .0458). Conclusions: Ki67/PCNA/AgNOR scores are positively correlated to grade in dogs with MCT. Further studies to correlate Ki67/PCNA/AgNOR/c‐Kit scores with clinical variables are ongoing.     

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.     

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high‐grade B‐cell lymphoma

Ki67 can discriminate between high‐ and low‐grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67‐positive cells), evaluated by flow cytometry, in 40 dogs with high‐grade B‐cell lymphoma, treated with a modified Wisconsin–Madison protocol (UW‐25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1–40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high‐grade B‐cell lymphoma.     

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.     

Canine subcutaneous mast cell tumors: cellular proliferation and KIT expression as prognostic indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers--including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)--as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.     

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.     

A phase II study to evaluate the toxicity and efficacy of alternating CCNU and high‐dose vinblastine and prednisone (CVP) for treatment of dogs with high‐grade,metastatic or nonresectable mast cell tumours

Safety and efficacy of a protocol of alternating 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (CCNU; 70 mg m^?2) and vinblastine (3.5 mg m^?2), and prednisone (1–2 mg kg^?1; CVP) in dogs with mast cell tumours (MCT) were evaluated. A total of 17 dogs had nonresectable MCTs and 35 received CVP as adjunctive treatment to locoregional control of metastatic MCTs or grade III MCTs. Neutropenia with fever occurred in 8% of dogs after treatment with vinblastine and in 2% after treatment with CCNU. Persistent elevation of serum alanine transaminase, suggestive of hepatotoxicity, occurred in 9% of the dogs. Response rate in dogs with nonresectable MCTs was 65%; five achieved a complete response (median, 141 days) and six achieved a partial response (median, 66 days). Overall median progression‐free survival (PFS) time in dogs treated in the adjuvant setting was 489 days. Dogs with grade III MCTs had shorter PFS compared with dogs with metastatic grade II MCTs (190 days versus 954 days; P < 0.001). Phase III studies are needed to provide reliable information about the comparative efficacy of this protocol.     

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.