Canine Mast Cell Tumors Research Articles

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre-incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre-incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.

RETRACTED ARTICLE: Cutaneous mast cell tumor (Mastocytoma): Cyto- histopathological and haematological investigations

Cutaneous mast cell tumours (MCTs) are the most common skin tumours 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. A cytologic diagnosis of mast cell tumor with evidence of prior hemorrhage was made, and the masses were surgically removed. Cytological evaluation of fine-needle aspirates from the cutaneous mass from the axillary comprised many well-differentiated, highly granulated mast cells with moderate numbers of eosinophils. Nuclei were varied in size and shape with high nuclear’to’cytoplasmic ratio, prominent nucleoli, marked atypical and mitotic figures. Microscopically, mass consisted of sheets of neoplastic round cells that formed nonencapsulated nodules in the dermis and infiltrated into the adjacent dermal collagen, and also there was diffuse subcutis invasion of round to pleomorphic tumor cells. Tumor cells had moderate to abundant cytoplasm, round to ovoid nuclei with scattered chromatin, and mitotic figures. In this tumor, cytoplasmic granules showed atypical metachromasia. In addition, eosinophils were scattered among the mast cells at the periphery of the nodules. The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules. Invasion of the deep subcutaneous fat or cutaneous muscles were a common feature of grade III tumour. Finally, a diagnosis of grade III cutaneous mast cell tumor was made.Virtual slidesThe virtual slide(s) of this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4755249151157024.

Additional Local Therapy With Primary Re‐Excision or Radiation Therapy Improves Survival and Local Control After Incomplete or Close Surgical Excision of Mast Cell Tumors in Dogs

To compare survival and local recurrence outcomes in dogs with mast cell tumors with incomplete or close margins treated with primary re-excision or radiation therapy of the primary site versus no additional local therapy. Retrospective case series. Dogs (n = 64). Outcomes of canine mast cell tumor cases that had incomplete or close surgical resection and presented to the Ontario Veterinary College Health Sciences Centre (2001-2010) were evaluated after additional local therapy (primary re-excision or radiation therapy) or no additional local therapy (comparison). Follow-up was performed through evaluation of medical records and telephone contact with referring veterinarians and owners. Tumors (n = 70) in 64 dogs were studied. Median survival times for the primary re-excision (2930 days) and radiation therapy (2194 days) groups were significantly longer than for the comparison (710 days) group. Local recurrence occurred in 13% of the re-excision group, 8% of the radiation therapy group, and 38% of the comparison group. Although local recurrence rate was not statistically significant for the re-excision group, time to local recurrence was statistically longer for both the re-excision and radiation groups. Adjunctive chemotherapy was not associated with improved survival or local control. There is significant improvement in survival and duration of local control when additional local therapy is performed after incomplete or close resection of mast cell tumors. These follow-up therapies should be recommended to owners when mast cell tumors are incompletely or closely resected.

Uterine mast cell tumor: a clinical and cytohistopathological study

BackgroundMast cells are one of the characteristic factors in angiogenesis, growth, and metastatic spread of tumors. Further studies are suggested to determine the type of these cells which might be useful in the assessment of biological nature of the tumor and its future treatment modality. Few studies have evaluated mast cell infiltration in visceral tumors, especially uterine tumors.Case presentationIn this study, age, sex, death rate, and histologic patterns were in agreement with those of previous reports on canine mast cell tumors. Cytopathology assays are widely used to prognosticate canine uterine mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the uterine. The anisocytosis and anisocytosis and giant cells were present in the tumor. Furthermore, the tumor had nuclear atypia with scattered multinucleated cells and prominent nucleoli and tumor were classified as poorly granulated. Under microscopic examination, we observed diffuse infiltration and proliferation of tumor cells from the uterine different area and the infiltrative characteristics and distribution patterns of neoplastic cells were observed. This tumor consisted of sheets and cords of uniform round cells with discrete cytoplasmic margins. Microscopically, the neoplastic masses were poorly-demarcated and lacked capsules and tumor cell usually showed a distinct cell boundary. Nevertheless, the neoplastic cells were located between collagen bundles forming small clusters and sheets and had large, centrally located, round to ovoid nuclei. In addition, eosinophils were scattered among the mast cells at the periphery of the masses. The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules.ConclusionBased on these findings, a diagnosis of poorly-differentiated mast cell tumor was made and data histologic grading was available for tumor. Neoplasm was poorly differentiated or gradeIII.

C-Kit expression, angiogenesis, and grading in canine mast cell tumour: a unique model to study c-Kit driven human malignancies.

Canine cutaneous mast cell tumour (CMCT) is a c-Kit driven tumour sharing similar c-Kit aberrations found in human gastrointestinal stromal tumour. CMCT is classified into three forms: well- (G1), intermediately (G2) (more benign diseases), and poorly (G3) differentiated (malignant) forms. We assess a correlation between c-Kit status, grading, and angiogenesis in CMCTs to explore their potential significance in humans. C-Kit receptor (c-KitR) expression, microvascular density (MVD), and mast cell granulated and degranulated status density (MCGD and MCDD, resp.) were analyzed in 97 CMCTs, by means of histochemistry, immunohistochemistry double staining, and image analysis system. Data showed that predominantly diffuse cytoplasmic- and predominantly focal paranuclear- (Golgi-like) c-Kit protein (PDC-c-Kit and PFP-c-Kit, resp.) expression correlate with high MVD, G3 histopathological grade, and MCDD. Moreover, predominant cell membrane-c-KitR (PCM-c-KitR) expression status correlates with low MVD, G1-G2 histopathological grade, and MCGD. These findings underline the key role of c-Kit in the biopathology of canine MCTs, indicating a link between aberrant c-Kit expression, increased angiogenesis, and higher histopathological grade. CMCT seems to be a model to study contributions of c-Kit activated MCs in tumour angiogenesis and to evaluate the inhibition of MCs activation by means of c-Kit tyrosine kinase inhibitors, currently translated in humans.

Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor

BackgroundMast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.ResultsThe canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.ConclusionsThis study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.

The Relevance of CD117-Immunocytochemistry Staining Patterns to Mutational Exon-11 inc-kitDetected by PCR from Fine-Needle Aspirated Canine Mast Cell Tumor Cells

Canine cutaneous mast cell tumors (MCT) are the lethal skin tumors. The biological behavior of the MCT cells is quite varied and unpredictable. Almost MCT dogs usually require a rapid diagnosis and therapy. However, MCT diagnosis and prognosis are still dependent on histopathology which is rather inconvenient, time-consuming, painful, and harmful for some cases. Indeed, MCT can be easily accessible using fine-needle aspiration (FNA). In this study, our biopsy specimens were classified as low- and high-grade MCT based on the novel 2-tier histopathologic grading system. We have demonstrated the usage of fine-needle aspirated MCT cells (FNA-MCT cells) from these specimens as a primary cell source to study the distribution of CD117-immunocytochemistry (CD117-ICC) staining patterns and the frequency of internal tandem duplication- (ITD-) mutant exon-11 of c-kit. The result has substantially shown that there were three staining patterns identified in the cells. Only paranuclear pattern was significantly increased in the cells from high-grade MCT. Altogether, the ITD-mutant exon-11 was also detectable only in these cells. Therefore, the result has supported our hypothesis that there was an increased opportunity to observe a higher CD117-ICC staining pattern and exon-11 mutation in high-grade MCT; even these two parameters may not precisely indicate a histopathological grade.

Immunophenotype of Neoplastic and Bystander Cells in Canine Cutaneous Mast Cell Tumours (CCMCTs)

Immunophenotype of Neoplastic and Bystander Cells in Canine Cutaneous Mast Cell Tumours (CCMCTs)

Evaluation of Histopathological Grading, Mitotic Index and KIT Labelling Patterns as Prognostic Indicators for Canine Cutaneous Mast Cell Tumours

Evaluation of Histopathological Grading, Mitotic Index and KIT Labelling Patterns as Prognostic Indicators for Canine Cutaneous Mast Cell Tumours

Expression of CD117 and C-KIT Mutations in Canine Cutaneous Mast Cell Tumours

Expression of CD117 and C-KIT Mutations in Canine Cutaneous Mast Cell Tumours

Breed related odds ratio and anatomic distribution of canine mast cell tumours in Austria. Retrospective study of cases in the years 2000-2010.

An increased risk of mast cell tumours (MCT) in certain breeds has been described repeatedly in the literature. The incidence of MCTs for registered breeds in Austria, an estimate of the risk by means of the odds ratios based on breed as well as the anatomic localisation of MCTs were examined. In the first part of the study, the ranking of breeds in Austria based on 147,802 dogs with known breed (including mixed breed) was determined, based on those dogs included in the laboratory data base from 2000 to 2010. In the second part of the study, 476 dogs were identified with MCTs and analysed by age, sex, Patnaik grade of MCT and breed distribution. The odds ratios with confidence intervals were calculated for all breeds with skin tumours. The age distribution showed a peak in the age group from 6.1 to 8.0 years; 70% of MCTs were localised to the head and trunk. No significant difference was found based on gender. The evaluation of the odds ratios showed that only four of the 20 of the most popular in Austria breeds (Boxer, Bernese Mountain Dog, Golden Retriever, Spaniel) had an increased risk; on the other hand, some breeds which have not been previously identified in the literature were indicated to have a significantly increased risk for MCT (e.g., Dogo Argentino, Tibetan Spaniel, Pyrenean Mountain Dog, Beauceron, and Austrian Smooth-haired Hound). Because disease risk may influence the popularity of some currently rare breeds, consultation with breeders and owners regarding the identification of the breeds newly identified in this study as an increased risk for development of mast cell tumours is indicated.

Histological Grading of Canine Mast Cell Tumour: is Two Better Than Three?

Histological Grading of Canine Mast Cell Tumour: is Two Better Than Three?

Concordance of c‐kit Mutational Status in Matched Primary and Metastatic Cutaneous Canine Mast Cell Tumors at Baseline

BackgroundMutation analysis of proto‐oncogene c‐kit (c‐kit) is advisable before starting treatment with tyrosine kinase inhibitors in dogs with mast cell tumor (MCT), including those with metastatic disease. Testing is usually performed on primary tumors, assuming that c‐kit mutation status does not change in metastasis.Hypothesis/ObjectivesTo give an insight into the mutational processes and to make a recommendation on the use of c‐kit mutational analysis in the clinical setting.AnimalsTwenty‐one client‐owned dogs with metastatic MCT.MethodsDogs undergoing resection or biopsy for both primary and matched metastatic MCT were prospectively enrolled. Total RNA or DNA was extracted from primary MCT and corresponding metastases. Exons 8, 9, and 11 were amplified by PCR and sequenced. Genetic features between primary MCT and metastases were compared. Their correlation with clinicopathologic features was investigated.ResultsConcordance (mutated or wild‐type) of mutational status, evaluable in 21 primary and matched metastatic (20 nodal and 1 splenic) MCTs, was 100%. Three new c‐kit mutations were identified. No significant correlation was detected between c‐kit mutation and clinicopathologic features.Conclusions and Clinical ImportanceProto‐oncogene c‐kit mutational status is conserved between any primary and its matched secondary tumor, suggesting that both can be used for c‐kit mutational testing. Targeted therapies might be also used to treat metastatic disease.

Imatinib Responsiveness in Canine Mast Cell Tumors Carrying Novel Mutations of &lt;i&gt;c-KIT&lt;/i&gt; Exon 11

ABSTRACTIn 2 individual cases of canine mast cell tumors, we identified 2 novel c-KIT mutations in exon 11: a 9-base pair (bp) deletion (c.1663-1671del) and a point mutation (c.1676T>A). The 9-bp deletion mutation caused a loss of 3 amino acids, corresponding to p.Gln555_Lys557del, and the point mutation resulted in the substitution of valine by aspartic acid (p.Val559Asp) in the juxtamembrane domain of the protein. Imatinib mesylate, a therapeutic agent for canine mast cell tumors, was used to treat both tumors. Complete remission was achieved at 33 and 14 days after administration, respectively. However, in both cases, the therapeutic response subsequently tapered with the duration of remission lasting 66 and 255 days, respectively. Although these 2 novel c-KIT mutations in exon 11 were not confirmed to be gain-of-function mutations, a further study may help clarify relevance between mutations identified in this report and responsiveness.

Bromodomain-Containing Protein 4 (BRD4): A Novel Marker and Drug Target Expressed In Neoplastic Cells In Advanced Mast Cell Neoplasms

Advanced mast cell (MC) neoplasms are characterized by uncontrolled growth and rapid expansion of neoplastic MC in various organ systems and a poor prognosis. In human patients, advanced systemic mastocytosis (SM) is rare and usually presents as aggressive SM (ASM) or mast cell leukemia (MCL). In canines, advanced MC tumors (MCT) are considered one of the most frequent skin tumors. Hence, so far, no effective treatment concept has been established for advanced MC neoplasms in humans or canines. This is due to the fact that neoplastic MC in advanced SM usually are resistant against various cytoreductive agents. Therefore, research is currently focusing on potential drug targets and the effects of novel targeted drugs on neoplastic MC. We have recently identified the bromodomain-containing protein-4 (BRD4) as a promising new drug target expressed by neoplastic cells in acute myeloid leukemia (Zuber et al, Nature 2011;478:524-528). In the present study, we examined BRD4 as a potential therapeutic target in human ASM and MCL, and canine MCT. As assessed by immunohistochemistry, neoplastic MC expressed substantial amounts of cytoplasmic and nuclear BRD4 in ASM and MCL, whereas in human indolent SM (ISM), MC expressed lower amounts of BRD4 or did not express cytoplasmic BRD4. The human MCL lines HMC-1.1 and HMC-1.2 as well as the canine mastocytoma cell lines NI-1 and C2, also expressed BRD4. In order to study the potential biological function of BRD4, we applied a BRD4-specific shRNA. The shRNA-induced knockdown of BRD4 was found to be associated with a markedly reduced proliferation of HMC-1 cells. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition in all MC lines examined, with higher IC50 values obtained in HMC-1.1 cells (IC50: 1.0 µM) and HMC-1.2 cells (IC50: 1.0 µM) compared to NI-1 cells (0.2 µM) and C2 cells (0.5 µM). As assessed by morphology and AnnexinV/PI staining, the growth-inhibitory effects of JQ1 on neoplastic human and canine MC lines were accompanied by apoptosis (Figure). The apoptosis-inducing effects of JQ1 was also demonstrable by staining for active caspase 3. Furthermore, JQ1 was found to downregulate the expression of the activation-related MC antigen CD63 and the proliferation-related antigen CD71 (transferrin receptor) in HMC-1 cells. In addition, JQ1 was found to inhibit proliferation in primary neoplastic MC obtained from patients with ASM or MCL (IC50 0.1-0.5 µM). We next screened for potential drug partners that would potentiate the anti-neoplastic effects of JQ1. Of all drugs tested, all-trans retinoic acid (ATRA) and PKC412 were identified as most potent drug partners of JQ1. These drugs were found to act highly synergistic with JQ1 in producing apoptosis and growth inhibition in HMC-1.1 and HMC-1.2 cells (Figure). Together, BRD4 is a novel marker and promising target in advanced MC neoplasms. Whether JQ1 or other BET bromodomain inhibitors are effective in vivo in patients with advanced SM or canine MCT remains to be elucidated. [Display omitted] HMC-1.2 cells were incubated in control medium (Co) or in various concentrations of JQ1 (black bars), ATRA (gray bars), or a combination of both agents at a ratio of 1:5 (white bars) at 37°C for 48 hours. Then, AnnexinV/propidium iodide (PI) staining was performed. The percentage of AnnexinV+ cells was determined by flow cytometry. Results represent the mean±S.D. of 3 independent experiments. Asterisk (*): p<0.05 compared to control (Co). Disclosures:Bradner:Tensha Therapeutics: Dr. Bradner is a scientific founder of Tensha Therapeutics, which is developing drug-like derivatives of the JQ1 bromodomain inhibitor as cancer therapeutics, through a license from the Dana-Farber Cancer Institute. Other. Horny:Novartis : Consultancy, Honoraria.

Production of stem cell factor in canine mast cell tumors

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.

Oncolytic Reovirus in Canine Mast Cell Tumor

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.

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.

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

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

Aggressive local therapy combined with systemic chemotherapy provides long‐term control in gradeIIstage 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.