**6. Treatment and prognosis**

#### **6.1. Conventional therapies**

Complete surgical removal with wide margin is the best way for solitary cutaneous MCTs. However, for dogs with multiple masses, metastasis, or sever invasive MCTs, surgical removal is sometimes incompetent. Chemotherapies have failed to overcome MCTs. However, to reduce tumor size, some combination chemotherapies have applied. Glucocorticoid is one of the most important drugs for treatment of MCTs. More than 70% of cutaneous MCTs in dogs respond well to oral administration of glucocorticoid [35–37]. Expression levels of glucocorticoid receptors in MCT cells have been reported to associate with glucocorticoid sensitivity [37]. Glucocorticoid shows strong antitumor effects on MCT cells with high expression of glucocorticoid receptors. Oral administra‐ tion of glucocorticoid must be an easy and effective chemotherapy for canine MCTs. Since side effects induced by glucocorticoid administration will sometimes be concerned, clinicians must pay attention on blood chemistry data and general conditions of dog patients. Glucocorticoid is usually applied as a part of multidrug chemotherapies for MCTs. Anticancer drugs, such as vincristine, vinblastine, cyclophosphamide, and CCNU, have been tested for combination chemotherapies for MCTs with or without glucocorti‐ coid [38–42]. However, very little information on the chemotherapeutic response of MCTs can be obtained. Since recent studies have been based on small numbers of cases and have often included MCTs of different pathologic grades and clinical stages, data must be carefully evaluated [43]. Recently, adjuvant chemotherapies have been proposed in treatment of various cancers and sarcomas. Since neo‐adjuvant administration with glu‐ cocorticoid usually reduces mass size of MCTs, wide surgical margins will be obtained [37]. On the other hand, postoperative adjuvant chemotherapy is suggested to kill MCT cells that remain at the affected site after incomplete excision. Several trials on adjunc‐ tive chemotherapy have been reported. However, most of the adjunctive chemotherapy does not appear to increase survival times. Although surgical removal with radiation has been tested for MCTs, remarkable improvement is not provided. No difference in overall survival rate has been observed between dogs with MCTs receiving and not receiving prophylactic irradiation of the regional lymph node [44].

#### **6.2. Molecular target therapies targeting KIT**

paracrine/autocrine manner [28]. In the analyses, high SCF production was confirmed in multiple clinical MCT samples [29]. It may explain the high response of clinical MCTs to KIT‐ specific molecular inhibitors even when the tumor cells express wild‐type KIT. This will be

**Analyzed exons Domain Exon Mutation Frequency (%) References** All Extracellular 8 417‐421ITD 8/202 (4.2%) [24]

Juxtamembrane 11 555–557mutation 7/202 (3.5%)

Tyrosine kinase 17 del826–828 1/202 (0.5%) All Extracellular 6, 7, 8 del102AA, Thr414Ala 1/47 (2.1%) [25]

Juxtamembrane 11 del558 1/47 (2.1%)

Tyrosine kinase 15 del714 1/47 (2.1%) 11 Juxtamembrane 11 ITD 8/88 (9.1%) [12]

 Juxtamembrane 11 ITD 8/60 (13.3%) [13] Juxtamembrane 11 ITD 7/68 (10.3%) [14] Juxtamembrane 11 ITD 24/118 (20.3%) [26] 8, 9, 11 Extracellular 8 421‐429del, ins3AA 1/21 (4.8%) [27]

8 Gln430Arg 1/202 (0.5%) 9 Ser479Ile 5/202 (2.5%) 9 Asn508Ile 3/202 (1.5%)

11 ITD 25/202 (12.4%)

8 417‐420ITD 3/47 (6.4%)

9,10 ins4AA 1/47 (2.1%)

11 Leu575Pro 1/47 (2.1%) 11 ITD 8/47 (17.0%)

11 del 4/88 (4.5%)

Recent approaches such as next‐generation sequencing will reveal even minor mutations or single nucleotide polymorphisms in neoplastic mast cells. In fact, Spector et al. [30] and Youk et al. [31] discovered a human mast cell leukemia‐specific mutation in several genes. As the cost of these approaches decreases, they will be introduced in the veterinary field, probably leading to the deep understanding of mast cell tumorigenesis among species. Another approach aiming at the control of tumor growth is modifying epigenetic status in tumor genome [32]. Regarding an epigenetic alteration in MCTs, Morimoto et al. [33] showed that DNA hypomethylation widely occurred in malignant, higher‐grade MCTs. Moreover, antitumor effects of AR‐42, a histone deacetylase inhibitor, on several MCT cell

further discussed in the following section.

**Table 1.** KIT mutations that have been reported in dog MCTs.

AA, amino acid.

Extracellular, transmembrane

84 Canine Medicine - Recent Topics and Advanced Research

Because aberrant activation of mutant KIT is one of the causes in mast cell tumorigenesis, anticancer effects of KIT inhibitors have been investigated. In fact, clinical trials that enrolled MCT‐diagnosed dogs have been undertaken to evaluate the efficacy of molecular targeting agents against KIT. We would like to overview the history of the research on KIT inhibitors and discuss therapeutic perspective fwor MCTs.


**Table 2.** Summary of a clinical trial of toceranib phosphate [26].

The first molecular inhibitor applied to human was the imatinib mesylate, which repress activations of KIT, platelet‐derived growth factor receptor (PDGFR), and Bcr‐Abl [45]. It was first administered to the patient of gastrointestinal stromal tumor with a mutation in the juxtamembrane domain of KIT [46]. At around the same time, KIT mutations in canine MCT were first discovered [21], suggesting the possibility that KIT inhibitors can be applied to dog MCTs. Actually, there have been several reports that show the inhibitory effects of imatinib mesylate on MCTs, especially for the tumor cells with KIT mutations in either the extracellular domain or juxtamembrane domain [22, 47]. Based on these results from basic researches, some clinicians administered imatinib mesylate to MCT‐diagnosed dogs and obtained partial response at least in some of them [47]. However, there was no rationale for the administration of imatinib mesylate to MCT‐diagnosed dogs through the clinical trials. In contrast to that, both masitinib mesylate and toceranib phosphate are approved by either the Food and Drug Administration (FDA) in the United States or European Medicines Agency based on the results from the clinical trials enrolling MCT‐diagnosed dogs [26, 48,49]. Basically, imatinib mesylate, masitinib mesylate, and toceranib phosphate are all ATP‐competitive inhibitors, and they suppress the activation of mutant KITs that require ATP binding for their activation.

Results of clinical trials for toceranib phosphate, which is a random double‐blind trials, are summarized in **Table 2** [26]. In this trial, more than 150 MCT patients were enrolled. After the six‐week treatment, either complete response (CR) or partial response (PR) was obtained 32 in 86 (37.2%) patients in the treatment group, while the proportion of CR/PR was only 7.9% (5 in 63 patients) in the placebo group. In addition, a group treated with toceranib phosphate following placebo‐escape, which administered toceranib phosphate after the placebo treat‐ ment, responded the agent, resulting in the CR/PR in 24 cases out of 58 (41.4%). At least in this trial, significant increase in severe adverse effects (grade III or IV) was not detected. In case of mastinib mesylate, a phase III trial was carried out in France, enrolling more than 130 MCT patients. One‐year survival and two‐year survival were 62.1 and 39.8%, respectively, in the treatment group, though the ones were 36.0 and 15.0%, respectively, in the placebo group [49].

Interestingly, both agents showed antitumor effects even on MCTs expressing wild‐type KIT (**Tables 2** and **3**). Though the agents suppress the activation of other receptor tyrosine kinases


**Table 3.** Summary of a clinical trial of masitinib phosphate [49].

such as platelet‐derived growth factor receptor or vascular endothelial growth factor receptor 2 [50, 51], aberrant activations of principal targets except KIT were not observed in our study using more than 30 clinical MCT tissue samples (unpublished data). Thus, it is likely that the data in **Table 2** indicate that tumor growth in no more than 30% of MCT was dependent on KIT signaling even though they express wild‐type KIT. We consider that these can be at least partly explained by SCF autoproduction from tumor cells as described above [28, 29]. Though further investigations are necessary, analyses to determine the KIT activation status will probably be a direct diagnostic agent to accurately predict the therapeutic efficacy of KIT targeting inhibitors.
