**5. Neoplastic transformation of mast cells**

#### **5.1. Overview**

Mast cell malignancies are observed among species, though the incidence of mast cell malig‐ nancies is much lower in human and rodents than in dogs [8, 9]. One of the well‐investigated mechanisms of mast cell tumorigenesis is mutations in the c‐*kit* gene [9, 10]. We would like to overview the current understandings of the mutant KIT contribution on mast cell tumorigen‐ esis as well as other tumor‐related transformations of mast cells that may correlate with their tumorigenesis in mast cells.

#### **5.2. KIT mutation‐dependent neoplastic transformation**

#### *5.2.1. KIT mutations in human and rodents*

KIT is a type III receptor tyrosine kinase of which ligand is stem cell factor (SCF) (**Figure 5**). It is consisted of the extracellular domain, transmembrane domain, juxtamembrane domain, and tyrosine kinase domain [16] (**Figure 5**). In human, aberrant proliferation of mast cell is observed in the patients of systemic/cutaneous mastocytosis, mast cell sarcoma, and mast cell leukemia [17]. Among them, most systemic mastocytosis occurs due to the mutations in the tyrosine kinase domain of KIT, especially a point mutation in Asp816 [12]. In general, SCF binding to KIT triggers the conformational changes in KIT and leads to the dimerization of

**Figure 5.** Schematic diagram of KIT protein. The numbers of exon correspond to the ones in dog KIT.

the protein, allowing the binding of adenosine triphosphate (ATP) and phosphorylation of tyrosine kinase domain [18]. Mutations in the tyrosine kinase domain alter the conformation to the one similar to its active form, thus resulting in the constitutive KIT activation even in the absence of either SCF binding, KIT dimerization, or ATP binding [19]. Neoplastic growth of mast cells is rarely observed in rodents, through currently available rodent‐derived mast cell lines. For example, RBL‐2H3 cells (derived from a Wistar rat) and P815 cells (derived from a DBA/2 mouse) express Asp817Tyr and Asp814Tyr, respectively, which correspond to the Asp816 mutation in human KIT [20]. As far as we know, other mechanisms that trigger rodent mast cell tumorigenesis have not been reported.

## *5.2.2. KIT mutations in dog*

**4.2. Diagnosis of MCTs**

82 Canine Medicine - Recent Topics and Advanced Research

**5. Neoplastic transformation of mast cells**

**5.2. KIT mutation‐dependent neoplastic transformation**

**5.1. Overview**

tumorigenesis in mast cells.

*5.2.1. KIT mutations in human and rodents*

Cytological or histological analyses through a fine needle aspiration or biopsy are required for the diagnosis of MCTs. Typically, round‐shape cells with round nuclei and with rich cytosol are observed. Mast cells have abundant cytosolic granules, and specific staining methods with toluidine blue or safranin O can identify them. However, MCTs with undifferentiated more malignant mast cells possess few granules. Confirmation of the swelling of draining lymph nodes and sometimes fine needle aspiration of the lymph node may help to determine the pres‐ ence of metastasis. Patnaik grading is mainly used pathological grading in the veterinary field because it is recognized as a good prognostic marker [15]. There are three pathological grades (grades I, II, and III), and higher grade indicates that the tumor is more malignant [15]. Several analyses have been revealed: the correlation between tumor grading and the c‐*kit* gene mutation, clearly showing that c‐*kit* mutations are more frequently observed in high‐grade tumor [12–14]. Therefore, analyzing c‐*kit* sequence can also be a prognostic marker for MCTs. In addition, ana‐ lyzing c‐*kit* gene is important in terms of selecting proper treatments because several molecular target inhibitors against KIT protein, a receptor encoded in the c‐*kit* gene, are currently available for the treatment of MCTs. Polymerase chain reaction that amplifies exon 11 and intron 11 region using tumor genome enables the detection of internal tandem duplications (ITDs) in the juxta‐ membrane domain, which is the most frequent type of c‐*kit* mutation. Recently, however, whole sequence of c‐*kit* mRNA is more common because the proportion of mutations in other region of KIT domain or other type of mutations in the juxtamembrane domain are not negligible. Recent reduction in the cost for sequencing analysis will probably boost this trend (see Section 5).

Mast cell malignancies are observed among species, though the incidence of mast cell malig‐ nancies is much lower in human and rodents than in dogs [8, 9]. One of the well‐investigated mechanisms of mast cell tumorigenesis is mutations in the c‐*kit* gene [9, 10]. We would like to overview the current understandings of the mutant KIT contribution on mast cell tumorigen‐ esis as well as other tumor‐related transformations of mast cells that may correlate with their

KIT is a type III receptor tyrosine kinase of which ligand is stem cell factor (SCF) (**Figure 5**). It is consisted of the extracellular domain, transmembrane domain, juxtamembrane domain, and tyrosine kinase domain [16] (**Figure 5**). In human, aberrant proliferation of mast cell is observed in the patients of systemic/cutaneous mastocytosis, mast cell sarcoma, and mast cell leukemia [17]. Among them, most systemic mastocytosis occurs due to the mutations in the tyrosine kinase domain of KIT, especially a point mutation in Asp816 [12]. In general, SCF binding to KIT triggers the conformational changes in KIT and leads to the dimerization of In contrast to human and rodents, KIT mutations in dog MCT are frequently observed in the juxtamembrane domain (**Table 1**). ITDs in the domain are the first discovered and the most frequent mutations in canine MCTs [21] (**Table 1**). Besides ITDs, other mutations in the juxtamembrane domain or extracellular domain have been also reported [21]. We recently demonstrated that most of these mutations in the extracellular or juxtamembrane domain cause aberrant KIT activation and neoplastic proliferation of mast cells by triggering ligand‐ independent dimerization (Ref. [22] and unpublished data). In contrast to the mutations in the tyrosine kinase domain, these mutations require ATP for the phosphorylation of the tyrosine kinase domain, providing a rationale for using ATP‐competitive small molecule inhibitors for suppressing the aberrant KIT activations [18, 23].

## **5.3. KIT mutation‐independent neoplastic transformation**

Few mechanisms of mast cell tumorigenesis except KIT mutations have been identified, but we recently demonstrated that MCT cells produce SCF and support their growth in a


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

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 further discussed in the following section.

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 lines as well as primary tumor cells have been demonstrated [34]. Thus, the characterization of epigenetic alteration is likely to be an effective approach to reveal MCT transformation.
