**1. Introduction**

The proto-oncogene *KIT*, firstly identified as a homolog of the feline sarcoma viral oncogene *v-kit* [1], encodes for a type 3 receptor tyrosine kinase (KIT) and it is expressed in a wide range of healthy cells [2,3]. Even if its precise role is not completely understood [4], *KIT* is also normally expressed in the

development of melanocytes [2] and it is involved in melanogenesis and melanocyte survival during migration from the neural crest to the cutis. It appears to be more involved in melanocyte migration rather than proliferation [5] with interesting implications since *KIT* mutant melanocytes could acquire elevated migration abilities. Indeed, given its key role, the gene coding for the KIT protein (*KIT*) received grea<sup>t</sup> attention in the field of human oncology, particularly in the study of neoplasms of melanocytic origin. The genomes of both human cutaneous and mucosal melanomas were deeply studied, and human mucosal melanomas (hMMs) showed a higher frequency of aberrations in the *KIT* gene rather than their cutaneous counterparts [3,6–9]. HMMs are affected by both structural alterations, such as copy number aberrations (CNAs), and single nucleotide polymorphisms (SNPs) of *KIT*, with CNAs in 26.3% of cases [6,8] and SNPs in 7–23% [4,6–13]. Therefore, *KIT* mutations have been proposed as an adverse prognostic factor [13]. On the contrary, in cutaneous melanomas, *KIT* is affected by CNAs in 6.7% of cases and by SNPs in 1.7% of cases [8].

Mutations identified in hMMs mainly affected exon 11 (Ex-11) [8,9], where four hotspots have been identified as driver mutations [7].

In humans, most tumors affected by *KIT* mutations [3,14] mainly show SNPs affecting Ex-11 (65% of GastroIntestinal Stromal Tumors, GISTs [3]), promoting the constitutive activation of the KIT receptor without binding to the specific ligand, and leading to uncontrolled proliferation and survival of neoplastic cells [14].

Given the increasing interest in pet dogs as a reliable spontaneous animal model for the study of non-UV-induced melanomas, several authors are also investigating canine melanomas arising in sun-protected sites such as canine oral melanoma (COM), the most frequent neoplasia of the oral cavity in dogs [15–17]. Several studies noted some similarities in can profiles between hMMs [10,18–21] and COMs [10,22–24]. In a recent comparative investigation, we were able to detect common chromosomal changes in 32 regions affecting human chromosomes (HSA) and the canine orthologous regions (CFA), with amplification in 35% of cases of the KIT-coding region located on CFA 13 [25].

However, regarding the SNP profile of COMs, the majority of the studies focused almost entirely on Ex-11, trying to compare the results obtained in human medicine without investigating other exons that play a key role in *KIT* activation in hMMs, such as exons 13 (Ex-13), 17 (Ex-17) and 18 (Ex-18) [6–8,13,26,27].

When considering the mutations reported in the available human literature, Ex-11 is altered in 67% of hMMs [6–9,12,13,20,28–32], while Ex-13 is affected in 15.2% [4,6–9,12,13,20,29–32], Ex-17 in 12.2% [4,6–9,13,20,29–32], and Ex-18 in 8.5% [6,7,13,29,30,32].

In veterinary medicine, only mutations affecting Ex-11 have been adequately investigated in the *KIT* gene, but no decisive results have been achieved, while a screening of Ex-13, Ex-17, and Ex-18 has not been performed [33,34].

The tissue expression of the KIT protein has also been investigated by immunohistochemistry (IHC) in various neoplasms, both in human and veterinary medicine (the latter particularly in pet dogs [35–38]). Although less documented in COMs, and with different ranges of intensities, 49–51% of neoplasms analyzed expressed the protein [34,39] in contrast to hMMs, where 74–89% of cases were reported to be positive [9,12,26].

These heterogeneous results, both in human and veterinary studies, also bear consequences in the use and effectiveness of therapeutic approaches to *KIT*-bearing mutation tumors.

In humans, the use of tyrosine kinase inhibitors (TKIs) has a better effect on patients affected by tumors bearing SNPs in *KIT* [8,13,27,40], particularly in GIST [41], some types of melanomas [11,20,40], chronic myeloid leukemia [42], and systemic mastocytosis [43].

Moreover, the disease control rate in human patients treated with imatinib, one of the most widely used TKIs, is better in patients bearing tumors with *KIT* point mutations (77%) compared to patients bearing tumors with *KIT* amplification (18%) [40], particularly when SNPs affect Ex-11 and Ex-13 [8,13,27]. For patients with SNPs in Ex-17 and Ex-18, which are not responsive to imatinib, responsiveness to treatment with MEK-1 inhibitors has instead been suggested [8].

TKIs are also successfully used in veterinary medicine for the treatment of Mast Cell Tumors (MCTs) with *KIT* point mutations in Ex-11 [34,44], and occasionally of GIST [45,46]. Moreover, although imatinib appears to be effective mostly for tumors with *KIT* point mutations, disease regression in dogs and cats treated with imatinib and affected by tumors without known *KIT* point mutations is rare [47].

In this study, we investigated the role of *KIT* in COMs at both the gene and protein level. Taking advantage of the cohort of COMs assembled for the array Comparative Genomic Hybridization (aCGH) study published in 2019 [25], we decided to deepen our knowledge of the possible role of the gene *KIT* and the protein KIT in our cohort of samples. We performed an accurate evaluation of the possible SNPs by sequencing Ex-13, Ex-17, and Ex-18, and by evaluating KIT expression in COMs. Ki67 was also evaluated by IHC due to its higher expression in metastatic hMMs with SNPs in *KIT* [13].

Therefore, the aim of this study was to evaluate and correlate the IHC expression of KIT and Ki67 with *KIT* somatic point mutations and amplification in COMs.

#### **2. Materials and Methods**

#### *2.1. First Case Selection*

Formalin-fixed, paraffin-embedded (FFPE) COMs were collected from different archives. For each patient, anamnestic data including size and location of the tumor were collected. Hematoxylin and eosin (H&E)-stained slides from each sample were checked by 3 experienced pathologists to confirm the diagnosis of COM and to assess the presence of healthy tissue near the pathological mass suitable for the nucleic acid extraction. The diagnosis of COM was made by adopting the criteria proposed by Smedley and colleagues [48], which were also used for pigmentation scoring, and uncertain cases (i.e., amelanotic specimens) were tested via IHC with anti-PNL-2 and anti-Melan-A antibodies. Only cases that were unanimously diagnosed as COM by all the reviewing pathologists were taken into consideration. At the end of phase one, forty cases met all the inclusion criteria and were selected for the following analyses.

#### *2.2. Nucleic Acid Extraction*

With the help of a microtome with disposable blades, 2–3 20 μm-thick slides were cut from each FFPE block. Healthy and cancerous tissues were separated using a clean scalpel and put in 2 different tubes. To avoid any possible contamination, all the instruments were cleaned between one sample and the other. From the paraffinized material, DNA was extracted using the All Prep DNA/RNA Extraction Kit (Qiagen, Hilden, Germany®®) using heptane as the deparaffinization agen<sup>t</sup> and following the manufacturer's instructions. The extracted DNA was then evaluated on a 1% agarose gel by electrophoresis and quantified with the NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA®®).

#### *2.3. aCGH Analysis and Second Case Selection*

Twenty of the 40 samples showed an adequate DNA yield and were submitted to an aCGH analysis as described in the work from Brocca and colleagues [25]. The aCGH allowed us to learn the aberrational status of CFA 13, and in particular of the portion containing the *KIT* gene. All the samples affected by a copy number gain in the *KIT* locus were selected for the study, together with an equal number of samples not affected by a copy number gain involving *KIT*. The latter were randomly chosen among the remaining samples submitted to the aCGH analysis to maintain a balanced number of samples between the 2 groups (Table 1).


**Table 1.** Selected samples included in the study, together with available clinical and histological data, immunohistochemistry (IHC) indexes, and amplification status. B: bad prognosis; F: female; FN: neutered female; G: good prognosis; M: male; MN: neutered male; NA: not available; \* mean number of positive cells in 5 high-power fields (hpf).

#### *2.4. Exon Amplification and Sequencing*

Primers were designed (Table 2) to amplify the exons considered in this study (Ex-13, Ex-17, and Ex-18). For each exon, reference sequences (CanFam3.1 annotation) were obtained from the online platform Ensembl [50], and primers were designed with the software Primer3 v4.1 [51]. Amplicon size and coding sequence covered for each exon are reported in Table 2.

**Table 2.** Primers designed for the amplification of exons 13, 17, and 18 of the *KIT* gene with the relative lengths. CDS: Coding DNA Sequence; F: forward; R: reverse.


A polymerase chain reaction was then performed for all samples with an initial denaturation step for 2 min at 94 ◦C, 42 cycles of 40 s at 94 ◦C (denaturing), 40 s at 60.5 ◦C (annealing), 50 s at 72 ◦C (extension), and 5 min of final extension at 72 ◦C.

The positive control was non-fragmented genomic DNA extracted from fresh-frozen canine cutis, while water was used instead of DNA as the negative control.

All amplicons obtained were separated on a 1.8% agarose gel via electrophoresis to assess the success of the PCR reaction, then purified with ExoSAP (Exonuclease I Shrimp Alkaline Phosphatase, Thermo Fisher Scientific®®). Using the corresponding forward (Ex-17) or reverse (Ex-13 and Ex-18) primer, amplicons were then sequenced with the Sanger method, and their sequences were visualized with the Chromas 2.6.5 software. Only sequences with high-resolution peaks (high signal-to-noise ratio), minimal baseline noise, and no trace of secondary sequence contamination were considered suitable for mutational analysis. For each sample, healthy and pathological sequences were matched and aligned with the Clustalw platform [52]. The reference sequence was used to identify SNP

positions. The use of pathological and healthy tissue from the same dog allowed for the discrimination of germline and somatic SNPs, and only the latter were taken into consideration for further analyses. The presence of a specific mutation in at least 15% of the samples was arbitrarily considered the minimum threshold.

#### *2.5. Immunohistochemistry and Immunohistochemical Assessment*

KIT expression was evaluated in each sample included in this study. For each FFPE block, a 4 μm-thick slide was cut, mounted on a polarized glass slide (TOMO®®, Matsunami Glass) and then tested with an anti-KIT rabbit polyclonal antibody diluted 1:300 (Dako®®, CD117 clone).

For each specimen, the expression of the Ki67 protein was also assessed with an anti-Ki67 mouse monoclonal antibody diluted 1:50 (Dako®®, MIB1 clone). Both antibodies were previously validated in the canine species [35,53].

Both procedures were performed with an automatic immunostainer (Ventana Benchmark GX, Roche-Diagnostic). To avoid the use of a bleaching reaction which could damage the integrity of the antigens, an ultraView universal alkaline phosphatase RED detection kit (Ventana Medical System Inc., Oro Valley, AZ, USA) was used (DAB chromogen in unbleached specimens is indeed not usable), and hematoxylin was used as a counterstain.

As positive controls, a canine MCT and canine cutis were used for KIT and Ki67 staining, respectively. As negative controls, antibody diluent was applied instead of the antibody.

The slides were visualized at 40× magnification using a D-Sight scanning machine and the D-Sight Viewer software (A. Menarini Diagnostics). The KIT index was evaluated by counting the mean number of positive cells in 5 consecutive high-power fields (hpf; 0.237 mm2) within the areas with clear positive staining, starting from the mostly positive field. If no positive cells were found in 3 consecutive hpf, the process was repeated in another IHC-positive area. This method was designed similarly to that used for the establishment of the Ki67 index as described by Bergin and colleagues [49], which was applied for the Ki67 index calculation. When more than one biopsy was present for each tumor (e.g., for margin evaluation), 5 hpf were selected for each specimen. Only areas with a cellular population representative of the tumor were selected, and areas a ffected by background, degeneration, scirrhous reaction, or necrosis were avoided. Neoplastic cells were considered positively KIT-labeled when they showed brightly red cytoplasmic and membrane staining, as exemplified in Figure 1A from the MCT control, and as described in the literature [35].

**Figure 1.** IHC for KIT and Ki67 with the RED labeling system. ( **A**) Mast cell tumor (MCT). Example of the mast cell tumor used as the control tissue for the KIT immunolabeling, with a high density of positive cells (100× magnification). (**B**,**C**) Canine oral melanoma (COM). (**B**) Example of a neoplastic area selected for the evaluation of the KIT index, which was 6.6 (100× magnification): the positive cells are discernible and scattered throughout the field. The details of the positive neoplastic melanocytes (arrow) are provided with higher magnification in the inset; melanomacrophages are also present within the inset (arrowheads). ( **C**) Example of a neoplastic area selected for the evaluation of the Ki67 index, which was 30 (100× magnification).

## *2.6. Statistical Analysis*

Statistical analyses were performed using MedCalc (MedCalc Statistical Software version 15.8). Data distribution was visually checked for normality. To verify mean differences among groups, either the Student's *t* test or the one-way ANOVA with Tukey's multiple comparisons were performed when data were normally distributed. The Mann–Whitney test or Kruskal–Wallis test was applied when data were not normally distributed. A Chi-square test and Fisher's exact test were used for analysis of the association between *KIT* amplification status and clinical features.

The Spearman's rank correlation analysis was applied to discover associations between variables. The level of significance was set at *p* < 0.05.
