**3. Results**

## *3.1. aCGH Analysis*

Data related to the aCGH analysis are published and extensively discussed in the paper from Brocca and colleagues [25]. Twenty samples were submitted for aCGH analysis, and a total number of 14 samples were selected for this study: seven harboring a KIT amplification, and seven without KIT amplification, which were randomly chosen among the remaining 13 samples to maintain a balanced number of samples between the two groups.

#### *3.2. Epidemiological Data*

The mean age of the 14 dogs selected for the study was 11.98, ranging from 10–17 years. Males represented 78.57% (11/14, one neutered) of the cases, while females represented the remaining 21.43% (3/14, one neutered). Male over-representation is in accordance with the literature [15,54]. The most represented breed was the cocker spaniel, with 28.57% (4/14), and the most common sites of the neoplasia (when specified) was the oral lip, with 57.14% (8/14, six of which on the upper lip). For the immunohistochemical evaluation of Ki67, the majority of the samples (85.71%, 12/14) showed an index >19.5, significant of a bad prognosis at one-year post-diagnosis [49]. This data is visible in Table 1. All samples came from surgical excision or incisional biopsy.

#### *3.3. Identification of Somatic Mutations*

The DNA from all 14 samples was successfully extracted from the fractions of healthy and pathological tissues, with the use of heptane as the deparaffinizing agen<sup>t</sup> to obtain a higher yield of extracted nucleic acids. From Nanodrop analysis, healthy tissues showed a lower amount of extracted DNA (*p* < 0.0001, data not shown), and the 260/280 and 260/230 ratios showed variable values, but all were considered of sufficient quality for the amplification steps. All primer pairs were firstly tested on canine control tissues at different temperatures with a gradient PCR, and all pairs showed successful and specific amplification of the selected exons. The amplification was successful in almost all samples for both the healthy and the pathologic tissues. A band at the expected length was obtained in 27/28 (96.4%) reactions (14 DNA samples from healthy tissues and 14 DNA samples from pathologic tissues) for exon 13, 27/28 (96.4%) for exon 17, and 28/28 (100%) for exon 18 (Table 3). Indeed, it was not possible to obtain an amplification product for exons 13 or 17 using the pathologic DNA extracted from sample 12 (Figure 2). All amplicons obtained were successfully sequenced and analyzed as described by three operators. In summary, 97.6% of the exonic sequences (82 sequences out of 84) examined were successfully amplified and Sanger-sequenced, and no somatic SNPs, insertions, or deletions were identified in any of the samples.


**Table 3.** Result of the PCR amplification obtained for each DNA fraction (healthy and pathologic) of the samples analyzed, and for each primer pair. H: healthy; P: pathologic. Ex: exon.

**Figure 2.** 1.8% agarose gel showing the amplicons obtained by PCR reaction for exon 13 (Ex-13) of the KIT gene. As shown, no amplified DNA was obtained from the pathological fraction of sample 12 (12\_P). +: positive control (non-fragmented canine genomic DNA); −: negative control (water).
