**4. Discussion**

The aim of this study was to deepen our understanding of the mutational landscape of the *KIT* gene in COMs, particularly in exons 13, 17, and 18, and to correlate the mutational profile of these exons with the amplification status of the gene itself, and with the IHC expression of the KIT protein.

This interest derives from the scarcity of currently available similar studies in the canine species, and from the possible use of pet dogs as a reliable model for the study of hMMs.

In our previous work [25] that aimed to improve our knowledge about the genomic DNA alterations that occur in COM, many genes related to MAPK and PI3K pathways were detected from the CNA analysis, together with a wide variety of genes coding for tyrosine kinases receptors, including *KIT*. Interestingly, the pathway enrichment analysis revealed the enhancement of pathways specifically related to cancer proliferation, but also a significant enrichment of those related to imatinib and drug metabolism. These results indicated that further investigation of the *KIT* alteration status was warranted.

In this work, we describe the first characterization of the mutational profile of exons 13, 17, and 18 of the *KIT* gene in COM, which were successfully PCR amplified and Sanger-sequenced. Taking advantage of the cohort of samples collected for the aCGH study [25], we were able to compare the exon sequences of healthy and pathologic tissues from COMs with known *KIT* amplification status. In particular, CFA 13 (comprising the *KIT* gene) was affected by a copy number gain in 7/20 samples of the original cohort (35%), and we considered it valuable to further analyze the DNA of these seven samples and to compare them with another randomly chosen seven samples that were not affected by the same copy number gain in CFA 13.

We developed highly-performing primer pairs and set up a reliable protocol for the amplification and sequencing of short genomic sequences extracted from FFPE blocks.

Since no SNPs were detected affecting the examined exons, our study suggests that *KIT* status in COMs does not resemble the mutational status reported in hMMs. This is in line with some of the latest Next Generation Sequencing-based veterinary studies [10,24]. Although Garrido and Bastian [4] suggested that CNAs and SNPs in hMM are mostly mutually exclusive, two different studies reported *KIT* amplification and coexisting SNPs in exons 11, 13, 17 and 18 in the same tumor [6,8].

In our study, none of the samples affected by a CFA 13 amplification had an SNP present, and no point mutations have been found at all, suggesting one possible molecular difference between COMs and hMMs.

The absence of point mutations in our cohort is consistent with the results reported in other recent studies of COMs, in which the *KIT* point mutations are considered a sporadic event [10,33,34,47], which highlights a potential significant molecular difference with hMMs [4,8,10].

The detection of a 35% prevalence of *KIT* amplification versus a 0% prevalence of *KIT* point mutations in our cohort of COMs corroborates the increasingly a ffirmed hypothesis that the main pathogenesis of COMs and hMMs is related predominantly to CNAs rather than SNPs [10,18–21].

Regarding the pathologic DNA from sample 12, we were not able to obtain an amplification reaction for exon 13 and 17, while the amplification of exon 18 was successful (Table 3). The reason for this could be the high melanin content of the sample. Indeed, melanin is an interferer of the PCR reaction and other molecular analysis when it is co-purified in the process of DNA extraction. Moreover, it has already been demonstrated that PCRs producing longer amplicons are more inclined to be inhibited by melanin than PCRs producing amplicons of shorter size [55]. In our case, melanin could have bound to the DNA polymerase enzyme, preventing the PCR reaction in the longer exons, i.e., exons 13 and 17, which both had a length close to 250 bp, but not in the shorter exon 18, which is approximately 200 bp long.

Here, we proposed a reproducible method for scoring KIT IHC positivity in COMs samples. To date, the IHC evaluation of KIT expression has been limited to a semi-quantitative evaluation, expressed as classes corresponding to an approximate percentage of immunoreactive cells on the total tumor area, often with wide ranges defining a single class [34,39]. In our opinion, this approach poorly describes the mutable status of expression of the protein. In support of this hypothesis and in contrast with other studies, our IHC results closely reflect the human literature: 84.6% of our samples were indeed positive, and therefore nearer to the percentage provided in human literature. In other veterinary studies, only about half of the COMs examined (49–51%) [34,39] were considered positive with the semi-quantitative scoring method. It is still unclear if this di fference is related to the di fferent scoring method or to a real di fference in KIT expression between hMMs and COMs, and further evaluation (or a re-evaluation of previous works) is necessary. We also semi-quantitatively scored our samples following the methods proposed in [34,39], and we noted an overestimation of negative cases when the percentage of positive cells was <10% (data not shown).

There was no significant di fference in KIT protein expression between *KIT* amplified and non-amplified samples. This could be due to the low number of cases analyzed, or to the fact that *KIT* gene amplification does not correspond to a higher KIT protein expression.

As reported by Lassam and Bickford [56], and by Montone and colleagues [57], an interesting observation regarding KIT in melanomas is the decrease (or even the loss) of KIT expression along with the progression of the neoplastic disease. This was observed in human cultured melanoma cells [56] and in cutaneous melanomas (from radial growth phase to vertical growth phase and metastatic melanoma) [57], which led to the hypothesis that the loss of KIT could represent a negative prognostic factor [56,57].

These findings support the hypothesis of Alexeev and Yoon [5], who proposed that for malignant melanocytes to acquire metastatic potential and escape from the epidermal boundaries [4], they necessarily have to lose KIT expression.

Indeed, a study from Newman et al. [39] found a significant association between the presence of KIT IHC positivity and patient survival, suggesting that the downregulation or loss of KIT could be related to increasing invasiveness in dogs as well.

Regarding the correlation with other tumor markers, Ma and colleagues [13] highlighted an increased Ki67 expression in metastatic hMM, while the lack of pigment was considered a negative prognostic factor by Prouteau and colleagues [58]. However, in our study, no statistically significant di fference was noted in either case.

Unfortunately, follow-up data were not available for this cohort of samples, making further evaluations impossible.
