*3.4. Immunohistochemistry*

One of the samples (sample 7 in Table 1) could not be evaluated for KIT expression since the DNA extraction procedure exhausted the paraffin block.

Positive immunolabeling was obtained in the MCT control (Figure 1A) and in all the cutaneous melanocytes (internal control) of the healthy tissues included along with COM specimens.

As expected, the anti-KIT antibody showed cytoplasmic and membranous labeling of neoplastic melanocytes with a bright multifocal positivity of a scarce to a moderate number of cells (Figure 1B). The positive cells were discernible and randomly distributed across the tumor tissue in the majority of samples; particular patterns of distribution were not noted. Only two samples (15.4%) did not show any staining for KIT expression.

The KIT index varied from 0.6 to 8.6 in positive samples, with a mean of 3.1 positive cells in 5 hpf. Positive staining for the anti-Ki67 antibody (Figure 1C) was obtained for all specimens and the Ki67 index was established as described [49]. The Ki67 index varied from 7.6 to 265, with a mean of 62.4 positive cells in 5 hpf. According to the Ki67 prognostic cut-off of 19.5, three samples were classified as having a good prognosis (G), and the remaining 11 as having a bad (B) prognosis. The results are summarized in Table 1. When samples were divided into two groups according to the KIT amplification status, no statistically significant differences were detected either for the KIT index (*p* = 0.56) or for the Ki67 index (*p* = 0.38). Similarly, no differences in terms of KIT and Ki67 expression were noted between males and females (*p* = 0.87 and *p* = 0.46, respectively), or according to the pigmentation level (*p* = 0.48 and *p* = 0.73, respectively). Finally, no correlation was found between the two indexes (*r* = 0.074, *p* = 0.81).
