*2.2. Histopathological Examination*

The tissue samples were fixed in 10% formalin and the method of paraffin embedding was used. For microscopic examination, hematoxylin–eosin stain (HE) was performed (Figures 1 and 2). We have determined the tumor diameter, depth of invasion, and the presence of ulceration. Depth of invasion was calculated as the perpendicular distance between the basement membrane and the deepest point of the infiltrative zone of the tumor according to the recommendations from the 8th Edition of the AJCC [41]. The values were expressed as millimeters.

**Figure 1.** Histopathological examination of cSCC. (**A**) Cell proliferation with giant nuclei and nuclear polymorphism (HE, ×40). (**B**) Proliferation of squamous cells which extend into the deeper layers of the skin, abundant inflammatory infiltrate (HE, ×40). (**C**) Massive inflammatory infiltrate distributed throughout the tumor mass and disseminated tumor cells with giant nuclei (HE, ×40). (**D**) Proliferation of squamous cells with nuclear pleomorphism and nuclear abnormalities and abundant inflammatory infiltrate (HE, ×90).

**Figure 2.** Histopathological examination of AK. (**A**) Disorganized growth, which disrupts differentiation (HE, ×10). (**B**) Dysplasia of basal keratinocytes (HE, ×90).

The Ki-67 antigen is a marker for cell proliferation and is widely used as a cancer activity marker [42]. We used the VECTASTAIN ABC KIT (PK-6101), specifically designed for immunohistochemical staining of tissues. The kit contains biotinylated IgG that was used to bind to the primary anti-Ki67 antibody. The cSCC samples were divided into three subgroups according to the Ki67 index as follows: low < 25%, intermediate 25–75%, and high > 75%.
