## 4 Discussion  
This analysis revealed that CD57+ natural killer cells predominated in preinvasive squamous cell lesions (\(p<0.001\)) and CD4+ cells were dominant in oral cavity and laryngeal SCCs (\(p=0.044\)). In addition, proliferation activity (Ki67+) positively correlated with SCC but not with preinvasive lesions (\(\rho=0.61\)). No significant differences were observed in the amount of the other immune cells (CD8+ T-suppressor/cytotoxic lymphocytes, CD15+ neutrophil granulocytes and CD68+ cells of the monocyte/macrophage lineages) as also for p16 and PD-L1 between preinvasive squamous cell lesions and SCC. Overall, these data are in agreement with the concept of the two-stage model of carcinogenesis in the oral and laryngeal mucosa and the cancer immunoediting process [15]. From preinvasive lesions to invasive SCC, the amount and the quality of immune cells changed and it is hard to define a clear \"black and white\" classification for which different cell types are procancer or anticancer. It is certain that T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, monocytes, macrophages and neutrophil granulocytes are key instruments in the immune response and tumor progression [16]. In our analysis, the negative correlation of CD57 (\(\rho=-0.69\)) with SCC is likely related to the fact that natural killer cells play a major role in the elimination phase [17], whereas the high CD4+ expression in SCC supports the evidence that CD4+ T cells \"are a critical cornerstone of optimal anti-tumor immunity\" [18]. Indeed, it is accepted that CD4+ T cells have a synergistic role with CD8+ T cells in the immune response. However, once the escape phase is activated, CD4+ cells seem to play a role in favoring tumor cell survival [18; 19]. The expression of PD-L1 has been considered a point of balance between the host immune response and cancer escape ability [20]. A vast body of literature indicates that PD-L1 is mainly expressed on the surface of tumor cells and antigen-presenting cells in head and neck SCC. Based on the randomized phase III CheckMate 141 trial [21] and nonrandomized phase Ib KEYNOTE-012 trial [22], immune checkpoint PD1/PD-L1 blockade therapy (nivolumab and pembrolizumab, respectively) become the standard of care in the recurrent/metastatic setting [4; 23]. In contrast, little is known on the role of PD1/PD-L1 pathway in preinvasive lesions. A recent meta-analysis study focused on the prevalence of PD-L1 expression in preinvasive lesions compared to invasive SCC in the head and neck region [20]. Aware of the high heterogeneity and the moderate quality of the included studies, PD-L1 expression was reported to be less frequent in preinvasive lesions than in invasive SCC. We were not able to confirm this result likely due to the low number and site heterogeneity of the examined cases. However, there is no doubt that much remains to be learned on the interactions between the multiple PD-L1-expressing cell types within the tumor microenvironment [24; 25]. We also assessed the p16 status without recording significant differences between preinvasive lesions and SCC. This absence of difference should not surprise. Despite a causal association between HPV infection and SCC development being well established over the years, nowadays, p16 detection is mandatory to correctly stage primary tumors at diagnosis, in particular in the oropharynx, and its expression is a surrogate marker for HPV-related lesions, both preinvasive and invasive [26; 27]. Therefore, p16 expression should not be considered as a marker for the differential diagnosis of preinvasive versus invasive lesion. Regarding the positive correlation between Ki67 and SCC in our series, it is well known that Ki67 expression is an important biological mechanism in oncogenesis. The significance of Ki67 in the head and neck preinvasive and invasive squamous cell lesions has been discussed over the last years, but at present it is highly correlated with the increased aggressiveness of the lesion [28; 29].