# Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy
## 2 In Vivo Confocal Laser Scanning Microscopy
### Main In Vivo CLSM Diagnostic Clues in Skin Cancers  
The incidence of skin cancers is rising throughout the world, with cutaneous melanoma being associated with 90% mortality related to skin malignancies. Melanoma prognosis is dependent on Breslow index, with increased thickness being associated with worse outcomes and lower survival rates [29]. Thus, an early diagnosis, supported by non-invasive skin imaging techniques, has a pivotal role [13; 30; 31; 32]. As previously mentioned, two melanoma-specific RCM key-features, represented by \"atypical cells\" and \"dermal-epidermal junction disarray\", have been described [1]. These features are basically shared by all melanoma type included lentigo maligna/ lentigo maligna melanoma (LM/LMM), superficial spreading (SSM) and nodular melanoma (NM). LM and LMM are mainly located on the face and other photo-exposed skin sites. RCM contributes to LM/LMM diagnosis through the identification of a disarranged epidermis and atypical cells (cells with atypia in terms of shape and size) at epidermal layer or infiltrating the hair follicle and a meshwork pattern with melanocytes aggregates (nests) at the DEJ [33]. In addition, the \"medusahead-like\" feature, showing elongated buddings bulging from the hair follicle and filled with atypical cells (with dendritic or pleomorphic appearance), represents another important diagnostic clue for LM/LMM [34], as well as folliciculotropism [35]. Interestingly, the identification of melanocytic nests with RCM, contribute to the differential diagnosis with other pigmented macules of the face [36]. To date, superficial spreading melanoma (SSM) represents the most common form of melanoma in Caucasians while NM is less represented [37; 38]. RCM variations corresponding to changes in each skin layer can be observed and a melanoma classification according to RCM features, corresponding to different molecular alterations, has been established [39; 40]. The prevalence of atypical cells with dendritic shape (above all in \"solar\" melanoma which develops in patients with low nevus count and sun-damaged skin) is observed in type 1 melanoma [14; 39], while roundish cells (above all in melanomas in subjects with numerous nevi and a history of intermittent sun exposure), as well as non-edged papillae at DEJ [39; 41], are typical of type 2 (Figure 2a,b). With increasing thickness of SSM, and also in some NMs, an increased degree of disarrangement within epidermis, cell pleomorphism, and melanocytic nests, organized in a cerebiform fashion, progressively infiltrating dermal papillae can be also observed (type 3) [39; 42] (Figure 2c,d). An additional feature of thick melanomas is represented by dark areas with irregular contours and sharp borders, containing amorphous material, corresponding to ulceration [43]. Furthermore, a combination of atypical cells with dendritic and roundish appearance has been described in type 4 melanomas [39]. Interestingly, RCM characteristics of melanoma metastasis have been described and they have been found to correlate with dermatropic and epidermotropic diagnoses with histopathology [44]. Non-melanoma skin cancers (NMSCs), including BCC and actinic keratosis/squamous cell carcinoma (AK/SCC), are the most common malignancy in populations with fair skin and intermittent or chronic exposure to UV radiation [45]. BCC is the most frequent skin tumor, characterized by local invasion, while about 20% is represented by SCC, with a worst prognosis related to the risk of potential metastatization [13]. BCC is typified by basaloid cords and or tumor islands with RCM [1]. Tumor islands are composed by highly packed cells with high refractivity and polarization, surrounded by clefting, corresponding to dark spaces around tumor nests [46] (Figure 3a,b). Other peculiar findings in BCCs are abundant inflammatory infiltrate (visible as bright cells with irregular contours and without nucleus, also known as melanophages) distributed inside and outside the tumor islands, increased number of dilated blood vessels [47].