Keywords

Fast diagnostic methods in melanoma have been gaining importance in the last few decades due its malignant nature and its rising incidence [1]. Being able to safely distinguish normal or dysplastic nevi from melanoma intraoperatively and immediately decide further therapeutic steps would potentially decrease the number of surgical procedures [2], as well as associated risk of complications. Possible use of ex vivo confocal laser scanning microscopy (ex vivo CLSM) in melanoma diagnostics, including fast immunofluorescence [3] and intraoperative tumor thickness measurement [4], as well as up-to-date experience (Table 11.1) together with examples of melanoma images (Figs. 11.1, 11.2, 11.3 and 11.4) are presented and discussed.

1 Basics of Melanoma

  • Definition: Malignant, invasive melanocytic skin tumor clinically presenting mostly as a deep brown to blue-blackish, brown-reddish, or even pigment-free (amelanotic melanoma) nodule or plaque with an early tendency to metastasize (strongly depending on tumor thickness). Different types of melanoma are distinguished on the basis of clinical and histological criteria (superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and others) [1].

  • Epidemiology: The incidence in a fair skinned population in Europe and North America is estimated to be 15/ 100,000 people annually [1].

  • Histopathology: Melanomas have various histologic features including presence of atypical melanocytes singly or as nests with varying shape and size. These atypical cells can invade local structures causing their destruction. Atypical mitosis in the tumor cells, trans-epidermal melanocytic migration (TEM), asymmetry of the tumor, ill-defined tumor, horizontal confluence of the nests, lack of maturation of tumor cells in the deep dermis, vascular and/or perineural invasion, as well as spread of melanoma nests along the epithelial adnexal structures. Ulceration of the tumor, solar elastosis in the dermis, and strong peritumoral inflammatory infiltrate may occur [5].

  • Immunofluorescence: First pilot studies on the combination of ex vivo CLSM and fluorescent-tagged antibodies have indicated new opportunities for fast and specific tissue examination [3, 6,7,8,9,10,11]. There is a particular interest in developing such techniques for the examination of melanocytic lesions to distinguish malignant from benign lesions. First report on the use of fluorescent-tagged S100-antibody and fluorescent-tagged Melan-A-antibody proved the possibility of such a concept and at the same time showed the complexity of such implementation within intraoperative setting [3]. Further studies on the use of immunofluorescence in the ex vivo CLSM are necessary.

  • Confocal tumor thickness MEASUREMENT: Ex vivo CLSM enables intraoperative measurement of tumor thickness. First pilot study showed promising results and very good correlations to the tumor thickness on histopathology tissue Sect. (4). Larger studies on confocal tumor thickness measurement are needed to validate these results.

Table 11.1 Melanoma features on FCM, DHE, and corresponding conventional H&E
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Fig. 11.1
figure 1

Superficial spreading melanoma examined in two modes of the ex vivo confocal laser scanning microscopy (a fluorescence, b DHE), and comparison with their corresponding H&E-stained image (c) presenting nests of varying size and shape consisting of atypical melanocytes in the epidermis and dermo-epidermal junction (long arrow), as well as strong inflammatory infiltrate (short, thick arrow), and solar elastosis (star) in the dermis. Melanocytic spread along the follicle is highlighted with an arrowhead. DHE = digital hematoxylin–eosin-like staining. H&E magnification = 10x

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Fig. 11.2
figure 2

Detailed view of a superficial spreading melanoma examined in two modes of the ex vivo confocal laser scanning microscopy (a fluorescence, b DHE) and comparison with their corresponding H&E-stained image (c), presenting atypical melanocytes clustered in nests as well as single cell proliferates in the epidermis and dermo-epidermal junction (long arrow) showing signs of transepidermal melanocytic migration DHE = digital hematoxylin–eosin-like staining. H&E magnification = 20x

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Fig. 11.3
figure 3

Dermal part of a nodular melanoma examined in two modes of the ex vivo confocal laser scanning microscopy (a fluorescence, b DHE) and comparison with their corresponding H&E-stained image (c), presenting dermal nests of varying size and shape consisting of atypical melanocytes (arrow) showing varying levels of fluorescence signal, as well as cellular and nuclear pleomorphism. DHE = digital hematoxylin–eosin-like staining. H&E magnification = 20x

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Fig. 11.4
figure 4

Melanoma metastasis examined in two modes of the ex vivo confocal laser scanning microscopy (a fluorescence, b DHE) and comparison with their corresponding H&E-stained image (c), presenting masses of atypical melanocytes (arrow) with multiple atypical mitoses. DHE = digital hematoxylin–eosin-like staining. H&E magnification = 20x

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