Confocal Microscopy in Skin Cancer

With RCM, actinic keratosis (AK) shows an irregular and thickened stratum corneum, which correlates to the ortho- and parakeratosis seen on histopathology [5]. Focally, dark nuclei are visualized on RCM within the stratum corneum, correlating with parakeratosis on histopathology. The stratum granulosum and spinosum display on RCM pleomorphism of keratinocytes—the cells are irregularly crowded, vary in size and shape; these findings create an irregular honeycomb pattern with lines that vary in width and level of brightness, and with dark nuclei that vary in size or that are obscured (Table 2). Within the papillary dermis, abnormal collagen bundles are seen that are wider than normal collagen bundles and that appear clumped, correlating with solar elastosis on histopathology. Small bright stellate dots, correlating to inflammatory cells, may also be detected within the dermis.

On RCM, SCC shows similar, albeit more pronounced findings compared to AK [6] (Fig. 2). Bright, round cells within the granular and spinous layers, whose nuclei are often obscured, are more frequently observed in SCC, correlating to dyskeratotic cells seen on histopathology. The honeycomb pattern is focally lost in the spinous and granular layers (“disarranged” pattern, Table 2). Dilated, round blood vessels (Table 2) running perpendicular to the horizontal confocal plane of imaging, in a looped or tortuous course, are observed within dermal papillae in most cases of SCC and less often in AK; these vessels may also be visualized as glomerular or dotted vessels on dermoscopy (Fig. 2a, d).

At present, RCM does not allow differentiation with high specificity between AK, SCC in situ and invasive SCC. It does, however, allow differentiation from other disease entities that occur on sun-damaged skin, such as BCC, solar lentigo, lichen planus-like keratosis, and melanoma, which is especially valuable in the facial skin [7]. Pigmented AK and SCC, however, represent potential diagnostic pitfalls because of overlapping dermoscopic and RCM features with lentigo maligna; the presence of gray dots in dermoscopy and of bright epidermal dendritic cells on RCM, corresponding to Langerhans cells, may result in an overdiagnosis of melanoma [8, 9].

The evaluation of genital skin diseases, including penile carcinoma in situ, is a new promising area of research in RCM [10].

A recent systematic review on the value of RCM for diagnosing actinic keratosis, actinic cheileitis, erythroplasia of Queyrat, Bowen disease, invasive SCC, and keratoacanthoma (KA) showed an overall range of sensitivity and specificity of RCM between 79 and 100% and 78–100%, respectively [11].

González et al. first described RCM features of BCC in 2002 [12]. The most striking feature of BCC with RCM is the presence of tumor islands (Table 2), sharply demarcated from the surrounding dermis by dark cleft-like spaces (Fig. 3). Bright tumor islands are typically seen in pigmented BCCs. The presence of tumor islands as hyporeflective areas (“dark silhouettes”) that are darker than the surrounding dermis or basal epidermis are mostly seen in non-pigmented BCCs (Fig. 3b). The tumor islands seen on RCM correlate on histopathology with aggregates of basaloid cells with peripheral palisading of nuclei and clefting. The bright and the darker tumor islands can also be seen in the same BCC reflecting the degree of pigmentation within the neoplastic aggregates. Bright dendritic structures and/or dendritic cells can be seen within refractile tumor islands and represent melanocytes in pigmented BCC (Fig. 3a); bright dendritic cells in the overlying spinous layer of the epidermis mostly represent Langerhans cells. Interestingly, when viewed by RCM at the en face plane, basal or spinous keratinocytes above tumor islands in superficial BCC appear to be focally elongated and distorted into alignment along an axis (streaming) (Table 2). The papillary dermis displays dense bright collagen bundles between the tumor islands, correlating to the fibrotic stroma associated with BCC. Dilated and tortuous linear blood vessels (Table 2) that run parallel to the horizontal plane of RCM imaging are also typically observed within the stroma. These vessels correlate with the arborizing or fine branching vessels seen on dermoscopy. In pigmented BCC, oval to triangular plump-bright cells with indistinct nucleus may be observed on RCM singly or in aggregates within the stroma; these correlate with melanophages on histopathology.

Subsequent studies validated the reliability of described RCM criteria of BCCs [13]; a recent meta-analysis of the diagnostic accuracy of RCM for BCCs showed a high summary estimate of sensitivity (97%) and specificity (93%) [14]. However, the number of infiltrative BCCs included in previous RCM studies was generally small, and the detection of this BCC subtype still remains challenging with RCM; the small cords and strands of neoplastic cells are difficult to detect within the bright dermal collagen. In addition, the limited depth of RCM imaging does not allow detection of more deeply infiltrating BCC.

The RCM features of melanocytic nevi were first described by Langley et al. [21]. Nevi typically display round to oval, bright, monomorphic melanocytes, and a regular epidermal architecture. According to their dermoscopic and histopathologic appearance, different nevus types may show variable findings with RCM [22]. In junctional nevi, melanocytes are distributed singly and in nests mostly at the tips and sides of the rete ridges. With RCM, dark round dermal papillae surrounded by a rim of monomorphous refractive cells (“edged papillae,” Table 3), corresponding to melanocytes and pigmented basal keratinocytes, are observed at the level of the DEJ (Fig. 4b, c). Small junctional dense nests of melanocytes are observed on RCM within the interpapillary spaces or protruding into the dermal papillae with connection to the basal layer of the epidermis (Fig. 4d and Table 3). When viewing a mosaic image of nevi (akin to low magnification) at the DEJ level, the edged papillae, homogeneously distributed throughout the lesion, result in a regular “ringed pattern,” which correlates with typical pigment network seen on dermoscopy (Fig. 4a, b). In compound and dermal nevi, histopathology shows large nests of melanocytes within the dermis, with or without concomitant junctional nests. On RCM, compact aggregates of cells (also called dense nests), which are round to oval in shape and show uniform brightness, are observed (Fig. 5). Distinct cellular outlines of melanocytes are not always detectable within individual nests. When viewed on mosaic RCM image at the DEJ level, these large dermal nests may appear as a “clod pattern”; the larger nests of melanocytes seen with RCM correlate to pigmented globules seen with dermoscopy (Fig. 5a, b) Nevi may show on RCM at the DEJ level elongated, tubular melanocytic nests that expand the interpapillary spaces (“junctional thickening,” Table 3) (Fig. 5c); these correlate on histopathology with confluent junctional nests of melanocytes. When viewed on mosaic RCM image at the DEJ level, junctional thickening may appear as a “meshed pattern”; this correlates on dermoscopy with thickened network (Fig. 6).

Spitz nevi, some Clark nevi, and uncommonly congenital nevi, can present conflicting RCM criteria that may sway the observer to the diagnosis of melanoma. Concerning RCM findings that can be seen in these nevi include melanocytes at suprabasal layers of the epidermis (i.e., in pagetoid pattern); large solitary melanocytes at the basal layer; dermal papillae that are not well demarcated (known as “non-edged papillae,” Table 3); and melanocytic nests that vary in size, brightness and cohesiveness (including “sparse nests,” Table 3). Of note, these nevi are often also equivocal for clinical and dermoscopic diagnosis. In these nevi, RCM examination cannot rule out the possibility of melanoma [23•]. Thus, excisional biopsy and histopathological analysis are judicious in such concerning melanocytic lesions.

In melanoma, bright-nucleated cells (corresponding to atypical melanocytes) and dendritic processes may be seen at suprabasal layers of the epidermis (i.e., in pagetoid pattern) (Fig. 7). Melanocytes disposed in pagetoid pattern may be shaped as round, triangular, spindle cells, or dendritic nucleated cells [24]; melanocytes of different shapes, size, and brightness observed concomitantly suggest cellular pleomorphism (Fig. 8a). Clusters of melanocytes in pagetoid pattern may be observed, in addition to solitary pagetoid melanocytes. An abnormal epidermal pattern is usually seen; the honeycomb pattern is not visualized, either focally or throughout the lesion (“disarranged honeycomb pattern”) (Table 3 and Fig. 7c). At the DEJ level, an abnormal architecture is also detected with RCM. Non-edged dermal papillae (Table 3) variable in size and shape may be observed; non-edged papillae are often separated by widened interpapillary spaces that may contain large reflecting nucleated cells (atypical melanocytes) (Fig. 8b). While junctional thickenings (on high magnification RCM) and meshed pattern (on RCM mosaic images) can be seen at the DEJ in nevi and in some melanomas, their pattern is different. In nevi, the meshed pattern would appear more regular, while in melanoma, the junctional thickenings which contribute to the lines of the mesh, would vary in thickens and in brightness (Fig. 8b). In addition, large melanocytes as solitary units can be observed in melanoma at the basal layer of the epidermis (Fig. 8b). Foci with loss of the dermal papillae, replaced by sheets of atypical melanocytes seen on RCM (Table 3), indicate a crowded proliferation of solitary melanocytes and flattening of the DEJ on histopathology. In melanomas with a dermal component, cell clusters seen on RCM at the level of the superficial dermis may appear at least focally as sparse or cerebriform aggregates (Fig. 9b, c and Table 3). The variable size, shape and brightness, and uneven distribution of melanocytic nests seen in melanoma on RCM mosaics (Fig. 9a) correlate on dermoscopy with the presence of irregular globules and dots. The finding on RCM of cell clusters or solitary bright-nucleated cells in the dermis in a melanoma suggests the presence of an invasive melanoma. Dermal aggregates of plump-bright cells without visible nucleus (correlating with melanophages) as well as bright dermal clusters of melanocytes are seen in areas showing blue-white structures on dermoscopy.

Pellacani et al. defined six RCM criteria for the diagnosis of melanoma; two major criteria (non-edged papillae, atypical cells at the DEJ) and four minor criteria (roundish pagetoid cells, pagetoid cells widespread throughout the lesion, cerebriform clusters and isolated nucleated cells within dermal papillae) contribute to a scoring algorithm (each major criterion 2 points, each minor criterion 1 point). A score ≥ 3 signifies the diagnosis of MM with a sensitivity of 97.3% and a specificity of 72.3% [25]. A two-step algorithm for the diagnosis of melanoma by RCM has also been proposed [26]. In the first step, the lesion is identified by RCM as melanocytic or non-melanocytic. In the second step, the lesion is diagnosed with RCM based on Pellacani’s scoring system as nevus versus melanoma. By using this algorithm, unnecessary biopsies may be reduced by more than 50% without missing a melanoma.

The above mentioned RCM features are well established for superficial spreading melanoma. However, different melanoma subtypes may show distinct combinations of RCM features. For example, in nodular melanoma, cerebriform dermal nests are more commonly seen, while melanocytes in pagetoid pattern or a disarranged honeycomb pattern of the epidermis are uncommon [27].

In melanoma on sun-damaged skin (lentigo maligna), a descent of atypical melanocytes along adnexal structures, aggregates of atypical melanocytes surrounding adnexal openings, sheets of atypical (predominantly dendritic) melanocytes and dendrites at basal/suprabasal layers, and cord-like rete ridges at the DEJ are typically observed [28]. A so-called ringed pattern is usually not observed due to the flattening of the DEJ in sun-damaged skin of the elderly (Fig. 10 and Table 3). The value of RCM as adjunct to dermoscopy in the diagnosis of skin neoplasms in this cosmetically sensitive area has repeatedly been shown; RCM correlates of dermoscopic patterns of facial lesions help discriminating lentigo maligna from pigmented non-melanocytic macules [29•].

Studies have demonstrated the potential benefit of RCM in the differentiation of benign pigmented genital skin lesions from melanoma, including nevi and melanotic macules [30]. The presence of bright epidermal dendritic cells, corresponding to Langerhans cells, in a considerable number of melanotic macules represented a possible diagnostic pitfall in the differentiation from melanoma.

Finally, RCM is particularly valuable in the evaluation of lightly pigmented or amelanotic melanomas that often lack diagnostic features on clinical and dermoscopic examination [31].

Limitations of RCM in the assessment of melanocytic skin lesions include the lack of nuclear staining, the limited depth of imaging, and the difficulty in distinguishing dendritic melanocytes in pagetoid pattern from Langerhans cells that may occasionally simulate pagetoid spread.