- Email: [email protected]
Glowing in the Dark
G lo wi ng in t he D a rk Use of Confocal Microscopy in Dark Pigmented Lesions Susana Puig Sardá, MD, PhDa,b,*, Rodolfo Suárez, MDa, Josep Malvehy Guilera, MD, PhDa,b KEYWORDS Reflectance confocal microscopy Melanoma Basal cell carcinoma Blue nevus
KEY POINTS Black color in melanoma is associated with melanin in upper epidermis. Under reflectance confocal microscopy (RCM), multiple bright atypical pagetoid cells or atypical cobblestone are visible in upper layers of the epidermis. Blue color in dermoscopy corresponds with pigment deep in the dermis. Blue color may correspond with bright nests or sheets of atypical cells in melanoma, spindle cells in blue nevus, or a tumor island wrapped by dendrites in pigmented basal cell carcinomas. Exogenous pigmentation can also be evaluated by RCM.
In vivo reflectance confocal microscopy (RCM) is a noninvasive imaging technique1–3 that provides images of the superficial layers of the skin at nearly histologic resolution generated thanks to backscatter of light by several structures. Melanosomes, which are highly reflective,4 are among the structures that generate white images in RCM. Thus, clinically dark lesions are easily visible by RCM because of melanin. In the RCM, light penetrates to a subsurface focus of the laser and reflects back from that focus out of the skin and into a detector in the microscope. Light that reflects superficial to the focus is eliminated, so if the focus is too deep, there will be no signal. For the same reason, if much melanin exists in superficial layers, like in black lamella (Fig. 1), no signal is generated from the deep epidermis or dermoepidermal layer. RCM can also detect pagetoid melanocytes in the epidermis, which correlate with black dots or dark blotches in dermoscopy.
The depth penetration of RCM is limited to 100 to 200 mm in human skin at the 830 nm laser wavelength used in the commercial system. The variability in the imaging penetration depth also depends on natural variations in the concentration of other reflective components comprising skin, such as keratin or collagen in addition to melanin.5 RCM criteria found in more superficial anatomic layers may be more readily identified, because there is decay in laser light intensity with increasing imaging depth and hence decrease in optical resolution. Because of the limitation in the penetration, those dark blue lesions, in which melanin is deep in dermis, are not always visible by RCM.6 Finally, some lesions can be clinically dark owing to substances or structures other than melanin structures, such us hemoglobin7 or exogenous materials.8 According to the reflective properties of the material, the images generated will be different. In the present article, the RCM criteria of several dark lesions are reviewed (Table 1).
a Melanoma Unit, Dermatology Department, Hospital Clinic Barcelona, IDIBAPS, University of Barcelona, Villarroel 170, Barcelona 08036, Spain; b CIBER de Enfermedades Raras, Barcelona, Spain * Corresponding author. Dermatology Department, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain. E-mail addresses: [email protected]; [email protected]
Dermatol Clin 34 (2016) 431–442 http://dx.doi.org/10.1016/j.det.2016.05.006 0733-8635/16/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved.
derm.theclinics.com
INTRODUCTION
432
Sarda´ et al
Fig. 1. Black melanoma. (A) Clinical image of a small melanoma on the knee of a woman in her 60s with personal and familial history of melanoma and carrier of a G101W mutation in CDKN2A. (B) Under dermoscopy, the lesion is showing a multicomponent pattern with presence of peripheral pigment network, focal globules, and central blotch. Predominant colors are black and blue. (C) Reflectance confocal microscopy (RCM) only reaches superficial layers of the epidermis showing a typical cobblestone pattern and presence of keratin. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with some nests and focal pagetoid growing. Hyperkeratosis with presence of pigmented corneocytes correlates with the typical cobblestone pattern seen in RCM.
Table 1 Most common cutaneous dark lesions with reported criteria with reflectance confocal microscopy Melanocytic
Non Melanocytic
Benign
Black nevus Blue nevus Spitz
Malignant
Melanoma Seborrhoeic-like melanoma Malignant blue nevus Blue nevus like cutaneous metastasis
Seborrheic keratosis Ink spot lentigo Angioma Angiokeratoma Pilomatricoma Ocronosis, argiria Tattoos Basal cell carcinoma
Confocal Microscopy in Dark Pigmented Lesions
Fig. 2. Black and blue melanoma. (A) Clinical image of a dark brown–blue lesion located on the trunk. (B) Dermoscopy shows an asymmetric lesion with multicomponent pattern with atypical pigment network, black dots/ globules and blue-gray dots. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing the presence of bright atypical cells (roundish and large) and the presence of atypical cobblestone pattern. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing.
DARK MELANOCYTIC LESIONS Even though most melanocytic lesions are brown, from light brown in fair skin to dark brown in dark skin, the presence of black color is less frequent and associated to specific diagnosis: melanoma, pigmented Spitz/Reed nevus, and black nevus (nevus with black lamella in dark skinned patients or after exposure to ultraviolet light). We also consider dark color the presence of dark blue, again, in the context of melanocytic lesions associated to few diagnosis, melanoma and blue nevus. Black color in melanoma: The presence of black color in melanoma is associated to the presence of melanin in upper epidermis, under RCM multiple bright atypical pagetoid cells (Figs. 2 and 3) are visible in upper
layers of the epidermis. Occasionally, in lesions with hyperkeratosis and transepidermal elimination of pigment in keratinocytes, a bright cobblestone is visible obscuring the dermoepidermal layers (see Fig. 3). If melanin is in atypical melanocytes in upper epidermis, then atypical cobblestone is present (with larger cells and the presence of large nuclei; Fig. 4), but if melanin is only present in keratinocytes of the upper layers of the epidermis, typical cobblestone can be present (see Fig. 1), obscuring deeper layers of the epidermis. In those cases, if we cannot see the dermoepidermal junction, melanoma cannot be ruled out by RCM. Blue color in melanoma: The presence of dark blue color in melanoma is associated with the presence of deeply located pigmented tumor.
433
434
Sarda´ et al
Fig. 3. Spitzoid melanoma. (A) Dark palpable fast growing lesion on the leg of a male in his 40s. (B) Under dermoscopy, the lesion is showing a star burst pattern with peripheral streaks and paracentral dark blotch and blue white area. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing pleomorphic pagetoid cells (roundish and dendritic) and an atypical cobblestone pattern with the presence of small round bright nucleated cells. (D) RCM of the dermoepidermal junction showing nonedge papilla, atypical basal cells and the obscuring of the basal layer.
If ortokeratosis is also present, then a whitish color is overlying the deep blue (Figs. 5 and 6). Under RCM, large bright nests (see Fig. 4) or sheets of atypical cells in the dermoepidermal junction (see Fig. 6) can be seen. Dark pigmented Spitz/Reed nevus. Starburst pattern in dermoscopy (see Fig. 3; Fig. 7) can be present in Reed/pigmented Spitz nevus but also in melanoma. Recently, Guida and coworkers9 analyzed the RCM findings present in both entities to identify those criteria that can be used to differentiate between them. In this study, striking cell pleomorphism within epidermis, widespread atypical cells at the dermoepidermal junction (see Fig. 3) and marked pleomorphism within nests were significantly associated with the diagnosis of malignant melanoma, whereas spindled cell (Fig. 8) and peripheral clefting were exclusively found and pathognomonic of spitz nevi (SNs).
Black nevus: In these nevi, melanin is present in keratinocytes of the upper layers of the epidermis showing in RCM typical cobblestone. In the dermoepidermal layers, if visible, a ringed pattern is present. In some cases, after exposure to ultraviolet radiation, many dendritic cells can be visible in upper epidermis (see Fig. 8). These lesions can show some of the criteria previously described in small melanomas.10 Blue nevi are characterized by the presence of dendritic melanocytes in dermis that produce melanin, even deep in the dermis. In some cases, a superficial component is present being the lesions dark blue. Under dermoscopy, blue homogeneous color without any other structures is present but, when cross-polarized dermoscopy is used, the blue can be less homogeneous and some irregularities visible inside. In those blue nevi with a preserved papillary dermis,
Confocal Microscopy in Dark Pigmented Lesions
Fig. 4. Superficial spreading melanoma with a black blotch. (A) Dark, slightly palpable lesion on the trunk of an elderly man. (B) Under dermoscopy the lesion is showing a multicomponent pattern with atypical pigment network, focal presence of dark brown/black dots, dark blotch and gray dots. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing a warty architecture with the presence of bright cords separated by black holes (sulci). The epidermal pattern is atypical cobblestone with the presence of roundish atypical cells. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing and large atypical nests in the junction.
RCM is not able to reach the lesion, but, if some nests are present at the papillary dermis, RCM shows a characteristic pattern11,12 with nests of bright dendritic cells (Fig. 9).
DARK NONMELANOCYTIC LESIONS Basal Cell Carcinoma Pigmented basal cell carcinoma may clinically mimic melanoma (Fig. 10). In a dark-skinned population,13 pigmented basal cell carcinoma is more frequent than nonpigmented basal cell carcinoma, and may present diagnostic difficulties. Dermoscopy is an easy, noninvasive technique that increases the specificity in the diagnosis of
both tumors14; however, RCM adds valuable criteria for the diagnosis of pigmented basal cell carcinoma,6,15,16 with the presence of tumor islands wrapped by bright dendrites and surrounded by clefting. These structures are visible at the dermoepidermal layer or papillary dermis. If the examination with RCM is too superficial, the presence of multiple dendritic cells in the upper layers of the epidermis may induce a misdiagnosis with melanoma. Segura and colleagues17 demonstrated that dendritic cells in the upper layers of the epidermis in pigmented basal cell carcinomas are Langerhans cells, whereas dendrites in the tumor island correspond with melanocytes colonizing the tumor cords or nests.
435
436
Fig. 5. Melanoma with blue whitish veil. (A) Clinical image of an asymmetric slightly palpable dark lesion. (B) Dermoscopy shows a melanocytic lesion with a multicomponent pattern, with atypical pigment network, streaks, and blue whitish veil. (C) Reflectance confocal microscopy of the dermoepidermal layer is showing nonedged papilla, atypical nests of large cells and atypical cells in the basal layer. (D) Hematoxylin and eosin staining (original magnification, 20) in the blue whitish area showing an atypical proliferation of very pigmented melanocytes in the dermis with orthokeratotic hyperkeratosis (the histopathologic background of the blue whitish veil).
Confocal Microscopy in Dark Pigmented Lesions
Fig. 7. Pigmented Spitz/Reed nevus. (A) Clinical image of a symmetric, elevated, and pigmented new lesion on the limb of a young man. (B) Dermoscopy shows a star burst pattern with peripheral pseudopods and projections, black dots and globules, atypical pigment network, black blotch and blue hue. (C) Reflectance confocal microscopy mosaic at upper epidermal layers showing a cobblestone pattern with presence of black holes. A detail of this mosaic is shown in the right lower corner showing an atypical cobblestone, dendritic pagetoid cells and small suprabasal nests at the periphery of the lesion. (D) Reflectance confocal microscopy mosaic at the dermoepidermal junction showing visible papilla, atypical bright cells, multiple nests and plump cells in papillary dermis.
Seborrheic Keratosis Pigmented seborrheic keratosis can be also difficult to differentiate from melanoma. Using dermoscopy as an additional tool, RCM also offers valuable criteria for the correct diagnosis. Under
RCM, a typical cobblestone in upper layers of the epidermis (Fig. 11) is shown together with the presence of follicular openings (black holes), follicular plugs (concentric bright keratin) and cysts (bright roundish structures) helps in the differential diagnosis.
= Fig. 6. (A) Clinical image of an elevated and ulcerated tumor with recent bleeding. (B) Dermoscopy of the nonulcerated area is showing blue whitish veil. (C) Reflectance confocal microscopy of the nonulcerated area is showing a sheet of atypical cells. (D) Hematoxylin and eosin staining (original magnification, 20) corresponding with the peripheral area of the tumor, showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing and presence of pigment in dermis and overlying orthokeratotic hyperkeratosis (the histopathological background of the blue whitish veil).
437
438
Fig. 8. Black nevus. (A) Dermoscopy of a 2-mm diameter lesion that appeared recently, after exposure to ultraviolet light, showing an atypical pigment network. (B) Reflectance confocal microscopy of upper epidermis is showing a pleomorphic proliferation of atypical cells, roundish and dendritic. (C) Hematoxylin and eosin staining (original magnification, 20) corresponding with an atypical proliferation of melanocytes in the dermoepidermal junction with heavily pigmented nests and inflammation in dermis.
Confocal Microscopy in Dark Pigmented Lesions
Fig. 10. Pigmented basal cell carcinoma. (A) Clinical image of a 4-cm black lesion on the lumbar area. (B) Dermoscopy shows a central area with blue whitish veil and some black globules and few erosions, and at the periphery the presence of maple leaf like areas (close to the B). (C) Reflectance confocal microscopy at the dermoepidermal junction shoving tumor cords wrapped with bright dendrites, palisading, peripheral clefting and presence of plump cells (inflammatory cells and melanophages) in the dermis. (D) Basaloid tumor island in superficial dermis showing palisading and clefting.
Ink Spot Lentigo In these dark lesions, RCM shows a cobblestone pattern in the epidermis and a ringed pattern in the dermoepidermal junction.
highly reflective superficial scale with abnormal architecture at the dermoepidermal junction that displays epidermal cells forming septa surrounding the prominent ecstatic vascular lacunae (Fig. 12).
EXOGENOUS PIGMENTATION Angiokeratomas and Thrombosed Hemangiomas In these lesions, the dark color is not owing to the presence of melanin, but to the presence of blood in dermis and superficial hyperkeratosis. On RCM,7 this lesion shows at the epidermal level
Ochronosis was the first exogenous pigmentation described with RCM8 showing dark curved silhouettes corresponding to the classical banana bodies described in histopathology. Later, silver deposits18 were also characterized by RCM.
= Fig. 9. Blue nevus. (A) Clinical image of a nodular lesion on the abdomen. (B) Blue homogeneous pattern under dermoscopy. (C) Reflectance confocal microscopy is showing a nested proliferation of dendritic bright melanocytes in dermal papilla. (D) Hematoxylin and eosin staining (original magnification, 40) showing a proliferation of dendritic and pigmented melanocytes in the dermal papilla.
439
440
Fig. 11. Clonal seborrheic keratosis. (A) Clinical image of a large pigmented lesion on the scalp of a man in his 60s. (B) Dermoscopy of 1 area of the lesion is showing homogeneous pigmentation with brown, blue, and black coloration. (C) Reflectance confocal microscopy is showing in a mosaic at upper epidermis the presence of a typical cobblestone interrupted by multiple follicular openings. (D) Hematoxylin and eosin staining (original magnification, 20) showing a proliferation of deep pigmented keratinocytes.
Fig. 12. Angiokeratoma. (A) Clinical image of a dark tumor in an acral site. (B) Under dermoscopy, the lesion shows black and blue color with the presence of a crust. (C) Reflectance confocal microscopy shows a typical honeycomb in the epidermis with the presence of ecstatic vascular structures in papillary dermis. (D) Hematoxylin and eosin staining (original magnification, 20) showing the prominent ecstatic vascular lacunae.
Confocal Microscopy in Dark Pigmented Lesions SUMMARY RCM criteria considered by users as more relevant for melanoma diagnosis may be frequently detected in dark lesions; most dark lesions present melanin that enhances the brightness of confocal criteria. One of the difficulties in the diagnosis of deep pigmented melanomas could be the presence of typical cobblestone pattern in upper epidermis owing to the presence of melanin in keratinocytes that obscure the dermoepidermal layer because decay in laser light intensity. On RCM, in blue nevi, bright dendritic cells can be visible at the papillary dermis. Pigmented basal cell carcinoma can be recognized easily on RCM if the papillary dermis is reached. There, tumor islands or cords with bright dendrites are recognized easily. If the RCM imaging is too superficial and the tumor in dermis is not reached, the presence of dendritic cells in the epidermis can be misleading towards a diagnosis of melanoma instead of pigmented basal cell carcinoma.
REFERENCES 1. Pellacani G, Vinceti M, Bassoli S, et al. Reflectance confocal microscopy and features of melanocytic lesions: an internet-based study of the reproducibility of terminology. Arch Dermatol 2009;145(10):1137–43. Available at: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/ elink.fcgi?dbfrom5pubmed&id519841401&retmode 5ref&cmd5prlinks. 2. Segura S, Puig S, Carrera C, et al. Development of a two-step method for the diagnosis of melanoma by reflectance confocal microscopy. J Am Acad Dermatol 2009;61(2):216–29. Available at: http://eutils.ncbi.nlm. nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed&id5 19406506&retmode5ref&cmd5prlinks. 3. Pellacani G, Longo C, Malvehy J, et al. In vivo confocal microscopic and histopathologic correlations of dermoscopic features in 202 melanocytic lesions. Arch Dermatol 2008;144(12):1597–608. Available at: http://eutils.ncbi.nlm.nih.gov/entrez/ eutils/elink.fcgi?dbfrom5pubmed&id519075142& retmode5ref&cmd5prlinks. 4. Huzaira M, Rius F, Rajadhyaksha M, et al. Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol 2001;116(6):846–52. 5. Leachman SA, Cassidy PB, Chen SC, et al. Methods of Melanoma Detection. Cancer Treat Res 2016;167: 51–105.
6. Casari A, Pellacani G, Seidenari S, et al. Pigmented nodular Basal cell carcinomas in differential diagnosis with nodular melanomas: confocal microscopy as a reliable tool for in vivo histologic diagnosis. J Skin Cancer 2011;2011:406859. Available at: http://www. ncbi.nlm.nih.gov/pubmed/21151507/nhttp://www.ncbi. nlm.nih.gov/pmc/articles/PMC2989703/pdf/JSC2011406859.pdf. 7. Alarcon I, Brito J, Alos L, et al. In vivo characterization of solitary angiokeratoma by reflectance confocal microscopy and high definition optical coherence tomography. J Am Acad Dermatol 2015; 72(1):S43–4. Available at: http://linkinghub.elsevier. com/retrieve/pii/S0190962214015618. 8. Gil I, Segura S, Martı´nez-Escala E, et al. Dermoscopic and reflectance confocal microscopic features of exogenous ochronosis. Arch Dermatol 2010; 146(9):1021–5. Available at: http://eutils.ncbi.nlm. nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed&id 520855704&retmode5ref&cmd5prlinks. 9. Guida S, Pellacani G, Cesinaro AM, et al. Spitz naevi and melanomas with similar dermoscopic pattern: can confocal microscopy differentiate? Br J Dermatol 2015;174(3):610–6. Available at: http://doi.wiley.com/ 10.1111/bjd.14286papers3://publication/doi/10.1111/ bjd.14286. 10. Pupelli G, Longo C, Veneziano L, et al. Small-diameter melanocytic lesions: morphological analysis by means of in vivo confocal microscopy. Br J Dermatol 2013;168(5):1027–33. Available at: http://doi.wiley. com/10.1111/bjd.12212. 11. Puig S, Di Giacomo T, Serra D, et al. Reflectance confocal microscopy of blue nevus. Eur J Dermatol 2012;22(4):552–3. 12. Collgros H, Vicente A, Dı´az AM, et al. Agminated cellular blue naevi of the penis: dermoscopic, confocal and histopathological correlation of two cases. Clin Exp Dermatol 2016. Available at: http:// doi.wiley.com/10.1111/ced.12798. 13. Ahluwalia J, Hadjicharalambous E, Mehregan D. Basal cell carcinoma in skin of color. J Drugs Dermatol 2012;11(4):484–6. Available at: http://eutils.ncbi. nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed& id522453586&retmode5ref&cmd5prlinks. 14. Argenziano G, Soyer HP, Chimenti S, et al. Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet. J Am Acad Dermatol 2003;48(5):679–93. Available at: http://eutils. ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom5pub med&id512734496&retmode5ref&cmd5prlinks. 15. Chuah SY, Tee SI, Tan WP, et al. Reflectance confocal microscopy is a useful non-invasive tool in the in vivo diagnosis of pigmented basal cell carcinoma in Asians. Australas J Dermatol 2015. [Epub ahead of print].
441
442
Sarda´ et al 16. Cuevas J, Goldgeier M, Nori S, et al. Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study. J Am Acad Dermatol 2004; 51(6):923–30. 17. Segura S, Puig S, Carrera C, et al. Dendritic cells in pigmented basal cell carcinoma: a relevant finding
by reflectance-mode confocal microscopy. Arch Dermatol 2007;143(7):883–6. 18. Garcı´a-Martı´nez P, Lo´pez Aventı´n D, Segura S, et al. In vivo reflectance confocal microscopy characterization of silver deposits in localized cutaneous argyria. Br J Dermatol 2016. Available at: http://doi. wiley.com/10.1111/bjd.14571.
KEY POINTS Black color in melanoma is associated with melanin in upper epidermis. Under reflectance confocal microscopy (RCM), multiple bright atypical pagetoid cells or atypical cobblestone are visible in upper layers of the epidermis. Blue color in dermoscopy corresponds with pigment deep in the dermis. Blue color may correspond with bright nests or sheets of atypical cells in melanoma, spindle cells in blue nevus, or a tumor island wrapped by dendrites in pigmented basal cell carcinomas. Exogenous pigmentation can also be evaluated by RCM.
In vivo reflectance confocal microscopy (RCM) is a noninvasive imaging technique1–3 that provides images of the superficial layers of the skin at nearly histologic resolution generated thanks to backscatter of light by several structures. Melanosomes, which are highly reflective,4 are among the structures that generate white images in RCM. Thus, clinically dark lesions are easily visible by RCM because of melanin. In the RCM, light penetrates to a subsurface focus of the laser and reflects back from that focus out of the skin and into a detector in the microscope. Light that reflects superficial to the focus is eliminated, so if the focus is too deep, there will be no signal. For the same reason, if much melanin exists in superficial layers, like in black lamella (Fig. 1), no signal is generated from the deep epidermis or dermoepidermal layer. RCM can also detect pagetoid melanocytes in the epidermis, which correlate with black dots or dark blotches in dermoscopy.
The depth penetration of RCM is limited to 100 to 200 mm in human skin at the 830 nm laser wavelength used in the commercial system. The variability in the imaging penetration depth also depends on natural variations in the concentration of other reflective components comprising skin, such as keratin or collagen in addition to melanin.5 RCM criteria found in more superficial anatomic layers may be more readily identified, because there is decay in laser light intensity with increasing imaging depth and hence decrease in optical resolution. Because of the limitation in the penetration, those dark blue lesions, in which melanin is deep in dermis, are not always visible by RCM.6 Finally, some lesions can be clinically dark owing to substances or structures other than melanin structures, such us hemoglobin7 or exogenous materials.8 According to the reflective properties of the material, the images generated will be different. In the present article, the RCM criteria of several dark lesions are reviewed (Table 1).
a Melanoma Unit, Dermatology Department, Hospital Clinic Barcelona, IDIBAPS, University of Barcelona, Villarroel 170, Barcelona 08036, Spain; b CIBER de Enfermedades Raras, Barcelona, Spain * Corresponding author. Dermatology Department, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain. E-mail addresses: [email protected]; [email protected]
Dermatol Clin 34 (2016) 431–442 http://dx.doi.org/10.1016/j.det.2016.05.006 0733-8635/16/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved.
derm.theclinics.com
INTRODUCTION
432
Sarda´ et al
Fig. 1. Black melanoma. (A) Clinical image of a small melanoma on the knee of a woman in her 60s with personal and familial history of melanoma and carrier of a G101W mutation in CDKN2A. (B) Under dermoscopy, the lesion is showing a multicomponent pattern with presence of peripheral pigment network, focal globules, and central blotch. Predominant colors are black and blue. (C) Reflectance confocal microscopy (RCM) only reaches superficial layers of the epidermis showing a typical cobblestone pattern and presence of keratin. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with some nests and focal pagetoid growing. Hyperkeratosis with presence of pigmented corneocytes correlates with the typical cobblestone pattern seen in RCM.
Table 1 Most common cutaneous dark lesions with reported criteria with reflectance confocal microscopy Melanocytic
Non Melanocytic
Benign
Black nevus Blue nevus Spitz
Malignant
Melanoma Seborrhoeic-like melanoma Malignant blue nevus Blue nevus like cutaneous metastasis
Seborrheic keratosis Ink spot lentigo Angioma Angiokeratoma Pilomatricoma Ocronosis, argiria Tattoos Basal cell carcinoma
Confocal Microscopy in Dark Pigmented Lesions
Fig. 2. Black and blue melanoma. (A) Clinical image of a dark brown–blue lesion located on the trunk. (B) Dermoscopy shows an asymmetric lesion with multicomponent pattern with atypical pigment network, black dots/ globules and blue-gray dots. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing the presence of bright atypical cells (roundish and large) and the presence of atypical cobblestone pattern. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing.
DARK MELANOCYTIC LESIONS Even though most melanocytic lesions are brown, from light brown in fair skin to dark brown in dark skin, the presence of black color is less frequent and associated to specific diagnosis: melanoma, pigmented Spitz/Reed nevus, and black nevus (nevus with black lamella in dark skinned patients or after exposure to ultraviolet light). We also consider dark color the presence of dark blue, again, in the context of melanocytic lesions associated to few diagnosis, melanoma and blue nevus. Black color in melanoma: The presence of black color in melanoma is associated to the presence of melanin in upper epidermis, under RCM multiple bright atypical pagetoid cells (Figs. 2 and 3) are visible in upper
layers of the epidermis. Occasionally, in lesions with hyperkeratosis and transepidermal elimination of pigment in keratinocytes, a bright cobblestone is visible obscuring the dermoepidermal layers (see Fig. 3). If melanin is in atypical melanocytes in upper epidermis, then atypical cobblestone is present (with larger cells and the presence of large nuclei; Fig. 4), but if melanin is only present in keratinocytes of the upper layers of the epidermis, typical cobblestone can be present (see Fig. 1), obscuring deeper layers of the epidermis. In those cases, if we cannot see the dermoepidermal junction, melanoma cannot be ruled out by RCM. Blue color in melanoma: The presence of dark blue color in melanoma is associated with the presence of deeply located pigmented tumor.
433
434
Sarda´ et al
Fig. 3. Spitzoid melanoma. (A) Dark palpable fast growing lesion on the leg of a male in his 40s. (B) Under dermoscopy, the lesion is showing a star burst pattern with peripheral streaks and paracentral dark blotch and blue white area. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing pleomorphic pagetoid cells (roundish and dendritic) and an atypical cobblestone pattern with the presence of small round bright nucleated cells. (D) RCM of the dermoepidermal junction showing nonedge papilla, atypical basal cells and the obscuring of the basal layer.
If ortokeratosis is also present, then a whitish color is overlying the deep blue (Figs. 5 and 6). Under RCM, large bright nests (see Fig. 4) or sheets of atypical cells in the dermoepidermal junction (see Fig. 6) can be seen. Dark pigmented Spitz/Reed nevus. Starburst pattern in dermoscopy (see Fig. 3; Fig. 7) can be present in Reed/pigmented Spitz nevus but also in melanoma. Recently, Guida and coworkers9 analyzed the RCM findings present in both entities to identify those criteria that can be used to differentiate between them. In this study, striking cell pleomorphism within epidermis, widespread atypical cells at the dermoepidermal junction (see Fig. 3) and marked pleomorphism within nests were significantly associated with the diagnosis of malignant melanoma, whereas spindled cell (Fig. 8) and peripheral clefting were exclusively found and pathognomonic of spitz nevi (SNs).
Black nevus: In these nevi, melanin is present in keratinocytes of the upper layers of the epidermis showing in RCM typical cobblestone. In the dermoepidermal layers, if visible, a ringed pattern is present. In some cases, after exposure to ultraviolet radiation, many dendritic cells can be visible in upper epidermis (see Fig. 8). These lesions can show some of the criteria previously described in small melanomas.10 Blue nevi are characterized by the presence of dendritic melanocytes in dermis that produce melanin, even deep in the dermis. In some cases, a superficial component is present being the lesions dark blue. Under dermoscopy, blue homogeneous color without any other structures is present but, when cross-polarized dermoscopy is used, the blue can be less homogeneous and some irregularities visible inside. In those blue nevi with a preserved papillary dermis,
Confocal Microscopy in Dark Pigmented Lesions
Fig. 4. Superficial spreading melanoma with a black blotch. (A) Dark, slightly palpable lesion on the trunk of an elderly man. (B) Under dermoscopy the lesion is showing a multicomponent pattern with atypical pigment network, focal presence of dark brown/black dots, dark blotch and gray dots. (C) Reflectance confocal microscopy (RCM) of the upper epidermis is showing a warty architecture with the presence of bright cords separated by black holes (sulci). The epidermal pattern is atypical cobblestone with the presence of roundish atypical cells. (D) Hematoxylin and eosin staining (original magnification, 20) showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing and large atypical nests in the junction.
RCM is not able to reach the lesion, but, if some nests are present at the papillary dermis, RCM shows a characteristic pattern11,12 with nests of bright dendritic cells (Fig. 9).
DARK NONMELANOCYTIC LESIONS Basal Cell Carcinoma Pigmented basal cell carcinoma may clinically mimic melanoma (Fig. 10). In a dark-skinned population,13 pigmented basal cell carcinoma is more frequent than nonpigmented basal cell carcinoma, and may present diagnostic difficulties. Dermoscopy is an easy, noninvasive technique that increases the specificity in the diagnosis of
both tumors14; however, RCM adds valuable criteria for the diagnosis of pigmented basal cell carcinoma,6,15,16 with the presence of tumor islands wrapped by bright dendrites and surrounded by clefting. These structures are visible at the dermoepidermal layer or papillary dermis. If the examination with RCM is too superficial, the presence of multiple dendritic cells in the upper layers of the epidermis may induce a misdiagnosis with melanoma. Segura and colleagues17 demonstrated that dendritic cells in the upper layers of the epidermis in pigmented basal cell carcinomas are Langerhans cells, whereas dendrites in the tumor island correspond with melanocytes colonizing the tumor cords or nests.
435
436
Fig. 5. Melanoma with blue whitish veil. (A) Clinical image of an asymmetric slightly palpable dark lesion. (B) Dermoscopy shows a melanocytic lesion with a multicomponent pattern, with atypical pigment network, streaks, and blue whitish veil. (C) Reflectance confocal microscopy of the dermoepidermal layer is showing nonedged papilla, atypical nests of large cells and atypical cells in the basal layer. (D) Hematoxylin and eosin staining (original magnification, 20) in the blue whitish area showing an atypical proliferation of very pigmented melanocytes in the dermis with orthokeratotic hyperkeratosis (the histopathologic background of the blue whitish veil).
Confocal Microscopy in Dark Pigmented Lesions
Fig. 7. Pigmented Spitz/Reed nevus. (A) Clinical image of a symmetric, elevated, and pigmented new lesion on the limb of a young man. (B) Dermoscopy shows a star burst pattern with peripheral pseudopods and projections, black dots and globules, atypical pigment network, black blotch and blue hue. (C) Reflectance confocal microscopy mosaic at upper epidermal layers showing a cobblestone pattern with presence of black holes. A detail of this mosaic is shown in the right lower corner showing an atypical cobblestone, dendritic pagetoid cells and small suprabasal nests at the periphery of the lesion. (D) Reflectance confocal microscopy mosaic at the dermoepidermal junction showing visible papilla, atypical bright cells, multiple nests and plump cells in papillary dermis.
Seborrheic Keratosis Pigmented seborrheic keratosis can be also difficult to differentiate from melanoma. Using dermoscopy as an additional tool, RCM also offers valuable criteria for the correct diagnosis. Under
RCM, a typical cobblestone in upper layers of the epidermis (Fig. 11) is shown together with the presence of follicular openings (black holes), follicular plugs (concentric bright keratin) and cysts (bright roundish structures) helps in the differential diagnosis.
= Fig. 6. (A) Clinical image of an elevated and ulcerated tumor with recent bleeding. (B) Dermoscopy of the nonulcerated area is showing blue whitish veil. (C) Reflectance confocal microscopy of the nonulcerated area is showing a sheet of atypical cells. (D) Hematoxylin and eosin staining (original magnification, 20) corresponding with the peripheral area of the tumor, showing an atypical proliferation of melanocytes in the dermoepidermal junction with pagetoid growing and presence of pigment in dermis and overlying orthokeratotic hyperkeratosis (the histopathological background of the blue whitish veil).
437
438
Fig. 8. Black nevus. (A) Dermoscopy of a 2-mm diameter lesion that appeared recently, after exposure to ultraviolet light, showing an atypical pigment network. (B) Reflectance confocal microscopy of upper epidermis is showing a pleomorphic proliferation of atypical cells, roundish and dendritic. (C) Hematoxylin and eosin staining (original magnification, 20) corresponding with an atypical proliferation of melanocytes in the dermoepidermal junction with heavily pigmented nests and inflammation in dermis.
Confocal Microscopy in Dark Pigmented Lesions
Fig. 10. Pigmented basal cell carcinoma. (A) Clinical image of a 4-cm black lesion on the lumbar area. (B) Dermoscopy shows a central area with blue whitish veil and some black globules and few erosions, and at the periphery the presence of maple leaf like areas (close to the B). (C) Reflectance confocal microscopy at the dermoepidermal junction shoving tumor cords wrapped with bright dendrites, palisading, peripheral clefting and presence of plump cells (inflammatory cells and melanophages) in the dermis. (D) Basaloid tumor island in superficial dermis showing palisading and clefting.
Ink Spot Lentigo In these dark lesions, RCM shows a cobblestone pattern in the epidermis and a ringed pattern in the dermoepidermal junction.
highly reflective superficial scale with abnormal architecture at the dermoepidermal junction that displays epidermal cells forming septa surrounding the prominent ecstatic vascular lacunae (Fig. 12).
EXOGENOUS PIGMENTATION Angiokeratomas and Thrombosed Hemangiomas In these lesions, the dark color is not owing to the presence of melanin, but to the presence of blood in dermis and superficial hyperkeratosis. On RCM,7 this lesion shows at the epidermal level
Ochronosis was the first exogenous pigmentation described with RCM8 showing dark curved silhouettes corresponding to the classical banana bodies described in histopathology. Later, silver deposits18 were also characterized by RCM.
= Fig. 9. Blue nevus. (A) Clinical image of a nodular lesion on the abdomen. (B) Blue homogeneous pattern under dermoscopy. (C) Reflectance confocal microscopy is showing a nested proliferation of dendritic bright melanocytes in dermal papilla. (D) Hematoxylin and eosin staining (original magnification, 40) showing a proliferation of dendritic and pigmented melanocytes in the dermal papilla.
439
440
Fig. 11. Clonal seborrheic keratosis. (A) Clinical image of a large pigmented lesion on the scalp of a man in his 60s. (B) Dermoscopy of 1 area of the lesion is showing homogeneous pigmentation with brown, blue, and black coloration. (C) Reflectance confocal microscopy is showing in a mosaic at upper epidermis the presence of a typical cobblestone interrupted by multiple follicular openings. (D) Hematoxylin and eosin staining (original magnification, 20) showing a proliferation of deep pigmented keratinocytes.
Fig. 12. Angiokeratoma. (A) Clinical image of a dark tumor in an acral site. (B) Under dermoscopy, the lesion shows black and blue color with the presence of a crust. (C) Reflectance confocal microscopy shows a typical honeycomb in the epidermis with the presence of ecstatic vascular structures in papillary dermis. (D) Hematoxylin and eosin staining (original magnification, 20) showing the prominent ecstatic vascular lacunae.
Confocal Microscopy in Dark Pigmented Lesions SUMMARY RCM criteria considered by users as more relevant for melanoma diagnosis may be frequently detected in dark lesions; most dark lesions present melanin that enhances the brightness of confocal criteria. One of the difficulties in the diagnosis of deep pigmented melanomas could be the presence of typical cobblestone pattern in upper epidermis owing to the presence of melanin in keratinocytes that obscure the dermoepidermal layer because decay in laser light intensity. On RCM, in blue nevi, bright dendritic cells can be visible at the papillary dermis. Pigmented basal cell carcinoma can be recognized easily on RCM if the papillary dermis is reached. There, tumor islands or cords with bright dendrites are recognized easily. If the RCM imaging is too superficial and the tumor in dermis is not reached, the presence of dendritic cells in the epidermis can be misleading towards a diagnosis of melanoma instead of pigmented basal cell carcinoma.
REFERENCES 1. Pellacani G, Vinceti M, Bassoli S, et al. Reflectance confocal microscopy and features of melanocytic lesions: an internet-based study of the reproducibility of terminology. Arch Dermatol 2009;145(10):1137–43. Available at: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/ elink.fcgi?dbfrom5pubmed&id519841401&retmode 5ref&cmd5prlinks. 2. Segura S, Puig S, Carrera C, et al. Development of a two-step method for the diagnosis of melanoma by reflectance confocal microscopy. J Am Acad Dermatol 2009;61(2):216–29. Available at: http://eutils.ncbi.nlm. nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed&id5 19406506&retmode5ref&cmd5prlinks. 3. Pellacani G, Longo C, Malvehy J, et al. In vivo confocal microscopic and histopathologic correlations of dermoscopic features in 202 melanocytic lesions. Arch Dermatol 2008;144(12):1597–608. Available at: http://eutils.ncbi.nlm.nih.gov/entrez/ eutils/elink.fcgi?dbfrom5pubmed&id519075142& retmode5ref&cmd5prlinks. 4. Huzaira M, Rius F, Rajadhyaksha M, et al. Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol 2001;116(6):846–52. 5. Leachman SA, Cassidy PB, Chen SC, et al. Methods of Melanoma Detection. Cancer Treat Res 2016;167: 51–105.
6. Casari A, Pellacani G, Seidenari S, et al. Pigmented nodular Basal cell carcinomas in differential diagnosis with nodular melanomas: confocal microscopy as a reliable tool for in vivo histologic diagnosis. J Skin Cancer 2011;2011:406859. Available at: http://www. ncbi.nlm.nih.gov/pubmed/21151507/nhttp://www.ncbi. nlm.nih.gov/pmc/articles/PMC2989703/pdf/JSC2011406859.pdf. 7. Alarcon I, Brito J, Alos L, et al. In vivo characterization of solitary angiokeratoma by reflectance confocal microscopy and high definition optical coherence tomography. J Am Acad Dermatol 2015; 72(1):S43–4. Available at: http://linkinghub.elsevier. com/retrieve/pii/S0190962214015618. 8. Gil I, Segura S, Martı´nez-Escala E, et al. Dermoscopic and reflectance confocal microscopic features of exogenous ochronosis. Arch Dermatol 2010; 146(9):1021–5. Available at: http://eutils.ncbi.nlm. nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed&id 520855704&retmode5ref&cmd5prlinks. 9. Guida S, Pellacani G, Cesinaro AM, et al. Spitz naevi and melanomas with similar dermoscopic pattern: can confocal microscopy differentiate? Br J Dermatol 2015;174(3):610–6. Available at: http://doi.wiley.com/ 10.1111/bjd.14286papers3://publication/doi/10.1111/ bjd.14286. 10. Pupelli G, Longo C, Veneziano L, et al. Small-diameter melanocytic lesions: morphological analysis by means of in vivo confocal microscopy. Br J Dermatol 2013;168(5):1027–33. Available at: http://doi.wiley. com/10.1111/bjd.12212. 11. Puig S, Di Giacomo T, Serra D, et al. Reflectance confocal microscopy of blue nevus. Eur J Dermatol 2012;22(4):552–3. 12. Collgros H, Vicente A, Dı´az AM, et al. Agminated cellular blue naevi of the penis: dermoscopic, confocal and histopathological correlation of two cases. Clin Exp Dermatol 2016. Available at: http:// doi.wiley.com/10.1111/ced.12798. 13. Ahluwalia J, Hadjicharalambous E, Mehregan D. Basal cell carcinoma in skin of color. J Drugs Dermatol 2012;11(4):484–6. Available at: http://eutils.ncbi. nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom5pubmed& id522453586&retmode5ref&cmd5prlinks. 14. Argenziano G, Soyer HP, Chimenti S, et al. Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet. J Am Acad Dermatol 2003;48(5):679–93. Available at: http://eutils. ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom5pub med&id512734496&retmode5ref&cmd5prlinks. 15. Chuah SY, Tee SI, Tan WP, et al. Reflectance confocal microscopy is a useful non-invasive tool in the in vivo diagnosis of pigmented basal cell carcinoma in Asians. Australas J Dermatol 2015. [Epub ahead of print].
441
442
Sarda´ et al 16. Cuevas J, Goldgeier M, Nori S, et al. Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study. J Am Acad Dermatol 2004; 51(6):923–30. 17. Segura S, Puig S, Carrera C, et al. Dendritic cells in pigmented basal cell carcinoma: a relevant finding
by reflectance-mode confocal microscopy. Arch Dermatol 2007;143(7):883–6. 18. Garcı´a-Martı´nez P, Lo´pez Aventı´n D, Segura S, et al. In vivo reflectance confocal microscopy characterization of silver deposits in localized cutaneous argyria. Br J Dermatol 2016. Available at: http://doi. wiley.com/10.1111/bjd.14571.