Dermatology: Practical and Conceptual Observation | Dermatol Pract Concept 2017;7(4):4 13 DERMATOLOGY PRACTICAL & CONCEPTUAL www.derm101.com In vivo intraoral reflectance confocal microscopy of an amalgam tattoo Oriol Yélamos1,2, Miguel Cordova1 , Gary Peterson1, Melissa P. Pulitzer3, Bhuvanesh Singh4, Milind Rajadhyaksha1, Jennifer L. DeFazio5 1 Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA 2 Dermatology Department, Hospital Clínic, Universitat de Barcelona, Barcelona, Spain 3 Pathology Department, Memorial Sloan Kettering Cancer Center, New York, NY, USA 4 Head and Neck Cancer Center, Memorial Sloan Kettering Cancer Center, Hauppauge, NY, USA 5 Dermatology Service, Memorial Sloan Kettering Cancer Center, Hauppauge, NY, USA Key words: reflectance confocal microscopy, amalgam tattoo, melanoma, oral, mucosa Citation: Yélamos O, Cordova M, Peterson G, Pukitzer MP, Singh B, Rajadhyaksha M, DeFazio JL. In vivo intraoral reflectance confocal microscopy of an amalgam tattoo. Dermatol Pract Concept 2017;7(4):13-16. DOI: https://doi.org/10.5826/dpc.0704a04 Received: July 11, 2017; Accepted: August 10, 2017; Published: October 31, 2017 Copyright: ©2017 Yélamos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded in part by NIH/NCI grant R01CA156773, NIH/NCI grant R01CA199673, NIH/NIBIB grant R01EB020029, the NIH/NCI Cancer Center Support Grant P30 CA008748 and the Beca Excelencia Fundación Piel Sana. Competing interests/Disclosures: Milind Rajadhyaksha is a former employee of and owns equity in Caliber Imaging and Diagnostics (formerly Lucid Inc.), the company that manufactures and sells the Vivascope confocal microscope. The Vivascope is the commercial version of an original laboratory prototype that he had developed at Massachusetts General Hospital, Harvard Medical School. The other authors have no disclosures or conflicts of interest to report. All authors have contributed significantly to this publication. Corresponding author: Jennifer DeFazio, MD, Dermatology Service, Memorial Sloan Kettering Cancer Center, 800 Veterans Highway, Hauppauge, NY, USA. Tel. +1 631-863-5118. Email: DeFazioJ@mskcc.org Introduction The majority of oral pigmentations are benign lesions such as nevi, melanotic macules, melanoacanthomas or amalgam tat- toos [1,2]. Conversely, mucosal melanomas are rare, but often lethal [2]. Reflectance confocal microscopy (RCM) allows imaging with cellular resolution and has excellent diagnostic accuracy to diagnose cutaneous melanoma [3]. However, RCM can be challenging to perform in the oropharynx using the current probes. The majority of oral pigmentations are benign lesions such as nevi, melanotic macules, melanoacan- thomas or amalgam tattoos. Conversely, mucosal melanomas are rare but often lethal; therefore, ex- cluding oral melanomas in this setting is crucial. Reflectance confocal microscopy is a non-invasive, in vivo imaging system with cellular resolution that has been used to distinguish benign from malignant pigmented lesions in the skin, and more recently in the mucosa. However, lesions located posteriorly in the oral cavity are difficult to assess visually and difficult to biopsy due to their location. Herein we present a patient with previous multiple melanomas presenting with an oral amalgam tattoo in the buccal mucosa, which was imaged using an intraoral telescopic probe attached to a commercially available handheld RCM. In this case report we describe this novel probe, the first RCM description of an amalgam tattoo and we discuss its differences with the findings described in oral melanomas. ABSTRACT mailto:DeFazioJ@mskcc.org 14 Observation | Dermatol Pract Concept 2017;7(4):4 During imaging, the patient was awake and tolerated the procedure well. Superficially, RCM showed an overall normal epithelium with focal areas of epithelial disarray (Figure 2a, b). Deeper, we identified increased vascularity (Figure 2c) and numerous large dendritic cells admixed with plump cells and bright dots (Figure 2d). In light of her past medical history, the lesion was biopsied to exclude a primary or metastatic mela- noma. Histopathologic analysis revealed fine black granular pigment within the dermis suggestive of an amalgam tattoo (Figure 2e). Conclusions In the last decade, RCM imaging has expanded its use beyond the skin and has been applied to the oral and geni- tal mucosa, specifically to distinguish mucosal melanomas from benign lesions [1,6,7]. Indeed, RCM features suggest- ing mucosal melanomas include suprabasal dendritic or large round cells, dendritic cells in the epithelial-connective tissue junction, and epithelial disarray [1,6,7]. In our case, RCM showed numerous suprabasilar dendritic cells along with epithelial disarray. However, these findings occurred focally, and we also noted numerous bright dots and plump cells suggesting a reactive lymphohistiocytic infiltrate. To better characterize these findings, immunohistochemical stains for melanocytes and Langerhans cells were per- formed. These showed normal numbers of melanocytes within the basal and suprabasilar epithelium (Figure 2f) Herein we present a patient with previous multiple mela- nomas presenting with an oral amalgam tattoo in the buccal mucosa, which was imaged using a novel intraoral telescopic probe attached to a commercially available handheld RCM. Case A woman in her 70s was referred by her dentist for a pig- mented lesion on the oral mucosa. She had a history of four cutaneous melanomas —three in situ and one invasive (Bres- low 0.25 mm)— excised four years prior. At physical exami- nation, she presented with a 3 mm asymptomatic bluish pap- ule on the left buccal mucosa (Figure 1a, asterisk). To evaluate this location, a handheld RCM (Vivascope3000, Caliber ID, Rochester, NY) fitted with a telescopic probe was used (Figure 1b). The probe was designed to be sufficiently small (12 mm) and long (~150 mm) to allow access inside the oral cavity [4]. The probe consists of a telescope and an objective lens of numerical aperture 0.7, providing a ~0.75 x 0.75 mm field of view, ~4 μm optical sectioning and ~1 μm lateral resolu- tion, allowing imaging to a depth of ~300 μm. Enclosing the lens there is a cap with a coverslip, which provides contact to the mucosa and keeps the tissue gently flattened and still during the imaging procedure (Figure 1b, arrowheads). Two caps are used: a shorter cap allows for imaging in the deeper epithelium/mucosal-submucosal junction and a longer cap in the superficial lamina propria. We acquired images and videos that were converted into videomosaics [5]. Figure 1. (a) Clinical image of pigmented lesion located on the left buccal mucosa (asterisk). Note the presence of a dental filling in the third left lower molar. (b) This lesion was imaged with a telescopic probe attached to a handheld reflectance confocal microscope. Attached to the probe is a small objective lens, which is enclosed in a small cap with a coverslip (arrowhead). The approach of using caps with different lengths allows for imaging at different depths in oral tissue. [Copyright: ©2017 Yélamos et al.] Observation | Dermatol Pract Concept 2017;7(4):4 15 No previous studies have reported the RCM findings of amal- gam tattoos. Although the presence of amalgam granules may not be visible with RCM since they are located deeper than 200-300 μm, the presence of bright dots (lymphocytes) and plump cells (macrophages), with suprabasal dendritic cells, is suggestive of a reactive process, such as an amalgam tattoo. To conclude, we have presented the first case of amal- gam tattoo imaged with RCM using a new telescopic probe. and numerous Langerhans cells extending into the upper epithelium (Figure 2g). Langerhans cells are difficult to distinguish from mela- nocytes on RCM [8], and have a low specificity on the oral mucosa since they occur in normal mucosa and in reactive processes such as amalgam tattoos [1]. Histologically amal- gam tattoos reveal small granules deposited between the col- lagen fibers and can present with a foreign-body reaction [2]. Figure 2. Reflectance confocal microscopy images (panels a – d) and its histopathologic correlates (panels e – f). Superficial confocal video- mosaic showing normal epithelial cells with prominent nucleoli (panel a, white rectangle and inset), and a focal area of epithelial disarray (panel b). Confocal videomosaic obtained at the epithelial junction showing increased vascularity (arrowheads, panel c) and an area with numerous large atypical dendritic cells (panel d). Hematoxylin and eosin stain of the lesion showed fine black granular pigment within the stroma in the dermis (panel e, original magnification x 40). Immunohistochemical stain for A103 showed scattered melanocytes in the basal layer and in the epidermis (panel g, original magnification x 20). Immunohistochemical stain for CD1a highlighted numerous Langerhans cells throughout the epidermis (panel f, original magnification x 20). [Copyright: ©2017 Yélamos et al.] 16 Observation | Dermatol Pract Concept 2017;7(4):4 3. Pellacani G, Guitera P, Longo C, Avramidis M, Seidenari S, Men- zies S. The impact of in vivo reflectance confocal microscopy for the diagnostic accuracy of melanoma and equivocal melanocytic lesions. J Invest Dermatol. 2007;127(12):2759-2765. 4. Peterson G, Zanoni DK, Migliacci J, Cordova M, Rajadhyaksha M, Patel S. Progress in reflectance confocal microscopy for im- aging oral tissues in vivo. Proceedings of SPIE Photonics West. 2016;9689. 5. Kose K, Cordova M, Duffy M, Flores ES, Brooks DH, Rajad- hyaksha M. Video-mosaicing of reflectance confocal images for examination of extended areas of skin in vivo. Br J Dermatol. 2014;171(5):1239-1241. 6. Debarbieux S, Perrot JL, Erfan N, et al. Reflectance confocal mi- croscopy of mucosal pigmented macules: a review of 56 cases in- cluding 10 macular melanomas. Br J Dermatol. 2014;170(6):1276- 1284. 7. Uribe P, Collgros H, Scolyer RA, Menzies SW, Guitera P. In vivo reflectance confocal microscopy for the diagnosis of melanoma and melanotic macules of the lip. JAMA Dermatol. 2017. 8. Hashemi P, Pulitzer MP, Scope A, Kovalyshyn I, Halpern AC, Marghoob AA. Langerhans cells and melanocytes share similar morphologic features under in vivo reflectance confocal micros- copy: a challenge for melanoma diagnosis. J Am Acad Dermatol. 2012;66(3):452-462. Although the presence of epidermal disarray and suprabasilar dendritic cells on RCM was suggestive of melanoma, the coexisting presence of bright dots and plump cells brings into consideration the differential diagnosis a reactive process such as an amalgam tattoo. However, since the RCM features of mucosal melanomas and other mucosal conditions are limited, larger studies are needed to increase the meaning of using this new probe with high-resolution images. Acknowledgements We would like to thank Dr. Marco Ardigò for his thoughtful feedback regarding this case. References 1. Maher NG, Solinas A, Scolyer RA, Guitera P. In vivo reflectance confocal microscopy for evaluating melanoma of the lip and its dif- ferential diagnoses. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;123(1):84-94. 2. Lundin K, Schmidt G, Bonde C. Amalgam tattoo mimicking mu- cosal melanoma: a diagnostic dilemma revisited. Case Rep Dent. 2013;2013:787294.