Dermatology: Practical and Conceptual Research Letter | Dermatol Pract Concept. 2023;13(1):e2023055 1 Combined Use of Dermoscopy, Reflectance Confocal Microscopy and Ex-Vivo Gene Expression Profiling to Detect a Micro-Melanoma Less Than 1 mm in Diameter Alexander Witkowski1, Joanna Ludzik1,2, Jina Chung2, Claudia Lee2,3, Sancy Leachman2, Giovanni Pellacani4 1 Department of Dermatology, Oregon Health and Sciences University, Portland, Oregon 2 Department of Telemedicine and Bioinformatics, Jagiellonian University Medical College, Krakow, Poland 3 School of Medicine, University of California Riverside, Riverside, California 4 Department of Dermatology, La Sapienza University, Rome, Italy Key words: reflectance confocal microscopy, dermoscopy, gene expression profiling, micro-melanoma, melanoma, skin cancer, early detection Citation: Witkowski AM, Ludzik J, Chung J, Lee C, Leachman S, Pellacani G. Combined use of dermoscopy, reflectance confocal microscopy and ex-vivo gene expression profiling to detect a micro-melanoma less than 1 mm in diameter. Dermatol Pract Concept. 2023;13(1):e2023055. DOI: https://doi.org/10.5826/dpc.1301a55 Accepted: June 3, 2022; Published: January 2023 Copyright: ©2023 Witkowski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding Author: Claudia Lee, Department of Dermatology, Oregon Health and Sciences University, United States. E-mail: leecla@ ohsu.edu Introduction Micro-melanomas, or melanomas < 2 mm in diameter, are increasingly reported making screening methods like the ABCD(E) acronym outdated. Early detection of melanoma remains the utmost important prognostic factor, therefore understanding how to utilize different diagnostic tools is necessary to optimize detection of melanoma at its earli- est, most treatable stage. Using a combination of imaging and molecular techniques, we detected and confirmed a micro-melanoma in situ measuring 0.65 mm in diameter. Case Presentation A 61-year-old female with no prior history of skin cancer presented for a skin examination. Clinical examination of her right cheek revealed a tiny, hyperpigmented dot not previously noticed by the patient. Dermoscopy showed sun-damaged skin with a dark brown macule and asymmet- ric perifollicular hyperpigmentation. Reflectance confocal microscopy (RCM) revealed dendritic cells concentrated around a single hair follicle, consistent with folliculot- ropism (Figure 1). The final diameter incorporated the 2 Research Letter | Dermatol Pract Concept. 2023;13(1):e2023055 farthest-reaching dendritic cell projections measuring at 0.65 mm. Initial histopathology showed a solar lentigo with- out atypia. Due to discordance with clinical-dermoscopic concern, deeper sections were requested and stained with Melan-A and SOX10 revealing irregular and heterogenous melanocytic nests and single melanocytosis, consistent with melanoma in situ (MIS) (Figure 2). Additionally, the sample underwent a diagnostic 23-gene expression profile (GEP) Figure 1. (A) Clinical image of pigmented skin lesion (arrow). (B) Zoomed digital dermoscopy image of the pigmented skin lesion present- ing with folliculotropism (circle). Sites of control punch biopsies in areas of increased pigmentation (triangles). (C) Reflectance confocal microscopy image (0.75 mm x 0.75 mm, measuring line bottom right corner: 0.25 mm) with presence of folliculotropism of pleomorphic melanocytes. Melanoma in-situ maximal diameter measured: 0.65 mm. Figure 2. Histopathology of the smallest reported melanoma. (A, B) H&E staining (A: 5X and B: 20X) showed an increased density of mostly solitary melanocytes at the dermal-epidermal junction. (C, D) SOX10 immunostaining (C: 5X and D: 20X) showed a continuous proliferation of melanocytes at the dermal-epidermal junction, consistent with melanoma in-situ. Research Letter | Dermatol Pract Concept. 2023;13(1):e2023055 3 test which returned a positive result (suggestive of malig- nancy). National Comprehensive Cancer Network (NCCN) guidelines make note of multiple ancillary diagnostic tests, which includes diagnostic GEP, that can help to differentiate equivocal melanocytic lesions and may be useful in cases of clinical discordance [1]. In the context of these ancillary find- ings final diagnosis of MIS was achieved. For control, four 5x5 mm RCM image sets of the epidermis, dermal- epidermal junction and upper dermis and two 2-mm punch biopsies were completed adjacent to the biopsy site in the most pig- mented areas and no atypical features were found confirm- ing the absence of residual tumor in nearby locations. Conclusions With the widespread adoption of dermoscopy, increasing numbers of micro-melanomas have been identified. Rely- ing on dermoscopy alone, the smallest melanoma recorded measured at 1.3 mm [2] and another case enhanced by total-body imaging system reported a 0.9 mm diameter melanoma [3]. RCM is also a valuable second-level ex- amination tool that combined with digital follow-up fa- cilitates recognition of early-stage disease. Some argue that such small lesions cannot be malignant and investing in its detection is wasteful, however Regio Pereira et al evaluated 86 melanomas <5 mm in diameter which found 44% were invasive melanomas [4] highlighting the impor- tance of investigating suspicious lesions regardless of size. Although our case demonstrated suspicious dermoscopy and RCM findings, initial histopathology reports were benign. Recognizing histopathology has limitations due to sampling bias, potentially missing up to 10% of mel- anomas [5], we requested additional staining and 23-GEP testing that ultimately revealed a diagnosis of MIS with a diameter of 0.65 mm. This case attests to the heightened diagnostic capabilities of multiple imaging and molecular tools when used in conjunction to confirm an early mela- noma, which may result in better cosmetic, financial and patient morbidity. References 1. Fried L, Tan A, Bajaj S, Liebman TN, Polsky D, Stein JA. 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