Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 1 Confocal Assessment of Pigmented-Mucosal Lesions: A Monocentric, Retrospective Evaluation of Lip and Genital Area Valeria Coco1, Simone Cappilli1,2, Alessandro Di Stefani1,2, Costantino Ricci3,4, Francesca Perino1, Lucia Di Nardo1, Caterina Longo5,6, Ketty Peris1,2 1 UOC di Dermatologia, Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Universitario A. Gemelli – IRCCS, Rome, Italy 2 Dermatologia, Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, Rome, Italy 3 Pathology Unit, Maggiore Hospital, AUSL Bologna, Bologna, Italy 4 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), S.Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy 5 Centro Oncologico ad Alta Tecnologia Diagnostica, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia, Italy 6 Department of Dermatology, University of Modena and Reggio Emilia, Modena, Italy Key words: diagnostic imaging, clinical dermatology, reflectance confocal microscopy, malignant melanoma, skin neoplasms Citation: Coco V, Cappilli S, Di Stefani A, et al. Confocal Assessment of Pigmented-Mucosal Lesions: A Monocentric, Retrospective Evaluation of Lip and Genital Area. Dermatol Pract Concept. 2024;14(1):e2024028. DOI: https://doi.org/10.5826/dpc.1401a28 Accepted: July 26, 2023; Published: January 2024 Copyright: ©2024 Coco 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: Simone Cappilli, MD, PhD candidate, UOC di Dermatologia, Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Universitario A. Gemelli - IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy Tel: +39 06-30154227 - Fax: +39 06-30154919 Email: simo.cappilli@gmail.com Introduction: Pigmentation of lip and/or genitalia is mainly due to the development of benign melanotic macules, with a less occurrence of melanocytic and other non-melanocytic lesions. Mucosal melanoma has worse prognosis compared with cutaneous counterpart, hence identification of atypical features for an early diagnosis is crucial. Objectives: The aim of this study was to report further data of confocal features characterizing pigmented mucosal lesions of genital area and of the lips and test the diagnostic role of the reflectance confocal microscopy (RCM)lip score. Methods: Clinical, dermoscopic and RCM images of histologically proven pigmented lesions, involv- ing the genital area (vulva or glans penis) and lip, were retrospectively reviewed. RCM images were evaluated for malignant criteria, and statistical analysis was conducted for categorical variables. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 Results: Seventy pigmented lesions were included in the study and divided into two groups based on the body area location: lip (17) and genital area (53). Architectural disarray (P = 0.002), dendritic (P = 0.031) and roundish cells in epidermis (P < 0.0001), interpapillary dendritic cells (P = 0.039) and junctional atypical cells (P = 0.002) were associated to genital melanoma. Melanoma involving the lip was characterized by roundish cells in epidermis, a criterion found in one labial benign lesion, only (P = 0.005). Main limitations of the study are the inclusion of low melanomas and the presence of epidermal dendritic cells in melanosis and melanoma, as a confusing factor in imaging. Conclusions: Dermatologists should consider confocal microscopy as an adjunctive tool to dermos- copy in the differential diagnosis of pigmented mucosal lesions, especially in presence of clinical and dermoscopic findings suspicious for malignancy. Introduction Pigmentation of lip and/or genitalia is mainly due to the development of benign melanotic macules (also called mel- anoses or mucosal pigmented macules), with a less occur- rence of melanocytic lesions (naevi and melanoma) and other non-melanocytic lesions (non-melanoma skin cancers, inflammatory conditions, infective diseases, or foreign-body pigmentations) [1,2]. Mucosal melanoma has worse progno- sis compared with cutaneous melanoma and lacks effective treatment options, hence identification of atypical features for an early diagnosis is crucial. Dermoscopy has improved diagnostic accuracy of pigmented mucosal lesions (PMLs), indicating multiple colors and structureless areas as strong indicators for malignancy [3,4], supported more recently, by reflectance confocal microscopy (RCM) in the identification of features more associated to mucosal melanoma, like atyp- ical pattern of the epithelium, pagetoid cells and disarranged papillae [2,5-9]. Recently, Uribe et al proposed a RCM lip score that can assist in the differential diagnosis of mela- notic macules and melanoma of the lip. Given the rarity of mucosal melanoma only 23 cases including genital and lip area, have been investigated using RCM, and incisional bi- opsy or surgical excision is still recommended for equivocal lesions [4]. Objectives The aim of this study was to report further data of confocal features characterizing PMLs of genital area and of the lips, and test the diagnostic role of the RCM lip score. Methods Clinical and Imaging Data A consecutive series of histologically proven PMLs, involv- ing the genital area (vulva or glans penis) and lip, collected at Dermatology Unit, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome, within 24 months (March 2016-February 2018) were retrospectively reviewed. Demo- graphic characteristics (patient age, gender, body site) were recorded, and clinical, dermoscopic and RCM images were acquired with digital imaging system (Dermaview DUAL, Tre T Medical snc, Cicciano, Italy; VivaScope® 1500, Cal- iber Inc). RCM mosaics (VivaBlock®, horizontal sections of 8x8 mm) were taken at the level of epidermis, epithelial- chorion junction (ECJ), and upper chorion, with VivaS- tack® (frames taken at incremental depths from epidermis to superficial chorion) acquired in areas of special interest. Two investigators (V.C., F.P.) jointly reviewed RCM images blinded for histopathological diagnosis, evaluating RCM fea- tures based on previously described criteria [1,8,9]. In case of disagreement, a third experienced dermatologist (A.D.S.) was consulted to reach a consensus. Additionally, the RCM lip score proposed by Uribe et al was considered in the eval- uation of pigmented lip lesions [9]. The patients have given written informed consent for their case details. All data were de-identified before use. Statistical Analysis Statistical analysis was performed using specific software (SAS Analytics software) and a descriptive evaluation for categorical variables, expressed as the absolute number of cases and percentage values, was conducted. Dowling-Degos disease was not included in the analysis due to the presence of peculiar and distinct RCM features. Fisher exact test was used for the comparison of different confocal parameters between malignant and benign lesions and their association with histological diagnosis. P value less than 0.05 was con- sidered as statistically significant. Results Seventy PMLs in 68 patients (52 [76.5%] females; mean age 76.5 [13-78] years) were included in the study and di- vided into two groups based on the body area location: lip Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 3 (17  pigmented lip lesions, PLLs) and genital area (53 pig- mented genital lesions, PGLs). Pigmented Genital Lesions Fifty-three PGLs (38/53, 74% in women) were evaluated: 29 melanoses, 18 melanocytic nevi, 3 seborrheic keratoses (SK), 2 mucosal melanomas, 1 Dowling–Degos disease. Architec- tural disarray (P = 0.002), presence of dendritic (P = 0.031) and roundish pagetoid cells in the epidermis (P < 0.0001), interpapillary dendritic cells (P = 0.039) and atypical cells at the DEJ (P = 0.002) were associated to genital melanoma (Table 1). Melanosis occurred on labia minora (15/22, 68%) and labia majora (7/22, 32%) in women while involved glans penis (3/7, 43%), foreskin of the glans (2/7, 29%) or skin (2/7, 29%) in men. Three melanoses had a multi- focal distribution, and 2 melanoses were associated to li- chen sclerosus. With RCM, melanoses displayed a regular honeycomb pattern with sparse dendritic cells in 21% of cases. Ringed pattern and draped pattern with homoge- neous distribution of papillae represented the main fea- tures at epidermal-chorion junction (ECJ) (76% and 55%), along with dendritic cells around papillae (41%), mainly exhibiting a fusiform shape (79%). Junctional atypical cells were seen in 14% lesions (14%) (Figure 1). Melanosis was associated to lichen sclerosus in two patients, reveal- ing in imaging non-specific pattern at epidermal-chorion junction and an inflammatory infiltrate with a prominent vascularization. Mucosal melanoma was localized on labia minora in a 21-year-old girl and on the glans penis in a 36-year-old man (Figures 1 and 2). RCM images showed honeycomb pattern with epidermal disarray and a widespread infiltration of pagetoid roundish and dendritic cells (>10 cells/mm2). At the ECJ a meshwork and ringed pattern co-existed, with atypi- cal peri- and inter-papillary dendritic cells (>10 cells/mm2). Junctional atypical cells (5-10 cells/mm2 and >10 cells/mm2) were detected in both cases along with melanocytic nests in the chorion. Melanocytic lesions included 6/18 (33%) compound nevi, 5/18 (28%) intradermal nevi, 4/18 (22%) AGN and 3/18 (17%) dysplastic nevi. Compound nevi were charac- terized by regular honeycomb pattern and a prevalent ring pattern (66%) at ECJ. Melanocytic nests in superficial cho- rion were observed in all lesions. Intradermal nevi showed a regular honeycomb pattern in the epidermis with a pre- dominance of nonedged, ringed or meshwork pattern at ECJ (75%), and melanocytic nests in superficial chorion. AGN mostly revealed honeycomb pattern with epidermal disarray and a sparse or widespread pagetoid infiltration in superficial layers (75%). Meshwork pattern represented the prevalent criterion (75%) at ECJ, along with peri- and inter-papillary dendritic cells (100% and 75% respectively) (Figure 2). In dysplastic nevi, RCM revealed a regular hon- eycomb pattern in more than half-cases (66%) with sparse dendritic and roundish cells in 33% of cases. DEJ pattern was heterogeneous showing the simultaneous presence of draped and meshwork pattern, or nonspecific pattern. Me- lanocytic nests were seen in the superficial chorion (100%). RCM of pSK showed epidermal bulbous projections and keratin-filled invaginations with plump bright cells and bright horn cysts. Ring pattern edged-papillae character- ized ECJ. Dowling Degos disease exhibited a honeycomb pattern in epidermis, junctional ring pattern or elongated and thick cord-like structures, corresponding to irregular, filiform epi- dermal elongation downward into the upper dermis. Keratin cysts were also seen. Pigmented Labial Lesions Seventeen pigmented labial lesions (PLLs) (14/17, 82% in women) were included in the study: 8 melanoses, 3 basal cell carcinomas (BCCs), 2 actinic keratoses (AKs), 1 naevus, 1 atypical nevus, 1 mucosal melanoma, 1 pSK. Although a single melanoma involved the lip, this malignant lesion was characterized by the presence of roundish cells in epidermis, a criterion found in one labial benign lesion, only (melano- sis) (P = 0.005) (Table 2). Melanoses mainly occurred on the lower lip and showed disarranged honeycomb pattern (7/8, 88%) with dendritic cells in half cases (4/8, 50%); DEJ was mainly character- ized by a draped pattern (5/8, 63%) and ringed pattern (4/8, 50%) with papillae homogeneously distributed (6/8, 75%); dendritic cells had a prevalent peri-papillary distribution (6/8, 75%) (Figure 3); plump bright cells were seen in 5/8 cases (63%) in papillary dermis. Melanoma was localized on the lower lip and RCM re- vealed a broadened honeycomb pattern with dendritic and roundish cells (<5 cells/mm2) in epidermis. DEJ was char- acterized by non-edged draped pattern, peri-papillary and inter-papillary dendritic cells (5-10 cell per mm2), showing stellate or fusiform shape (Figure 3). Compound naevus was found on the upper lip and showed on RCM a regular honeycomb pattern in epidermis, non-edged ringed pattern at DEJ and dermal melanocytic nests. Atypical nevus was located on the lower lip and nu- merous cyto-architectural atypia were seen on RCM: dis- arranged honeycomb pattern with dendritic cells (>10 per mm2) in epidermis, nonspecific pattern at DEJ, numerous peri- and inter-papillary dendritic cells (>10 per mm2). Me- lanocytic nests and plump bright cells were observed in pap- illary dermis. BCCs were clearly detected by the presence of bright tu- mor islands and peri-tumoral vessels in papillary dermis. 4 Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 Table 1. Comparison between benign and malignant pigmented genital lesions. Benign pigmented lesions1 Malignant lesions2 (n=50) (n=2) P Value Sex F n (%) 38/50 (76.0) 1/2 (50.0) 0.405 M n (%) 12/50 (24.0) 1/2 (50.0) Age (years) [Mean(±SD)] 44.3(17.6) 28.6 (10.3) 0.219 Localization Women Labia majora 14/50 0/2 >.99 Labia minora 24/50 1/2 >.99 Men Glans 7/50 1/ (50.0) >.99 Foreskin 2/50 0/1 >.99 Penis 2/50 0/1 >.99 Pubis 2/50 0/1 >.99 Architectural Pattern Honeycomb n (%) 49/50 (98.0) 2/2 (100.0) 0.959 Cobblestone n (%) 1/50 (2.0) 0/2 (0.0) Epidermal Disarray 0.002 No n (%) 43/50 (86.0) 0/2 (0.0) Yes n (%) 7/50 (14.0) 2/2(100.0) Broadened HP 0.166 No n (%) 43/50 (86.0) 1/2 (50.0) Yes n (%) 7/50 (14.0) 1/2 (50.0) Presence of dendritic cells in the epidermis n (%) 14/50 (28.0) 2/2 (100.0) 0.031 Density of cells <5 cells/mm2 n (%) 5/14 (35.7) 0/2 (0.0) 0.230 5-10 cells/mm2 n (%) 4/14 (28.6) 0/2 (0.0) >10 cells/mm2 n (%) 5/14 (35.7) 2/2 (100.0) Distribution of cell [tot. obs. (%)] 12/50 (24.0) 2/50 (4.0) Localized n (%) 4/12(33.3) 0/0(0.0) 0.014 Sparse n (%) 7/12(58.3) 0/0(0.0) Widespread n (%) 1/12(8.3) 2/28(100.0) Roundish cells in epidermis 4/50 (8.0) 2/2 (100.0) <0.0001 DEJ DEJ architecture§ Ringed Pattern 32/50 (64.0) 2/2 (100.0) 0.294 Benign pigmented lesions1 Malignant lesions2 (n=50) (n=2) P Value Draped Pattern 19/50 (38.0) 1/2(50.0) 0.732 Meshwork Pattern 5/50 (10.0) 1/2(50.0) 0.083 Clod Pattern 3/50 (6.0) 0/2 (0.0) 0.721 Nonspecific Pattern 4/50 (8.0) 1/2(50.0) 0.048 Distribution of papillae Homogeneously 36/50 (72.0) 1/2(50.0) 0.501 Nonhomogeneously 14/50 (28.0) 1/2 (50.0) Papillae Edged papillae 33/50 (66.0) 1/2(50.0) 0.578 Nonedged papillae 15/50 (30.0) 1/2(50.0) Mixed 2/50 (4.0) 0/2 (0.0) Presence of peripapillary dendritic cells 20/50 (40.0) 2/2(100.0) 0.092 Density of cells <5 cells/mm2 7/20 (35.0) 0/2 (0.0) 0.204 5-10 cells/mm2 6/20 (30.0) 0/2 (0.0) >10 cells/mm2 7/20 (35.0) 2/2 (100.0) Presence of interpapillary dendritic cells 15/50 (30.0) 2/2 (100.0) 0.039 Density of cells <5 cells/mm2 6/15 (40.0) 0/2 (0.0) 0.365 5-10 cells/mm2 2/15 (13.3) 0/2 (0.0) >10 cells/mm2 7/15 (46.7) 2/2 (100) Morphology of dendritic cells§ [tot. obs. (%)] 22/50 (44.0) 2/2 (100.0) fusiform 17/22 (77.3) 2/2 (100.0) 0.449 stellate 7/22 (31.8) 1/2 (50.0) 0.602 triangular 7/22 (31.8) 1/2 (50.0) 0.602 Presence of atypical cells at DEJ 7/50 (14.0) 2/2 (100.0) 0.002 Density of cells <5 cells/mm2 2/7 (28.6) 0/5(0,0) 0.669 5-10 cells/mm2 3/7 (42.99 ½(50.0) >10 cells/mm2 2/7 (28.6) ½(50.0) Papillary dermis Papillary nests 18/50 (36.0) 2/2(100) 0.068 Tumor Islands 0/50 (0.0) 0/50 (0.0) Vessels visible 5/50 (10.0) 0/2 (0.0) 0.638 Plump bright cells 14/50 (28.0) 0/2 (0.0) 0.381 1 Benign pigmented lesions: melanosis, atypical melanocytic lesion, naevus, pigmented seborrheic keratosis, atypical nevus of genital type 2 Malignant lesions: melanoma Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 5 Figure 1. (A-D) Melanosis of a 45-year-old woman and (E-H) invasive melanoma (Breslow tumor thickness, 0,2 mm) of a 30-year-old woman: (A) Multiple pigmented macules in the labia minora, exhibiting with dermoscopy (B) parallel lines, clods and brown color, and (C) with RCM focal dendritic cells in epidermis (yellow circle); (D) Histopathology features of increased basal keratinocyte pigmentation mostly restricted to tips of rete ridges, mild increase in the number of melanocytes (no cytologic atypia and/or nests) at dermo-epidermal junction, melanin pigment incontinence and melanophages in the upper chorion/lamina propria (H&E stain, original magnification × 200); (E) Asym- metric pigmented macule of the skin adjacent to the clitoris (F) showing structureless pattern, circles, and multiple colors with dermoscopy; (G) Confocal revealing junctional non-specific pattern with numerous atypical cells (yellow circle) and melanocytic nests (red arrows); (H) histopathology section exhibiting a radial proliferation of atypical melanocytes at epidermal basal layer and upper lamina propria with focal pagetoid spread (H&E stain, original magnification × 150). Figure 2. (A-D) In situ melanoma of a 40-years-old man and (E-H) atypical melanocytic nevus of genital type of a 15-year-old woman: (A) brown irregular macule on the glans penis (B) exhibiting reticular lines and circles with dermoscopy; (C) Confocal unveils inter- and peri-papillary atypia with sheets of atypical cells at the epithelial-chorion junction (yellow arrow); (D) Histopathology showing a radial pro- liferation of contiguous, mildly atypical melanocytes at the basal layer of epidermis, with focal pagetoid spread in the supra-basal layer (H&E stain, original magnification × 200); (E) Dark pigmented irregular macule on labia majora displaying (F) globules and clods, reticular lines and brown color on dermoscopy; (G) confocal image of pagetoid spreading with dendritic and roundish cells in epidermis (yellow circles), with (H) histology showing irregularly shaped and sized nests, large and roundish, with bizarre balloon or cannonball appearance (H&E stain, original magnification × 200). 6 Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 Table 2. Comparison between benign and malignant pigmented labial lesions. Benign pigmented lesions1 Malignant lesions2 (n=16) (n=1) P Value Sex F 13/16 (81.3) 1/1 (100.0) 0.633 M 3/16(18.7) - (0.0) Mean Age (years) 50.0 (19.0) 42 Localization Upper lip 5/16 (31.3) 0/1 (0.0) 0.801 Lower lip 11/16 (68.7) 1/1 (100.0) Epidermis Architectural pattern Honeycomb 15/16 (93.7) 1/1 (100.0) 0.797 Cobblestone 1/16 (6.3) 0/1 (0.0) Epidermal Disarray 6/16 (37.5) 0/1 (0.0) 0.446 Broadened HP 8/16 (50.0) 1/1 (100.0) 0.331 Presence of Dendritic cells in the epidermis 6/16 (37.5) 1/1 (100.0) 0.218 Roundish cells in epidermis 1/16 (6.3) 1/1 (100.0) 0.005 DEJ DEJ architecture [tot. Obs(%)] 15/16 (93.7) 1/1 (100.0) Ringed Pattern 6/15(40.0) 0/1 (0.0) 0.309 Draped Pattern 4/15 (26.7) 1/1(100.0) Nonspecific Pattern 5/15 (33.3) 0/1 (0.0) Distribution of papillae Homogeneously 10/16(62.5) 1/1 (100.0) 0.446 Benign pigmented lesions1 Malignant lesions2 (n=16) (n=1) P Value Nonhomogeneously 6/16 (37.5) 0/1 (0.0) Papillae§ [tot. Obs(%)] 15/16 (93.7) 1/1 (100.0) Edged papillae 4/15 (26.7) 0/1 (0.0) 0.587 Nonedged papillae 7/15 (46.6) 1/1 (100.0) Nonvisible papillae 4/15 (26.7) 0/1 (0.0) Presence of Peripapillary dendritic cells 9/16 (56.2) 1/1 (100.0) 0.388 Presence of Interpapillary dendritic cells 5/16 (31.2) 1/1 (100.0) 0.163 Morphology of dendritic cells§ [tot. Obs (%)] 9/16 (56.2) 1/1 (100.0) fusiform 8/9 1/1 (100.0) 0.998 stellate 3/9 1/1(100.0) triangular 3/9 0/1(0.0) Presence of atypical cells at DEJ 2/16(12.5) 0/1 (0.0) 0.707 Papillary dermis Papillary nests 2/16 (12.5) 0/1 (0.0) 0.707 Tumor Islands 3/16 (18.7) 0/1 (0.0) 0.633 Vessels visible 6/16 (37.5) 0/1 (0.0) 0.446 Plump bright cells 10/16 (62.5) 0/1 (0.0) 0.218 1 Benign pigmented lesions: melanosis, naevi, atypical melanocytic naevus, pigmented seborrheic keratosis 2 Malignant lesions: melanoma AKs disclosed disarranged honeycomb pattern and den- dritic cells in epidermis; nonspecific pattern was observed at DEJ with peri- and inter-papillary dendritic cells. Images of SK revealed a regular honeycomb pattern with bulbous projections and invaginations in epidermis, plump bright cells at DEJ and in the upper dermis. Conclusions Diagnosis of PMLs may be challenging with clinical/dermo- scopic examination alone [4,5]. Melanosis is the most fre- quent cause of mucosal pigmentation, although a skin cancer, an inflammatory condition, foreign-body pigmentation and pigmented cicatricial scar may rarely occur in clinical setting [1,2]. In this study 70 PMLs were assessed based on a large series of RCM criteria. Epidermal disarray, pagetoid cells, junctional atypia was statistically associated to a diagnosis of mucosal melanoma, supporting results of a recent review that identified pagetoid large cells, high density of basal den- dritic cells and loss of chorion normal architecture as the major features of mucosal melanoma [10]. In our series, dendritic cells in epidermis were detected in mucosal melanoma and in considerable proportion of melanoses (6/29, 21%), potentially representing a confound- ing factor and a major problem for differential diagnosis. Lamier et al defined “irregular dendritic type”, a subtype of melanoses showing RCM overlapping features with mucosal melanoma: epidermal atypical cells, junctional atypia and focal loss of regular architecture. In presence of epidermal atypia, the density of dendritic cells may represent an im- portant clue, since ≥10 cells/mm2 are more suggestive of mu- cosal melanoma [8,10]. Roundish cells at DEJ, seen in 4/29 Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 7 Figure 3. (A-D) Melanosis of a 16-year-old man and invasive melanoma (Breslow tumor thickness, 0,2 mm) of a 42-year-old woman: (A) pigmented macule on the lower lip (B) with structureless pattern, parallel lines and brown color on dermoscopy; (C) Confocal images of the junction displaying draped pattern edged-papillae with papillary small bright cells (red arrows), (D) histologic section showing increased basal keratinocyte pigmentation and a mild increase of non-atypical melanocytes at the dermo-epidermal junction (H&E stain, original mag- nification × 200); (E) Brownish macule on the lower lip with (F) dermoscopic features of brown structureless areas (G) confocal findings of non-edged papillae, atypical cells at the junction (yellow circle); (H) Histopathology section exhibiting a proliferation of atypical melanocytes at epidermal basal layer and upper lamina propria with focal pagetoid spread (H&E stain, original magnification × 200). melanoses (14%), are generally also detected in lentiginous pattern of in situ mucosal melanoma, originating from an atypical melanocytic hyperplasia ie the so-called “intraepi- dermal atypical melanocytic proliferation of uncertain sig- nificance (IAMPUS)” [11]. Following a continuous model, in its early phase, melanoma may show minimal cytological atypia without clear architectural disarrangement, mimick- ing a melanosis. Thus, RCM can be useful in the monitoring of atypical PMLs over time detecting minimal worrisome changing. AGN represent a subset of benign nevi occurring in young adults that show worrisome clinical features such as dark pigmentation, irregular borders and large size, and sus- picious dermoscopic findings, like mixed pattern and mul- tiple colors [4,12]. Herein, AGN were found in young girls (14-18-years-old) exhibiting clinical features of dark brown color, fast growth and large size (>1cm in diameter). RCM was not useful for their correct recognition due to relevant cyto-architectural irregularities, leading therefore to surgical excision. It is noteworthy that diagnosis of AGN is also chal- lenging with histopathology, because of the presence of ar- chitectural disorder, nested pattern and pagetoid spreading, with various degree of cellular atypia [12]. Pigmentary changes following inflammatory conditions, like occurring with lichen sclerosus, may be a cause of con- cern often requiring histopathological examination [13,14]. Confocal displayed classical features of melanosis in our two cases, leading to a conservative approach. Results from PLLs revealed roundish cells in epidermis as the unique criterion significantly associated with melanoma (P = 0.005). Calculating the RCM lip score of Uribe et al, it was ≥ 4 in atypical/malignant lesions and lower in benign le- sion, providing further evidence of its utility, as already sug- gested by Gomez-Martin et al, evaluating 51 PLLs, of which 5 mucosal melanomas [1]. In our series 2/8 melanoses (25%) were considered false positives obtaining a LIP score ≥ 4, due to disarray and dendritic cells in epidermis and junctional atypical cells. Such discrepance could be related to the pres- ence of epidermal inflammation, as confirmed by histopa- thology revealing numerous Langerhans cells in suprabasal layers [1,9]. Diagnosis of benign (SK, AK) and malignant (BCC) ke- ratinocyte skin lesions was simplified by the recognition of common confocal criteria [15-17]. Main limitation of the study is the inclusion of only 3 cases of mucosal melanoma, of which 1 melanoma of the lip, probably related to rarity of this entity in clinical setting. Additionally, the detection in RCM of dendritic bright cells in the epithelium of melanosis and melanoma, may represent a confusing factor for diagnosis, although density, shape and location of this cells may add important clues. Dermatologists should consider confocal microscopy as an adjunctive technique to dermoscopy, for the differen- tial diagnosis of PMLs showing clinical and dermoscopic findings suspicious for malignancy, and for long-term sur- veillance of atypical melanocytic lesions. Anyhow, PMLs 8 Original Article | Dermatol Pract Concept. 2024;14(1):e2024028 9. Uribe P, Collgros H, Scolyer RA, Menzies SW, Guitera P. In vivo reflectance confocal microscopy for the diagnosis of mel- anoma and melanotic macules of the lip. JAMA Dermatol. 2017;153(9):882-891. DOI: 10.1001/jamadermatol.2017.0504. PMID: 28467525. PMCID: PMC5710424. 10. De Pascalis A, Perrot JL, Tognetti L, Rubegni P, Cinotti E. Review of Dermoscopy and Reflectance Confocal Microscopy Features of the Mucosal Melanoma. Diagnostics (Basel). 2021;11(1):91. DOI: 10.3390/diagnostics11010091. PMID: 33429900. PM- CID: PMC7827612. 11. Elder DE, Bastian BC, Cree IA, Massi D, Scolyer RA. 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