The impact of in vivo reflectance confocal microscopy on the diagnostic accuracy of lentigo maligna and equivocal pigmented and nonpigmented macules of the face.

Limited studies have reported the in vivo reflectance confocal microscopy (RCM) features of lentigo maligna (LM). A total of 64 RCM features were scored retrospectively and blinded to diagnosis in a consecutive series of RCM sampled, clinically equivocal, macules of the face (n=81 LM, n=203 benign macules (BMs)). In addition to describing RCM diagnostic features for LM (univariate), an algorithm was developed (LM score) to distinguish LM from BM. This comprised two major features each scoring +2 points (nonedged papillae and round large pagetoid cells > 20 microm), and four minor features; three scored +1 point each (three or more atypical cells at the dermoepidermal junction in five 0.5 x 0.5 mm(2) fields, follicular localization of atypical cells, and nucleated cells within the dermal papillae), and one (negative) feature scored -1 point (a broadened honeycomb pattern). A LM score of > or = 2 resulted in a sensitivity of 85% and specificity of 76% for the diagnosis of LM (odds ratio (OR) for LM 18.6; 95% confidence interval: 9.3-37.1). The algorithm was equally effective in the diagnosis of amelanotic lesions and showed good interobserver reproducibility (87%). In a test set of 29 LMs and 44 BMs, the OR for LM was 60.7 (confidence interval: 11.9-309) (93% sensitivity, 82% specificity).

Subungual melanoma: a study of 124 cases highlighting features of early lesions, potential pitfalls in diagnosis, and guidelines for histologic reporting.

Subungual melanoma (SUM) is an uncommon variant of melanoma that is often difficult to diagnose, both clinically and pathologically. In an attempt to provide pathologic clues to diagnosis, especially in early lesions or small biopsies, and to provide practical advice to pathologists in reporting, the clinicopathologic features of 124 cases of SUM were reviewed, the largest series reported to date. The features of 28 cases of subungual melanoma in situ (MIS), comprising 4 cases of MIS and 24 cases where areas of MIS were present adjacent to dermal-invasive SUMs, were compared with those of a similar number of acral nevi to identify useful distinguishing features. The median age of the patients was 59 years and the most common site was the great toe (24%). Nine percent of cases were AJCC stage 0, 14% were stage I, 41% were stage II, 32% were stage III, and 4% were stage IV at initial diagnosis. The commonest histogenetic subtype was acral lentiginous (66%), followed by nodular (25%) and desmoplastic (7%). The majority of tumors were locally advanced at presentation with 79% being Clark level IV or V. The median Breslow thickness was 3.2 mm. The median mitotic rate was 3 per mm and 33% of cases demonstrated primary tumor ulceration. Seven of 29 patients (24%) who underwent a sentinel lymph node biopsy had nodal disease. Multivariate Cox-regression analysis showed higher disease stage to be the only significant predictor of shortened survival. In comparison to acral nevi, MIS more frequently showed lack of circumscription, a prominent lentiginous growth pattern, predominance of single cells over nests, moderate-to-severe cytologic atypia, a dense and haphazard pagetoid intraepidermal spread of melanocytes, and the presence of junctional/subjunctional lymphocytes ("tumor infiltrating lymphocytes"). Tumor infiltrating lymphocytes have not been highlighted previously as a feature of subungual MIS and represent a useful diagnostic clue. Guidelines for the reporting of SUMs are also presented. Knowledge and recognition of the pathologic features of SUMs and the important features that distinguish them from nevi should reduce the frequency of misdiagnosis.

Low prevalence of RAS-RAF-activating mutations in Spitz melanocytic nevi compared with other melanocytic lesions.

Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a 'halo' SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low-level microsatellite instability at one locus, D10S214. The low rate of RAS-RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS-RAF-MEK-ERK-MAPK pathway. Germline DNA from members of 111 multiple-case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.

Dermoscopy and its role in diagnosing melanocytic lesions: a guide for pathologists.

Dermoscopy (surface microscopy) is a clinical technique which uses a hand-held magnifying instrument, usually with liquid at the skin-instrument interface, to examine pigmented lesions on the skin surface. A magnification of x 10 is usually used. Dermoscopy assists in deciding if the lesion should be excised or biopsied, requires monitoring or can be safely left in situ. The technique provides a bridge between the naked eye appearance of a lesion and the histopathological examination. Multiple dermoscopic features have been described and many of their histological correlates have been determined. Dermoscopic diagnosis usually involves a two-step procedure. The first step is to decide if the lesion is melanocytic or not. If melanocytic, the second step is to decide if the lesion is benign or malignant. Multiple algorithms have been developed to help in this decision. Dermoscopic criteria have been developed for melanoma and naevi. Several non-melanocytic pigmented lesions can be diagnosed with dermoscopy, including pigmented basal cell carcinoma, seborrhoeic keratoses, haemangioma and lichen planus-like keratosis.

UV light from 290 to 325 nm, but not broad-band UVA or visible light, augments the formation of melanocytic nevi in a guinea-pig model for human nevi.

We have previously described a guinea-pig model where pigmented nevi similar to human nevi can be produced by application of low-dose topical 7,12-dimethylbenzanthracene (DMBA) followed by solar-simulated light. Five groups of guinea-pigs were used to test the effect of various spectral bands of solar-simulated light on low-dose DMBA-induced melanocytic nevi. Animals were irradiated with either UVB to near UVA2 (290-325 nm), UVA, visible light, full solar spectrum or no irradiation three times per wk for 12 mo to determine the broad-band effect of nevi-inducing irradiation. There was a significant increase in nevi/animal in the UVB-treated group (mean 1.53) compared with all groups (versus UVA 0.3, p<0.001; versus visible light 0.24, p<0.001; versus full spectrum (UVB+UVA+visible) 0.68, p=0.02; versus control (nil irradiation) 0.37, p=0.01). No differences in skin thickness were found between any group (p=0.11). In conclusion, we present a report of the active waveband of melanocytic nevi induction; where UVB to near UVA2 is the likely responsible waveband. Furthermore, because there was a significant decrease in nevi/animal receiving the full solar spectrum compared with the UVB group, it is possible that broad-band UVA and or visible light may be inhibitory wavebands for nevi induction.

Use of multiple cytometric markers improves discrimination between benign and malignant melanocytic lesions: a study of DNA microdensitometry, karyometry, argyrophilic staining of nucleolar organizer regions and MIB1-Ki67 immunoreactivity.

Confident separation of benign naevi and malignant melanoma can sometimes be very difficult using conventional microscopy. This study evaluated the combined diagnostic abilities of multiple cytometric markers in separating various types of naevi from melanomas. The lesions studied included 27 benign compound naevi, 20 dysplastic naevi, 10 Spitz naevi and 24 melanomas. The cytometric features investigated were: (i) nuclear DNA content and chromatin compactness, measured by video imaged DNA microdensitometry; (ii) nuclear morphology, measured by nuclear morphometry (karyometry); (iii) transcriptional activity of nucleolar organizer regions, measured as the number and size of argyrophilic staining of nucleolar organizer regions (AgNORs); and (iv) cellular proliferative activity detected by quantifying the immunoreactivity of MIB1-Ki67 antigen. These variables were evaluated in the superficial, middle and deep zones of each lesion. Using multivariate discriminant analysis, a total diagnostic effectiveness of 97% could be achieved in separating the benign and malignant melanocytic lesions by co-evaluating variables for DNA microdensitometry, karyometry and AgNORs. A diagnostic effectiveness of 100% could be achieved if further co-evaluation with MIB1-Ki67 immunoreactivity was performed. Our study suggests that co-evaluation of multiple cytometric markers can improve the diagnostic abilities of individual techniques in separating benign naevi from malignant melanomas. This may be of particular significance in the diagnosis of melanocytic lesions whose biological behaviour cannot be confidently predicted by their histological features using conventional microscopy.

Autonomous histopathological regression of primary tumours associated with specific immune responses to cancer antigens.

Spontaneous histopathological regression of cancer has been reported. The involvement of the immune system in such regression has been advocated, leading to the theory of immunological surveillance against cancer. A prediction of this theory is that common tumour antigens can be recognized upon repeated exposure by cell-mediated immunity, which leads to tumour regression and the subsequent appearance of tumour antigen-loss variants. However, no direct evidence has been provided in non-viral-induced experimental animal models of primary malignancy or in human primary cancer. This study examined two groups of melanoma patients where histopathological regression of the primary tumour was observed. Many of the 23 patients with multiple (> or =3) primary melanomas showed significant regression of their last melanoma (median 33%, mean 40) compared with matched melanomas from patients with a single primary melanoma (median 0%, mean 12) (p=0.0080), or compared with their first primary melanoma (p=0.0013). Regression was consistent with an 'immunization effect' seen in murine tumour transplantation studies, where inoculation with > or =3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in the expression of the melanoma common tumour antigen MART-1 in the last primary tumour from multiple melanoma patients (median 8%, mean 24) versus matched single melanoma patients (median 79%, mean 68) (p=0.0041) and in the last versus first tumour in multiple primary patients was found (p=0.0083). Metastases from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 loss (median 0%, mean 11) compared with matched metastases from patients with non-regressing primary melanoma (median 51%, mean 50) (p=0.0013). MART-1 antigen-loss variants observed in the multiple primary and occult primary patients correlated with the presence of peripheral blood MART-1-specific cytotoxic T lymphocytes (CTLs) (p=0.03). No similar effects were observed with two other melanoma antigens, gp100 and CD63. Thus, in two groups of human melanoma patients, evidence is provided for histopathological tumour regression associated with cancer immune surveillance.

Argyrophilic staining of nucleolar organizer region count and morphometry in benign and malignant melanocytic lesions.

Differentiation between malignant melanomas (MMs) and benign nevi based on histologic features can sometimes be difficult. This study evaluated the diagnostic effectiveness of argyrophilic staining of nucleolar organizer regions (AgNORs) in separating benign nevi from MMs by assessing 27 compound nevi (CN), 20 dysplastic nevi (DN), 10 Spitz nevi (SN), and 24 MMs. Both AgNOR count and morphology variables were measured from the superficial, middle, and deep zones of the lesions using video image analysis. Malignant melanomas had a significantly greater AgNOR number per nucleus, mean AgNOR area per nucleus, and variation in AgNOR area per nucleus compared with all types of benign nevi (p < 0.05). In multivariate discriminant analysis using a combination of four AgNOR counts and morphometric parameters, all CN and DN, 8 of 10 SN, and 23 of 24 MMs could be correctly classified as benign or malignant. The results suggest that both AgNOR count and morphology help to separate benign and malignant melanocytic lesions and that the combination of both sets of parameters improves their discriminating ability.

Differentiating benign nevi from malignant melanoma using DNA microdensitometry and karyometry and maturation: a zonal comparison, correlation and multivariate analysis.

OBJECTIVE: To evaluate the diagnostic effectiveness of cytometric features of DNA microdensitometry, karyometry (nuclear morphometry) and maturation and their combinations in separating benign nevi from malignant melanomas. STUDY DESIGN: Tumor cells were measured from each of the superficial, middle and deep zones of 81 melanocytic lesions using video image analysis for nuclear DNA content, chromatin compactness, and nuclear size and shape variables. There were 27 banal compound melanocytic nevi, 20 dysplastic compound nevi, 10 Spitz nevi and 24 malignant melanomas (MM). Maturation of cells with depth into the dermis was also studied by comparing cells from superficial to deep zones. RESULTS: MM showed distinct characteristics of DNA microdensitometry, karyometry and maturation as compared to all groups of benign nevi. There were overall close correlations between nuclear DNA content variables and nuclear size parameters in the total group of 81 lesions. However, there were fewer significant correlations between the various indices in the group of melanomas alone. Using multivariate discriminant analysis, up to 97% of the lesions could be correctly separated as benign or malignant by a combination of five key microdensitometric, karyometric and maturation parameters. CONCLUSION: DNA microdensitometry, karyometry and maturation parameters have independent abilities in identifying individual malignant melanomas. Coevaluation of various cytometric features and maturation profiles offers better diagnostic ability in separating benign nevi from MM.

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation.

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.

Spitz naevus versus Spitzoid melanoma: when and how can they be distinguished?

Spitz naevus is a benign melanocytic lesion that shares many histological features with melanoma. While Spitz naevi characteristically occur in children and young adults and melanomas in the middle-aged and elderly, either tumour can occur in patients of any age. In many cases, the histopathological diagnosis of Spitz naevus is straightforward, particularly in small lesions displaying many or all of the typical histological features and occurring in young patients. Tumours that deviate from the classic description, however, cause difficulties in diagnosis. In this review, we highlight histopathological features of Spitz naevi and those that may be useful in distinguishing Spitz naevi from melanomas. We find that the presence of good symmetry, Kamino bodies, and uniformity of cell nests or sheets from side-to-side favours a Spitz naevus. The presence of abnormal mitoses, a dermal mitotic rate of >2/mm2, and mitotic figures within 0.25 mm of the deep border of the lesion favours a melanoma. Immunohistochemical stains for HMB45 and Ki67 sometimes provide additional useful information. Despite this, in some cases it may not be possible to give an unequivocal diagnosis. Recommendations for the reporting of such cases are provided. New techniques have also demonstrated chromosomal, molecular and genetic differences between Spitz naevi and melanomas. This report highlights these new data and speculates on their possible future role in the diagnosis of borderline lesions.