BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin

BRAF mutations have recently been detected with a high frequency (66%) in cutaneous melanoma. All those mutations are activating, with a single substitution (T1796A) at codon 599 (V599E) accounting for over 90%. To investigate the stage in which those mutations occur in the currently proposed sequential malignant transformation of melanocytes, 22 benign melanocytic nevi, 23 melanocytic atypical nevi, and 25 primary cutaneous melanoma from 63 different patients were examined for BRAF mutations using DNA extracted from microdissected formalin-fixed and paraffin-embedded tissues, and a two-round PCR-RFLP-based strategy. A subset of samples was sequenced for mutation confirmation. Sixteen benign (73%) and eleven atypical (52%) melanocytic nevi, and thirteen melanoma (56%) demonstrated BRAF mutations at codon 599, and no statistically significant differences were detected among all three types of lesions. No mutations were demonstrated in microdissected epidermal keratinocytes adjacent to melanocytic lesions having BRAF mutations. No correlation was detected between BRAF mutational status and age, sun exposure, and Clark's level in malignant melanoma. However, comparing only atypical nevi and melanoma lesions the frequency of BRAF mutation is significantly greater in male (78%) than female (35%) patients (P = 0.0194). The previously described T1796A point mutation was detected in 17 of 18 mutated samples, and a novel mutation consisting of a substitution of valine for lysine (GT1795-96AA) was detected in one melanoma case. Our findings of a high frequency of BRAF mutations at codon 599 in benign melanocytic lesions of the skin indicate that this mutation is not sufficient by itself for malignant transformation.     

Distinguishing benign and malignant melanocytic lesions with the AgNOR method

A silver staining technique has recently been devised to aid in the differentiation between benign and malignant melanocytic lesions. This study showed a statistically significant difference between the staining of silver-nucleolar organizer regions (AgNORs) in melanocytic nevi and that of AgNORs in malignant melanomas.     

AgNORs in benign, dysplastic, and malignant melanocytic skin lesions

Nucleolar organizer regions (NORs) are loops of DNA situated on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22; they can be demonstrated in formalin-fixed paraffin-embedded sections by a one-step silver technique, the resulting black structures being termed AgNORs. We have applied the technique to 30 benign "banal" nevi (BN), 30 dysplastic nevi (DN), and 30 malignant melanomas (MM). AgNORs in 200 nuclei were scored and the means calculated. Counts were as follows: BN showed a mean of 1.3 AgNORs per nucleus within a range of 1.1-1.6; DN showed a mean of 1.2 within a range of 1.0-1.6; and MM showed a mean of 2.1 within a range of 1.2-4.2. A significant difference existed between counts for MM and those for BN and DN, despite some overlap. There was no statistically significant difference between BN and DN. Although still within the field of research, the AgNOR technique may prove to be of value in helping to differentiate MM from DN, but is unlikely to be of help in separating DN from BN.     

BRAF kinase gene V599E mutation in growing melanocytic lesions

Mutations in the BRAF-gene are found in benign and malignant melanocytic lesions, >90% being a V599E mutation. This mutation results in constitutively active kinase function and increased colony formation in vitro. The biological impact of this mutation in vivo is still debated. To address this question, we used our digital epiluminescence image archive and retrospectively selected 49 melanocytic lesions, which did not meet the criteria of melanoma at the initial presentation. Mean 12 months later these lesions were excised because of increased size or changed structure and BRAF(V599E) mutations were analyzed. Among 36 growing lesions, BRAF(V599E) mutations were found in 16 (11 melanomas and 5 nevi). Among 13 lesions with structural changes, BRAF(V599E) mutations were found in 4 (3 melanomas and 1 nevus). Thirty-five randomly selected additional lesions with no changes during follow-up served as controls, all nevi by histology, and two of them showed a BRAF(V599E) mutation. Statistics revealed odds for the presence of the BRAF(V599E) mutation being seven times higher in lesions with structural changes and 13 times higher in growing lesions as compared with lesions without changes. This raises the question if the V599E mutation determines lesions at risk developing into melanoma and if not, what are the mechanisms controlling growth stop in benign lesions?     

Stereological estimation of nuclear volume in benign melanocytic lesions and cutaneous malignant melanomas

Recent developments in stereology have made it possible to obtain unbiased, objective estimates of the volume weighted mean nuclear volume (vV) from ordinary histological sections, without any assumptions concerning nuclear shape. The aim of this study was to gather baseline data of nuclear vV in melanocytic cutaneous tumors and to compare these with estimates of nuclear volume fraction and with traditional two-dimensional morphometric estimates of nuclear profile area, nuclear density, and mitotic index. Routinely processed, paraffin embedded tissue specimens from 47 malignant melanomas and 76 noninvasive melanocytic cutaneous tumors were investigated retrospectively. vV clearly distinguished between noninvasive (average vV = 122 microns 3) and invasive lesions (average vV = 246 microns 3). Most of the patients with malignant melanomas showing an overlap of nuclear vV with benign lesions had a favorable prognosis. No significant differences in vV could be demonstrated among different noninvasive tumor types. Two-dimensional estimates only distinguished benign from malignant tumors with considerable overlap and with significantly varying influence from other factors among different benign lesional types. The variance associated with estimates of vV disclosed a linear relationship to vV, whereas the relative variation seemed to be constant. Tumor heterogeneity was without significant influence on estimates of nuclear vV. It is concluded that nuclear vV is a superior, efficient, and sensitive estimator for distinguishing between melanocytic cutaneous tumors showing different biological behavior, well-suited for objective malignancy grading.     

Confocal scanning laser microscopy of benign and malignant melanocytic skin lesions in vivo

BACKGROUND: The ability of physicians for early diagnosis of cutaneous melanomas is less than perfect, prompting research into noninvasive methods for diagnosis. OBJECTIVE: Our purpose was to evaluate confocal scanning laser microscopy (CSLM) for noninvasive imaging of benign and malignant melanocytic lesions in vivo. METHODS: Forty pigmented skin lesions (including adjacent normal skin as control) in vivo were imaged with near-infrared CSLM. The confocal images were correlated to histopathology. RESULTS: Nuclear, cellular, and architectural detail in the epidermis and superficial dermis is imaged with high resolution and contrast. Melanin causes the cytoplasm of pigmented cells to appear bright. Melanocytic nevi had cohesive nests of uniformly circular cells and increased microvascular blood flow. Melanomas had a polymorphous cytologic structure, containing atypical, pleomorphic cells in disarray and irregular dendritic cells. CONCLUSION: CSLM is capable of identifying distinct patterns and cytologic features of benign and malignant pigmented skin lesions in vivo. CSLM may be useful to noninvasively discriminate benign and malignant lesions in vivo.     

Expression of the proliferation and apoptosis-associated CAS protein in benign and malignant cutaneous melanocytic lesions

We have examined the expression of the cellular apoptosis susceptibility protein, a nuclear transport factor that plays a role in apoptosis and cell proliferation, in benign and malignant melanocytic lesions. Tissue samples of 55 formalin-fixed, paraffin-embedded melanoma (primary n=32, metastatic n=23) and of 27 control cases (junctional dermal, compound, Spitz, Reed, blue nevi, balloon-cell nevus, lentigo maligna) were analyzed by immunohistochemistry with anti-cellular apoptosis susceptibility antibodies. The percentage of cellular apoptosis susceptibility-positive cells as well as the intensity on a four-point scale was evaluated. In normal skin, expression of cellular apoptosis susceptibility was primarily found in the basal cell layer of the epidermis. Benign melanocytic lesions that stained positive for cellular apoptosis susceptibility (13 of 27) showed a homogeneously distributed staining pattern with a mean of 5+/-12% cellular apoptosis susceptibility positive cells. Five out of 7 lentigo maligna melanoma, 11 out of 12 superficial spreading melanoma and all acrolentiginous (n=7) and nodular (n=6) melanoma showed immunoreactivity of medium (++) to high ( ) intensity. Vertical growth phases of primary cutaneous melanoma stained stronger than horizontally growing cell clusters. All metastases (n= 23) stained strongly positive, the staining pattern being inhomogeneous. Cellular apoptosis susceptibility detection in clinical stages according to UICC showed an increase from 43+/-34% cellular apoptosis susceptibility positive cells in stage I, to 53+/-26% in stage II, 68+/-24% in stage III and 72+/-24% in stage IV, respectively. Because the expression of cellular apoptosis susceptibility correlates predominantly with advanced stages of melanoma, staining with anti-cellular apoptosis susceptibility antibodies may be useful for diagnosis of melanoma and possibly as an immunohistochemical prognostic factor in cutaneous melanocytic lesions.     

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.     

Epidermal consumption in benign and malignant melanocytic neoplasms

Consumption of the epidermis associated with effacement of the rete ridge pattern has been cited as a useful criterion in the diagnosis of melanoma, but the significance of consumption in the absence of rete ridge effacement is unknown. We evaluated 701 melanocytic neoplasms for presence and ‘grade’ of consumption by melanocytic nests relative to diagnosis, body location, gender and age. We defined 1+ consumption as collections of melanocytes occupying greater than two thirds of the viable epidermis, with or without loss of the rete ridge pattern. Nests extending to the bottom of, within, and through the granular layer were graded 2+, 3+ and 4+, respectively. Consumption was more frequent and higher grades were found in melanomas followed by Spitz nevi compared with conventional melanocytic nevi (p < 0.001). Melanomas with higher Breslow thickness showed higher grades (p < 0.05). In conventional nevi, consumption occurred most frequently in back (13.7%), acral (11.9%) and scalp (9.8%) locations. Consumption without the requirement for rete ridge effacement occurs more frequently and at higher grades in melanoma. Higher grades correlate with higher Breslow thickness. Consumption is also common in Spitz nevi and occurs at lower grades in conventional (non‐Spitz) nevi, especially on the back, the scalp and at acral sites.     

A zonal comparison of MIB1-Ki67 immunoreactivity in benign and malignant melanocytic lesions

Differentiation between malignant melanomas and benign nevi can sometimes be difficult by conventional histopathology, and additional diagnostic markers may be helpful. This study investigated the immunoreactivity of the cell proliferation marker MIB1-Ki67 in 23 compound nevi, 17 dysplastic nevi, 8 Spitz nevi (SN), and 24 malignant melanomas (MMs) and evaluated its ability in separating benign nevi from MMs. In each lesion, the average number (percentage) of MIB1-positive nuclei (%MIB1-Mean) and the maximal number (percentage) of MIB1-positive nuclei (%MIB1-Max) were determined from each of the superficial, middle, and deep dermal zones of the lesion as well as from the entire lesion. The %MIB1-Max was determined from subjectively selected area(s) of high count. Malignant melanomas had a significantly greater %MIB1-Mean and %MIB1-Max than all benign nevi in all individual zones and in the entire lesion (p < 0.05). Discriminant analysis showed that the %MIB1-Mean and %MIB1-Max counted from the whole lesions had better discriminating abilities than from the individual zones. By using the %MIB1-Mean from all zones, all lesions except 1 SN and 3 MMs could be correctly classified as benign or malignant. When using the %MIB1-Max from all zones, all but 2 SN could be correctly separated as benign or malignant. Thus, MIB1-Ki67 immunoreactivity closely correlates with the benignancy or malignancy of melanocytic lesions and may assist in the differentiation of benign nevi from MMs.     

HMB-45 staining in benign and malignant melanocytic lesions. A reflection of cellular activation.

The antibody HMB-45 used as an immunohistochemical reagent has often been labeled as a marker for melanoma, even though some benign lesions have been noted to show positive staining reactions with this reagent. Biopsy specimens from 225 benign and malignant melanocytic lesions were examined after immunoperoxidase staining for S-100 protein and HMB-45. The lesions studied included common acquired nevi, spindle cell and epithelioid cell nevi (Spitz nevi), cellular blue nevi, deep penetrating nevi, congenital nevi, nevi from hormonally reactive areas (genital), malignant melanoma, and desmoplastic malignant melanoma. A positive reaction for HMB-45 was seen in the dermal component in a high percentage of each of these types of lesions except for the common acquired nevi and the desmoplastic malignant melanomas that were uniformly negative for HMB-45 in the dermal component. HMB-45 correlates with melanosome production and thus a melanocytic origin of HMB-45-positive cells. HMB-45 may correlate best with factors that stimulate melanocytic proliferation and production of melanosomes.     

Comparison of pHH3, Ki-67, and survivin immunoreactivity in benign and malignant melanocytic lesions

Differentiating malignant melanoma from benign melanocytic lesions can be challenging. We undertook this study to evaluate the use of the immunohistochemical mitosis marker phospho-Histone H3 (pHH3) and the proliferation markers Ki-67 and survivin in separating malignant melanoma from benign nevi. Sixty-six melanocytic lesions (18 malignant melanomas, 8 Spitz nevi, 20 dysplastic nevi, and 20 compound nevi) were stained with antibodies to pHH3, Ki-67, and survivin. No pHH3 expression was detected in the dermis of compound and dysplastic nevi. Rare mitoses were observed in the superficial dermis in 3 of 8 Spitz nevi (37%). Staining for pHH3 was higher in malignant melanomas [average 25 per 10 high-power field (HPF), range 2-75 per 10 HPF] than in Spitz nevi (average 0.5 per 10 HPF, range 0-2 per 10 HPF) and was heterogeneously distributed in the malignant melanomas compared with a superficial dermal location in Spitz nevi. There was no cytoplasmic staining for survivin in any of the 66 melanocytic lesions and no nuclear staining in any of the benign ones. Survivin nuclear staining was present in 12 of 18 cases of malignant melanoma (67%) with an average index of 7% (range 0%-15%). In benign melanocytic lesions, the Ki-67 index was less than 5% (range 0%-4%) and staining was present close to the dermo-epidermal junction compared with an average index of 27% in melanomas (range 5%-50%) and a generally heterogeneous pattern of staining throughout the dermis. pHH3 and Ki-67 can be useful adjuncts to histopathology to separate malignant melanoma from benign nevi. pHH3 is especially useful to highlight mitoses and to rapidly assess the mitotic activity in melanocytic lesions.     

The dermoscopic classification of atypical melanocytic naevi (Clark naevi) is useful to discriminate benign from malignant melanocytic lesions

BACKGROUND: The dermoscopic classification is a useful tool for handling patients with atypical naevi (Clark naevi). OBJECTIVES: To investigate if the dermoscopic classification of atypical naevi is of any value to discriminate benign from malignant melanocytic lesions. METHODS: Consecutive patients (n = 205) were included with 254 suspicious melanocytic lesions, confirmed by histopathology at the Pigmented Lesions Clinic of the Department of Dermatology, University Medical Center, University of Tuebingen, Germany. In this retrospective study, dermoscopic images of benign and malignant melanocytic lesions were classified according to the dermoscopic classification of atypical naevi (reticular, globular, homogeneous or combinations of two of these) and pigmentation (uniform, central hyper- or hypopigmentation, eccentric peripheral hyper- or hypopigmentation, or multifocal hyper- or hypopigmentation). The three-structure type (reticular, globular and homogeneous) was additionally defined. RESULTS: Reticular, homogeneous and reticular-homogeneous types were significantly more frequent in naevi than in melanomas, whereas the three-structure type was significantly more frequent in melanomas (P < 0.001). A sensitivity of 86.7%, specificity of 87.7% and diagnostic accuracy of 87.4% was obtained. Uniformly pigmented and centrally hyperpigmented types were significantly more frequent in naevi than in melanomas, whereas eccentric peripheral hyperpigmented and multifocal hyper- or hypopigmented types were significantly more frequent in melanomas (P < 0.001). CONCLUSIONS: The dermoscopic classification of atypical naevi (Clark naevi) is useful to discriminate benign from malignant melanocytic lesions. The three-structure type and eccentric peripheral hyperpigmentation were significantly more frequently found in malignant than in benign melanocytic lesions. The knowledge of these two dermoscopic types should be helpful for the management of patients presenting with multiple melanocytic lesions.     

Selective expression of FLIP in malignant melanocytic skin lesions

FLIP (FLICE Inhibitory Protein) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits cell death mediated by all death receptors including Fas and TRAIL. FLIP has recently been shown to favor tumor growth and immune escape in mouse tumor models. We analyzed FLIP expression by immunohistochemistry in a panel of 61 benign and malignant human melanocytic skin lesions. FLIP expression was undetectable in all but one benign melanocytic lesion (31/32, 97%). In contrast, FLIP was strongly expressed in most melanomas (24/29 = 83%). Overexpression of FLIP by transfection in a Fas- and TRAIL-sensitive human melanoma cell line rendered this cell line more resistant to death mediated by both TRAIL and FasL. Selective expression of FLIP by human melanomas may confer in vivo resistance to FasL and TRAIL, thus representing an additional mechanism by which melanoma cells escape immune destruction.