Diagnostic tissue elements in melanocytic skin tumors in automated image analysis

In tissue counter analysis, digital images are divided into subregions (elements), and the digital information in each element is used for statistical analysis. In this study, we assessed the morphologic details of tissue elements that have turned out to be of diagnostic significance in the discrimination of benign common nevi and malignant melanoma. After creation of a data set based on a total of 12,000 cellular elements obtained from 100 benign common nevi and 100 malignant melanomas, classification and regression tree (CART) analysis was performed to differentiate between cellular elements of nevi and melanoma. In a second step, the slides were re-evaluated by the decision tree; cellular elements suggestive either for benign common nevi or for malignant melanoma were highlighted on zoomed images of the whole sections, and the individual elements were displayed in galleries. Eight groups of elements (so-called terminal nodes) seemed to indicate benign common nevi, whereas seven terminal nodes were suggestive for malignant melanoma. The elements of nodes suggestive for benign nevi largely contained nevus cells with amphiphilic cytoplasm intermingled with fibrillary material, whereas the elements of the nodes suggestive for malignant lesions often showed hyperchromatism, perinuclear halos, heavy pigmentation, or a lymphohistiocytic infiltrate. Tissue counter analysis automatically detects tissue elements that are in accordance with morphologic criteria used in conventional histopathology for diagnostic discrimination.     

Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors

In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the examination of skin morphology in real time at a resolution equal to that of conventional microscopes. The aim of the study was to test the applicability of CLSM to the diagnostic discrimination of benign nevi and melanoma. five independent observers without previous experience in CLSM received a standardized instruction about diagnostic CLSM features. Subsequently, 117 melanocytic skin tumors (90 benign nevi and 27 melanoma), imaged using a commercially available, near-infrared, reflectance confocal laser scanning microscope, were evaluated by each observer. Overall, sensitivity of 88.15% and specificity of 97.60% was achieved by the five observers. Logistic regression analysis revealed that mainly cytomorphology, architecture and keratinocyte cell borders should be taken into account for diagnostic decisions. Remarkably, using the presence or absence of monomorphic melanocytes as a single diagnostic criterion, the classification results with a sensitivity of 98.15% and a specificity of 98.89% were superior to the intuitive, integrative judgement of the observers. This first sensitivity and specificity study with CLSM has yielded promising results. CLSM provides new and useful information to the clinician diagnosing melanocytic skin tumors.     

Morphometric diagnosis of melanocytic skin tumors

Checking consecutively sampled routine sections of 206 melanocytic lesions with a maximum vertical diameter of at least 1 mm (133 benign dermal nevi, 20 Spitz's nevi, 53 primary malignant melanomas), we measured the morphometric features of at least 60 nuclei each from the superficial and the deep dermal tumor portion using a computer-assisted interactive image analysis system. Furthermore we calculated the so-called maturation parameter (MP) in each case as the ratio of the mean nuclear area in the deep portion and the superficial portion. When we compared the results with those obtained in a training set, we found that the lowest evidence for the discrimination of benign and malignant melanocytic lesions resulted from the application of the mean values of the nuclear area in the superficial layer (efficiency = 62.1%). The efficiency was higher when we used the mean values of the nuclear area in the deep layer (96.1%) and the maturation parameter (85.4%). By applying the mean nuclear area in the deep portion and the maturation parameter simultaneously, we gained the highest efficiency, specificity, and sensitivity for the distinction between benign dermal nevi and malignant melanomas (0.968, 0.955, 1) as well as for the distinction between Spitz's nevi and malignant melanomas (0.986, 0.950, 1). Our study shows that morphometry provides reliable diagnostic results in routinely sampled melanocytic skin tumors.     

Diagnostic problems in skin tumors

Several starting-points for improvement of diagnostic accuracy in skin tumors are illustrated by examples: Frequencies and direction of diagnostic errors, as well as the value of diagnostic criteria need further investigations. Frequent "customary" diseases need more attention in medical textbooks, compared with less frequent "interesting" syndromes. Erroneous denominations and historical false classifications which are handed down in the literature demand correction. The possibility continues, that apparently clearly defined entities may decay; they may be replaced by new entities in consequence of new perceptions.     

Tubulin expression in melanocytic skin tumors. An immunohistochemical study

The microtubulus system as a part of the cellular cytoskeleton contributes to cell movement. Microtubulus assembly and disassembly is considered to be essential for tumor invasion and serves as a target for tumor chemotherapy. Using immunohistochemical methods, we investigated the distribution of tubulin in normal skin and 34 melanocytic skin tumors. In normal skin, tubulin was strongly expressed in dermal nerves, melanocytes, fibroblasts within the papillary dermis and in myoepithelial cells. In melanocytic skin tumors, nevus cells and melanoma cells stained positive, particularly at the periphery of the lesions, where there were single cells and small nests. The main difference between benign and malignant melanocytic tumors was found in the stromal cells: In melanocytic nevi, the stromal fibroblasts were entirely tubulin negative; whereas, adjacent to the invasive edge in primary and metastatic malignant melanoma, the stroma fibroblasts were strongly positive. Our results show that tubulin is regularly expressed in melanocytic skin tumors and may serve as a prerequisite for cell movement. The pronounced expression of tubulin in fibroblasts surrounding malignant melanocytic skin lesions reflects a stromal alteration that might contribute to tumor invasion.     

Melanotic pigmentation in excision scars of melanocytic and non-melanocytic skin tumors

The appearance of pigmented lesions in melanoma surgical scars is a frequent finding that in some instances may cause confusion with a melanoma persistence. Nevertheless, only a few papers have dealt with this subject in the dermatologic literature. The melanoma surgical scars of 60 consecutive patients were reviewed with special attention to the presence of pigmentation and its clinical characteristics. Simultaneously, the scars of 60 consecutive patients who had been subjected to excision of a non-melanoma skin tumor were also studied. Biopsies were performed in representative clinical cases of pigmented lesions arising on the scars of both groups, as well as in non-pigmented scars, and processed for hematoxylin-eosin and immunohistochemistry. Pigmented lesions were present in a similar percentage in both groups (30% in melanoma scars (18/60) and 25% in non-melanoma scars (15/60)). Clinically, three types of clinical pigmentation were observed: lentigine-like lesions; pigmented streaks in scars after direct closure; and diffuse pigmentation in grafts. Histologically, two patterns emerged: one with lentiginous epidermal hyperplasia, hyperpigmentation, and a normal or moderately increased number of melanocytes; and a second one characterized by melanocytic hyperplasia of a variable degree. The scar process itself, irrespective of the tumor excised, seems to be responsible for the pigmentation. We suggest the existence of an induction process of scar tissue acting on melanocytes of the overlying epidermis.     

Spitz样肿瘤研究进展

Spitz痣、非典型Spitz肿瘤和Spitz样黑素瘤的临床及组织学特征有重叠, 三者鉴别极具挑战。该文结合Spitz样肿瘤的临床和组织学特征, 综述其免疫组化特点和荧光原位杂交检测方法在本组疾病中的研究进展。     

Tissue microarray analysis of methylthioadenosine phosphorylase protein expression in melanocytic skin tumors

BACKGROUND: Using tissue microarrays, we investigated whether methylthioadenosine phosphorylase (MTAP) protein expression is associated with clinicopathologic variables in benign and malignant melanocytic skin tumors. OBSERVATIONS: Cytoplasmic MTAP expression was detected in 227 (72.1%) of 315 informative cases. Expression was significantly reduced in primary malignant melanomas and in melanoma metastases compared with benign nevi (P<.001 for both). No difference was noted in MTAP expression between primary malignant melanomas and melanoma metastases. In primary malignant melanomas, a Ki67-labeling index less than 5% was associated with MTAP expression (P = .04), suggesting that loss of MTAP expression is associated with proliferation. No other variables had significant associations with MTAP expression. Lymph node metastases demonstrated significantly higher MTAP expression compared with skin metastases (P = .01). In the overall cohort, MTAP expression was not associated with prognosis. Among 26 patients with MTAP-positive melanomas and tumor recurrence, 18 patients who received interferon therapy had a significant benefit compared with 8 patients who did not receive interferon therapy (P = .009). This was not seen in the patients with MTAP-negative tumors.Conclusion Methylthioadenosine phosphorylase protein expression may be a predictive marker of interferon therapy resistance in patients with melanoma and disease progression.     

Kl 67 immunostaining in melanocytic skin tumors. Correlation with histologic parameters

A total of 145 melanocytic tumors (nevus, 38; primary malignant melanoma, 72; metastatic malignant melanoma, 35) were stained with Ki 67 monoclonal antibody using a three-step immunoperoxidase technique. For each case, mean numerical density and maximum numerical density of Ki 67 positive nuclei (number per mm3) were quantitatively evaluated using interactive image analysis. Maximum numerical densities revealed highly significant differences. Within the group of primary malignant melanomas, there was a significant correlation between proliferative activity and maximum tumor thickness. Further, a 'Ki 67-prognostic index' was assessed in each case of primary malignant melanoma, calculating the product of the Breslow index and maximum numerical density/1000 (103 +/- 12; range 1-694). In a prospective, short-term evaluation of primary malignant melanomas, there was a significant difference concerning 'Ki 67-prognostic index' between disease-free survival and occurrence of metastases. After a follow-up time of 24 months, only 63% of the patients with a 'Ki 67-prognostic index' greater than 25 were disease-free, whereas no patient with a 'Ki 67-prognostic index' less than 25 was found to have metastases. We conclude: assessment of the maximum numerical density of Ki 67 reflects the degree of malignancy in melanocytic skin tumors; within primary malignant melanomas, maximum numerical density of Ki 67 positive cells correlates with well-established prognostic parameters (tumor thickness, level of invasion, mitotic rate); assessment of the 'Ki 67-prognostic index' may be of additional prognostic value for patients with primary malignant melanoma.     

Modified dermoscopic algorithm for the differentiation between melanocytic and nonmelanocytic skin tumors

BACKGROUND: The use of dermoscopy (dermatoscopy, epiluminescence microscopy, surface microscopy) improves the clinical diagnostic accuracy of skin tumors by applying different algorithms or scores. The first step in the dermoscopic evaluation is the differentiation between melanocytic and nonmelanocytic skin tumors. OBJECTIVE: To evaluate the diagnostic accuracy of the established dermoscopic algorithm (EDA) and the modified dermoscopic algorithm (MDA) for melanocytic versus nonmelanocytic skin tumors. METHODS: Two hundred forty-nine patients with melanocytic and nonmelanocytic skin lesions were included. Dermoscopic images of the tumors were taken with 10-fold magnification, followed by surgery and histopathology at the departments of Dermatology at the universities of Tuebingen, in Germany, and Naples, in Italy. Each lesion was classified using the EDA and MDA. In the MDA, accessory nipples and dermatofibromas were considered in particular. RESULTS: With the EDA, 225 of 249 (90.4%) skin tumors were correctly classified in one of the six groups. With the MDA, 237 of 249 (95.2%) were correctly classified. Improvement was achieved in 12 (4.8%) better classified skin tumors. In both algorithms, no melanoma was classified as a nonmalignant melanocytic tumor. All melanomas were classified in the group of melanocytic tumors and one melanoma was classified in the group of basal cell carcinomas. CONCLUSION: Both dermoscopic algorithms for the differentiation between melanocytic and nonmelanocytic skin tumors were simple and effective when applied step by step. The MDA is an improvement on the EDA with the classification of accessory nipples and dermatofibromas.     

Cytoskeleton and motility: an immunohistological and computer simulation analysis of melanocytic skin tumors

Tumor cell motility and tumor cell proliferation are supposed to be essential for tumor invasion. The cytoskeleton, which consists of different components, is considered to be important for maintaining cell shape and facilitating cell movement. Numerous data are available about tumor cell motility in vitro , but the behavior of tumor cells in vivo is as yet poorly understood. In the present study, estimates of tumor cell motility and proliferation were statistically derived from morphological tumor patterns in human melanocytic skin tumors, and their relationship to expression of certain cytoskeletal components was evaluated. Over-expression of vimentin within tumor cells correlated with low actual tumor cell motility and proliferation, indicating a structurally stabilizing function of these filaments. An overexpression of actin was found within tumor cells of high motility and proliferation, suggesting the contribution of cytocontractile elements to active tumor cell locomotion in situ. Concerning the cytoskeleton of the stromal cells, expression of actin, myosin and tubulin correlated with a high number of motile tumor cells and high mitotic counts. Thus increased tumor cell motility seems to be associated with cytoskeletal changes not only of the tumor cells themselves but also of the surrounding stromal cells.
Fink-Puches R, Smolle J. Cytoskeleton and motility: an immunohistological and computer simulation analysis of melanocytic skin tumors.     

Genetic alterations in melanocytic tumors

In the last decade, significant progress has been made in our understanding of the genetic alterations in melanocytic tumors. The most exciting finding is the discovery of oncogenic BRAF mutations in both malignant melanoma and melanocytic nevi. This finding indicates that activation of the mitogen-activated protein kinase pathway may be a critical initiating step of melanocytic neoplasia, and that the fundamental difference between melanoma and nevi may lie in the inhibitory machinery for this oncogenic signaling. In addition, different genetic alterations identified in melanomas at different sites and with different levels of sun exposure have been shown, indicating that there are several distinct genetic pathways in the development of melanoma. Different patterns of genetic alterations have also been identified among different kinds of melanocytic nevi. While acquired nevi and small congenital nevi show a high frequency of BRAF mutations regardless of their anatomic localization, the mutations were rare in medium-sized congenital nevi and giant congenital nevi. Spitz nevi show no BRAF mutations, while a subset of cases show HRAS mutations, often associated with a copy number increase of chromosome 11p. The clear differences in genetic aberration patterns have significant clinical implications in the diagnosis and treatment of melanocytic tumors.     

Expression of galanin in melanocytic tumors

IntroductionGalanin is a neuropeptide with wide-ranging effects, especially within the endocrine and nervous systems. Galanin and its receptors are present in human skin. Galanin is expressed in different neural, endocrine and neuroendocrine tumors and, on the other hand, several neuropeptides, particularly α-MSH, seem to play a role in the pathogenesis of melanoma.ObjectiveTo investigate the expression of galanin in cutaneous melanomas and melanocytic nevi and correlate it with α-MSH expression and several prognostic factors for melanoma.Material and methodsWe performed an observational and retrospective study of the immunohistochemical expression of galanin and α-MSH in samples of cutaneous melanomas diagnosed in the last 5 years in the San Jorge Hospital, Huesca (Spain). Different types of melanocytic nevi were also analyzed.ResultsA total of 130 pigmented lesions were studied: 38 primary cutaneous melanomas, 6 cutaneous melanoma metastases and 86 melanocytic nevi. Immunostaining with galanin and α-MSH was significantly higher in melanomas than in melanocytic nevi (p < 0.001), although spindle cell and blue nevi showed significant expression of α-MSH. More than 50 % of nodular melanomas and 90 % of superficial spreading melanomas were positive for galanin and α-MSH, and the latter also showed the highest percentage of positive cells for galanin (mean 35.09 ± 28.16) as well as for α-MSH (mean 67.64% ± 35.38). A positive correlation of 71 % was found for immunostaining of both neuropeptides in melanomas. No significant correlation was observed between galanin expression and age, gender, location of the lesions, Breslow index, Clark level and mitotic index.ConclusionOur study shows the expression of galanin in cutaneous melanoma and its significant correlation with α-MSH immunostaining.     

Increased tenascin expression in melanocytic tumors

Tenascin is a large extracellular matrix glycoprotein which is widely distributed in normal, hyperplastic and neoplastic tissues. Its function is unknown but it has been associated with the epithelial-stromal interactions, such as cell adhesion and movement which take place, e.g. in morphogenesis, cellular proliferation and neoplasia. In this study, we investigated tenascin expression in 70 benign, dysplastic and malignant melanocytic tumors by using immunohistochemistry and monoclonal anti-tenascin L43DB7C8 antibody on paraffin sections. In all types of benign nevi, both intradermal, compound and functional, there was a moderate expression of tenascin at the dermoepidermal junction and in the papillary dermis. In dysplastic nevi, the fibrotic areas in the papillary clermis also showed a moderate staining for tenascin. Invasive malignant melanomas showed the strongest expression of tenascin. In addition to the staining at the dermo-epidermal junction and in the papillary clermis, there was a variable expression of tenascin in the reticular dermis. Intracyloplasmic tenascin was detected both in primary melanomas and melanoma metastases. In conclusion, we have shown that tenascin expression is moderately increased in benign and dysplastic melanocytic tumors and greatly increased in malignant melanomas and melanoma metastases. The function of tenascin may be related to the cellular-stromal interactions and it is possibly associated with the proliferation and spread of the melanocytic tumors.     

High-resolution laser Doppler perfusion imaging aids in differentiating between benign and malignant melanocytic skin tumours

Malignant melanomas are characterized by heterogeneity and asymmetry as well as by a higher density of blood vessels than benign pigmented tumours. The aim of this study was to evaluate the benefit of high-resolution laser Doppler perfusion imaging (LDPI) in the differential diagnosis of pigmented skin tumours. One-hundred-and-eighty-nine patients were examined with the LDPI, 22 with malignant melanomas, 39 with clinically suspicious dysplastic melanocytic naevi and 27 with basal cell carcinomas. Following examination, the tumours were excised and examined histologically. A control group of 101 melanocytic naevi showed clinically and, with epiluminescence microscopy, definitely benign criteria. These naevi were not excised. In malignant melanomas there was a 3.6+/-1.5 times higher perfusion than in healthy skin. The corresponding figures for clinically suspicious melanocytic naevi and basal cell carcinomas were 2.2+/-1.1 and 2.0+/-0.7, respectively. The increase in flow in malignant melanomas was significantly higher than in clinically suspicious melanocytic naevi and basal cell carcinomas (p < 0.001). All malignant melanomas showed at least 1.8 times higher flow values than healthy skin. When this value is taken as the basis for the diagnosis "benign or malignant", the LDPI proved a sensitivity of 100% and a specificity of 85%. If only the distinction between malignant melanomas and clinically suspicious naevi is considered, the specificity is reduced to 48%. There was no correlation between tumour thickness and increase in the mean perfusion of malignant melanomas (r = 0.14; p = 0.5). High-resolution LDPI can be used as an additional automatic screening method.