DNA ploidy and nuclear morphometry for the classification of dysplastic nevi

OBJECTIVE: To examine the diagnostic value of DNA ploidy and nuclear morphometric features in sporadic dysplastic nevi as compared to those in compound nevi and melanoma. STUDY DESIGN: DNA ploidy profiles plus seven direct and three derived nuclear features were obtained in a series of 120 melanocytic skin neoplasms (30 dysplastic nevi [DN], 30 melanomas [MM], 60 compound nevi [CN]) and the results compared. RESULTS: DNA ploidy separated melanomas from benign melanocytic skin neoplasms with 96.5% accuracy in classifying the grouped cases. The derived nuclear shape factor Form PE and nuclear axis ratio were the most successful discriminants separating DN from MM but allowed only 73.3% correct classification of cases. Separation of DN from CN was best achieved using Form PE and mean nuclear area (74.4% correctly classified). Results from compound nevi in subjects < 25 years of age fell between those for DN and MM. CONCLUSION: Quantitative nuclear cytologic characteristics in sporadic dysplastic nevi span a range seen in common nevi through to those in thin melanomas. Cytologic changes in sporadic dysplastic nevi overlap those seen in other melanocytic skin neoplasms. Therefore, other reproducible morphometric features need to be assessed in order to further refine the histopathologic diagnosis of this entity.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

Discriminant analysis of image karyometric variables in benign and malignant melanocytic skin lesions

OBJECTIVE: To perform a quantitative analysis to identify which of 7 nuclear morphometry-related variables are of diagnostic value in distinguishing benign from malignant melanocytic skin lesions. STUDY DESIGN: At the Institute of Pathology, University of Nis, formalin-fixed, paraffin-embedded skin biopsies from 23 cases of benign nevi (18 intradermal and 5 junctional) and 25 cases of primary nodular malignant melanomas were retrieved. Specimens were routinely stained with hematoxylin and eosin and analyzed using a computer-assisted interactive image analysis system. Nuclear area, equivalent diameter, volume of equivalent sphere, perimeter, mean chord, circularity and integrated optical density were estimated after manual editing of binary images. RESULTS: In univariate analysis, 6 features were found to be significantly different between the benign and malignant groups (P < .0001); all measured nuclear variables (except circularity) were higher in malignant melanomas. No significant differences were found among lesions with respect to nuclear shape. Using discriminant function analysis, a correct diagnosis was achieved in 95.8% of benign nevi cases and 84.0% of malignant melanoma cases. The best discriminant variable was nuclear area. CONCLUSION: Image analysis is diagnostically relevant to the evaluation of melanocytic lesions of the skin. The area of the nucleus appeared to have potential for differentiating benign from malignant tumors and can be estimated in the course of routine histology.     

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Karyometry of melanocytic lesions: quantitative assessment of the 'maturation to the depth'

40 cases each of malignant melanoma. Spitz's nevus and benign intradermal nevus were examined using an interactive image analysis system. 60 consecutive nuclei were evaluated in the upper and lower portion of the melanocytic lesions. Besides basic karyometric data, a 'maturation parameter' (MP) expressing the previously described 'maturation to the depth' was assessed in each individual case, by calculating the ratio of the nuclear area in the deep portion and in the superficial portion. When the three parameters (superficial nuclear area, deep nuclear area and maturation parameter) were evaluated separately (k-nearest neighbour method), the efficiency of the superficial nuclear area was only 19%, compared with 72% for the deep nuclear area and 94% for the maturation parameter. Combination of the maturation parameter and the deep nuclear area provided an efficiency of 98% with a sensitivity of 98% and a specificity of 97%. The results indicate that the maturation parameter is superior to conventional karyometric data in the differentiation between benign and malignant melanocytic lesions.     

Ki-67 immunostaining and stereologic estimation of nuclear volume in melanocytic skin tumors

OBJECTIVE: To investigate the proliferative activity and mean nuclear volume (MNV) of melanocytic skin tumors. STUDY DESIGN: Proliferative activity, assessed by immunostaining for the Ki-67 monoclonal antibody (reactive with all actively cycling cells), and MNV, estimated by means of a stereologic method, were determined in 60 cutaneous melanocytic tumors, including 28 primary malignant melanomas (PMM), 13 compound nevi (CN), 11 dysplastic nevi and 8 metastatic malignant melanomas. RESULTS: Both MNV and Ki-67 expression differed significantly between CN and other melanocytic tumors and showed a good correlation with Clark's level (a well-established prognostic parameter in PMM). CONCLUSION: The association of proliferative activity and quantitative nuclear features may be helpful in the interpretation of the degree of malignancy in melanocytic skin tumors.     

Collagenase-3 (MMP-13) expression in cutaneous malignant melanoma

BACKGROUND: Matrix metalloproteases (MMPs), enzymes with the ability to degrade the extracellular matrix, play an important role in tissue invasion by cutaneous malignant melanoma (CMM). One specific MMP, collagenase-3 (MMP-13), is thought to have a key function in the activation of MMP. AIMS: To evaluate the expression of MMP-13 in CMM and assess its possible relationship to clinical and pathological parameters. METHODS: MMP-13 expression was analyzed in 51 paraffin-embedded tumor samples from patients with invasive CMM, ten samples from in situ melanomas, and in eight samples from benign lesions (three dermal melanocytic nevi, three compound melanocytic nevi and two atypical melanocytic nevi) using immunohistochemical techniques. The median follow-up period in patients with invasive CMM was 50 months. RESULTS: Benign lesions were consistently negative for MMP-13, whereas three of the ten in situ melanomas (30%) and 23 of the 51 invasive CMMs (45%) showed positive immunostaining for MMP-13. The percentage of MMP-13-positive tumors correlated significantly and positively with the mitotic index (p=0.002) in invasive CMM. However, our results did not show any significant association between tumoral MMP-13 expression and relapse-free survival in patients with invasive CMM. CONCLUSIONS: MMP-13 appears to be a factor associated with tumor aggressiveness in CMM. It seems to eliminate an important barrier not only against tumoral invasion but also against proliferation.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

Nucleolar organizer regions in pigmented skin lesions. Value in the differential diagnosis of Spitz nevi.

Forty-one cases of typical melanocytic skin lesions (15 intradermal nevi, 14 Spitz nevi and 12 malignant melanomas) were used to investigate the value of staining of nucleolar organizer regions (NORs) in the differential diagnosis of such pigmented lesions. Histologic sections were stained by the silver colloid (Ag) method, with and without the prior use of a melanin blocking agent. There were statistically significant differences in the mean numbers of AgNORs per nucleus between the groups of lesions studied (1.658 for intradermal nevi, 3.0042 for Spitz nevi and 6.669 for malignant melanomas). Sections treated with potassium permanganate (melanin blocking agent) prior to staining showed an obvious increase in the AgNOR scores in all groups; this increase was highest for Spitz nevi. Although AgNOR staining allows a distinction to be made between intradermal nevi and malignant melanomas, the striking overlap between the counts for Spitz nevi and malignant melanomas precludes the use of this technique as the sole method for establishing the diagnosis of malignancy. Other clinical and morphologic data are especially required to make the diagnosis of Spitz nevi.     

Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.     

Evaluation of tubulin β‐3 as a novel senescence‐associated gene in melanocytic malignant transformation

Malignant melanoma might develop from melanocytic nevi in which the growth‐arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth‐arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β‐3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β‐3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β‐3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β‐3 as a candidate marker.     

Tissue Factor Expression and Serum Level in Patients with Melanoma does not Correlate with Disease Progression

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

DNA ploidy-characteristics of human malignant melanoma analysed by flow cytometry and compared with histology and clinical course

Flow cytometric analysis of nuclear DNA content is valuable for indicating ploidy- and proliferation abnormalities in surgically removed human malignant melanomas. In 35 primary cutaneous melanomas, 20 metastases of melanoma in skin and lymph nodes, and 16 nevi the DNA distribution was analyzed by flow cytometry and compared with a variety of histological parameters and the subsequent clinical course. Heteroploid DNA distributions with increased polyploid or aneuploid fractions were found in 26 primary melanomas (74%), 14 metastases (70%), and 4 nevi (25%) indicating tumor clones with an abnormal nuclear DNA content. Three or more cell clones in a single biopsy was found in 10 primary melanomas, 2 metastases, and 1 nevus. The frequency of heteroploidy was significantly higher in primary and secondary melanomas than in nevi (p less than 0.001) and was correlated significantly with a high mitotic activity (p less than 0.002), marked nuclear pleomorphism (p less than 0.01), large nucleoli (p less than 0.01) and a thickness of the primary melanoma of more than 2.25 mm (p less than 0.02). Such histologic findings in malignant melanomas have been shown previously to be correlated with a bad prognosis. No significant correlation was found between heteroploidy and the histologic type of melanoma or the level of invasion. A 2-year clinical follow-up showed that more patients died from melanoma if the DNA distribution in the primary or secondary melanoma was heteroploid (6/26; 23% and 8/13; 62% respectively) than if it was diploid (0/9; 0% and 2/5; 40% respectively). However, the differences were not statistically significant. It is concluded that heteroploidy 1) is not an absolute criterion of malignancy, 2) is significantly correlated with histologic features indicating marked cellular anaplasia, and 3) is apparently correlated with a bad prognosis.     

Discriminating nevus and melanoma on paraffin-embedded skin biopsies using FTIR microspectroscopy

FTIR microspectroscopy, in combination with cluster analysis, has been used to characterise skin tissues, in order to discriminate cancerous from non-cancerous ones. The main objective of this in vitro study was to demonstrate the applicability of infrared spectral imaging to separate, on paraffinised biopsies, pigmented nevi (benign skin lesions) from melanomas (malignant skin lesions). Infrared spectra were collected from paraffin-embedded samples of nevi and melanomas, without deparaffinisation. Despite the important contribution of the paraffin in these spectra, it was possible to find meaningful and discriminating spectral regions. Spectral imaging was first performed to localize different skin layers (dermis and epidermis). Spectra extracted from the images were subjected to hierarchical classification algorithm, which allowed the discrimination of melanomas from the nevi, using selected spectral windows that correspond to vibrations of DNA and melanin content. The diversity of skin lesions and direct accessibility to the skin make this organ an interesting field of investigation using this technique.     

Expression of cyclins A and E in melanocytic skin lesions and its correlation with some clinicopathologic features

Cyclins play a fundamental role in the cell cycle. Recent studies have focused on their role in the development of various malignancies. The objective of this study was to evaluate and compare the expression of cyclins A and E in common nevi, dysplastic nevi and malignant melanomas, and to investigate the relationship between cyclin expression and some pathological parameters such as tumor thickness, ulceration, regression, and mitotic rate, as well as several clinical and phenotypic parameters such as skin phototype, hair and eye color, number of nevi, personal or family melanoma history, and personal history of nonmelanoma skin cancer (NMSC). A total of 102 melanocytic skin lesions, including 30 common nevi, 38 dysplastic nevi and 34 melanomas, were examined. Expression of cyclins was detected by immunohistochemistry and quantified as a percentage of immunostained cell nuclei in each sample. Significant differences in expression of both cyclins were found between all lesion types: the median percentage of cyclin A-positive nuclei was 8.2% in melanomas, 3.4% in dysplastic nevi, and 0.95% in common nevi (p < 0.001). The corresponding percentages for cyclin E were 9.5%, 4.25% and 1.44% (p < 0.001). Expression of both cyclins was significantly higher among patients with a personal history of NMSC. Cyclin A was also significantly overexpressed in patients with a high total nevus count (TNC) compared to moderate and low TNC. Expression of cyclins did not significantly correlate with the other clinicopathologic features investigated. These findings indicate the possible involvement of cyclins A and E in the pathogenesis of malignant melanoma. Our results also show a potential diagnostic significance of these cyclins as markers allowing discrimination between dysplastic nevi and melanoma.     

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun‐exposed sites. Most occur in the oral cavity, with a subset having intra‐epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ‐line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c‐kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.