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Early detection and treatment of melanoma, the most aggressive skin cancer, improves the median 5-year survival rate of patients from 25% to 99%. Melanoma development involves a stepwise process during which genetic changes drive histologic alterations within nevi and surrounding tissue. Herein, a comprehensive analysis of publicly available gene expression data sets of melanoma, common or congenital nevi (CN), and dysplastic nevi (DN), assessed molecular and genetic pathways leading to early melanoma. The results demonstrate several pathways reflective of ongoing local structural tissue remodeling activity likely involved during the transition from benign to early-stage melanoma. These processes include the gene expression of cancer-associated fibroblasts, collagens, extracellular matrix, and integrins, which assist early melanoma development and the immune surveillance that plays a substantial role at this early stage. Furthermore, genes up-regulated in DN were also overexpressed in melanoma tissue, supporting the notion that DN may serve as a transitional phase toward oncogenesis. CN collected from healthy individuals exhibited different gene signatures compared with histologically benign nevi tissue located adjacent to melanoma (adjacent nevi). Finally, the expression profile of microdissected adjacent nevi tissue was more similar to melanoma compared with CN, revealing the melanoma influence on this annexed tissue., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

PRAME (PReferentially expressed Antigen in MElanoma) is a cancer testis antigen that is frequently expressed in melanoma compared to benign melanocytic proliferations and nevi. However, the interpretation of the intensity and distribution of PRAME immunostaining is not standardized a lot, which makes interpretation difficult. PRAME-stained histological slides of superficial spreading melanomas (SSM) and dysplastic nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. t -tests and ROC AUCs were performed with SPSS. A p -value of <0.05 was used for statistical significance, and a ROC AUC score of >0.8 was considered a good result. A cut-off score was defined in an evaluation cohort and subsequently analyzed in an independent validation cohort. In total, 81 PRAME-stained specimens were included. The evaluation cohort included 32 (50%) SSM and 32 (50%) DN, and the mean of PRAME-positive cells/mm 2 for the entire lesion was 455.3 (SD 428.2) in SSM and 60.5 (SD 130.1; p < 0.001) in DN. The ROC AUC of PRAME-positive cells of the entire lesion was 0.866, and in the epidermis it was 0.901. The defined cut-off score to distinguish between DN and SSM was 97.67 cells/mm 2 . In the validation cohort, 16 out of 17 cases (94.1%) were correctly classified by the cut-off score. The computer-aided assessment of PRAME immunostaining is a useful tool in dermatopathology to distinguish between DN and SSM. Lesions with a moderate expression and indifferent morphologic features will remain a challenge for dermatopathologists.

Background and Objectives: Epidemiologic data show significant differences in melanoma incidence and outcomes between sexes. The role of hormonal receptors in the pathogenesis of melanocytic lesions remains unclear, thus we performed this study aiming to assess estrogen receptors expression in different melanocytic lesions. Materials and Methods : We performed a cross-sectional study that included 73 consecutively excised melanocytic lesions. Estrogen receptor alpha (ERα), beta (ERβ), and G-protein coupled estrogen receptor (GPER) expression was analyzed in melanocytes and keratinocytes of common nevi, dysplastic nevi, melanoma, healthy skin margin, and in sebaceous and sweat gland cells. Results : ERβ expression was higher in dysplastic nevi margin melanocytes compared to common nevi ( p = 0.046) and in dysplastic nevi keratinocytes compared to melanoma keratinocytes ( p = 0.021). ERβ expression was significantly higher in margin melanocytes compared to melanoma melanocytes ( p = 0.009). No difference in ERβ expression was shown between melanocytes of three types of lesions. GPER expression was higher in nuclei and cytoplasm of dysplastic nevi ( p = 0.02 and p = 0.036 respectively) and at the margin compared to melanoma. GPER expression was lower in sebaceous glands of tissue surrounding common nevi ( p = 0.025) compared to dysplastic nevi. GPER expression was higher in skin margin tissue melanocytes ( p = 0.016 nuclear, p = 0.029 cytoplasmic) compared to melanoma melanocytes. There were no differences in ERα expression between the melanocytic lesions. Conclusion: Further large-scale studies are warranted to investigate the potential role of ERβ and GPER in the pathogenesis of melanocytic lesions.

Background. Dendritic cells could be involved in immune surveillance of highly immunogenic tumors such as melanoma. Their role in the progression melanocytic nevi to melanoma is however a matter of controversy. Methods. The number of dendritic cells within epidermis, in peritumoral zone, and within the lesion was counted on slides immunohistochemically stained for CD1a, CD1c, DC-LAMP, and DC-SIGN in 21 of dysplastic nevi, 27 in situ melanomas, and 21 invasive melanomas. Results. We found a significant difference in the density of intraepidermal CD1c+ cells between the examined lesions; the mean CD1c cell count was 7.00/mm 2 for invasive melanomas, 2.94 for in situ melanomas, and 13.35 for dysplastic nevi. The differences between dysplastic nevi and melanoma in situ as well as between dysplastic nevi and invasive melanoma were significant. There was no correlation in number of positively stained cells between epidermis and dermis. We did not observe any intraepidermal DC-LAMP+ cells neither in melanoma in situ nor in invasive melanoma as well as any intraepidermal DC-SIGN+ cells in dysplastic nevi. Conclusion. It was shown that the number of dendritic cells differs between dysplastic nevi, in situ melanomas, and invasive melanomas. This could eventually suggest their participation in the development of melanoma., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Background/aim: Melanoma is the most aggresive malignant tumor of the skin. Contradictory data was published on vascular endothelial growth factor (VGEF) in tumor samples and its role in skin melanoma progression and prognosis. The aim of this study was to investigate the significance of VEGF expression as a prognostic parameter in melanoma., Methods: The experimental group included 81 patients with primary skin melanomas treated from 2009 to 2013 at the Military Medical Academy, Belgrade. The control group included 20 patients with dysplastic and 20 with benign naevi. Stratification was done according to gender, age, clinical and patological stage, localization, histologic type, Clark's, Breslow, mitotic count, regression and ulceration, tumor infiltrating lymphocytes and metastatic spread.Immunohistochemical staining was performed on skin biopsies using DAKO anti-VEGF antibodies (Ab), LSAB+HRP, DAB and microvawe antigen (Ag) retrieval in DAKO pH 9.0 solution. For statistical data analysis was done with ANOVA, Bonferroni, Mann Whitney and Wilcox on test., Results: The mean intensity of VEGF staining was statistically significantly higher in melanomas than in benign or dysplastic naevi. Furthermore, the highest recorded values were in Ia and IV clinical stages. The majority of melanomas with high intensity of VEGF staining were in pT1a pathological stage. Melanomas with the highest mitotic count (> 6) had a significantly higher intensity of VEGF staining than those with < 2 mitoses. The higest intensity of staining was in melanomas without significant lymphocytic infiltrate and the lowest was in those with brisk lymphocytic infiltrate, thus a statistical difference was siginifant. The mean intensity of VEGF staining was highest in melanomas with lymphovascular invasion. There was no statistically significant difference between VEGF and any other parameter., Conclusion: VEGF in primary skin melanomas plays an important role in tumor progression and is linked to the absence if tumor infiltrating lymphocytes and the presence of lymphovascular invasion. More detailed studies have to be done on VEGF prognostic value in melanoma on a larger number of patients.

KAI1/CD82 is a member of the transmembrane 4 superfamily, which was first identified as a metastasis suppressor for prostate cancer. The expression of KAI1 was found to be reduced in many types of cancers, including prostate, breast, ovarian, cervical and endometrial cancer. However, the role of KAI1 in melanoma pathogenesis is not known. In this study, we investigated the expression level of KAI1 in a large set of melanocytic lesions at different stages. We found that the expression of KAI1 is significantly decreased during melanoma progression. In fact, KAI1 expression is drastically reduced in primary melanoma compared with dysplastic nevi (P = 1.8×10(-4)) and further reduced in metastatic melanoma compared with primary melanoma (P = 9.4 × 10(-15)). Furthermore, decreased KAI1 staining is strongly correlated with a worse 5 year and 10 year patient survival. Multivariate Cox regression analysis showed that KAI1 is also an independent prognostic factor for both 5 year and 10 year survival. Moreover, we found that overexpression of KAI1 significantly inhibited melanoma cell migration through suppression of Rho-associated kinase-mediated formation of stress fiber. Our data also suggested that overexpression of KAI1 significantly inhibited melanoma cell invasion by reducing the activity of metalloproteinase-2. In addition, we found that suppression of melanoma cell migration by KAI1 is mediated by another tumor-suppressor protein called inhibitor of growth 4 through the regulation of p65. Taken together, our data suggest that KAI1 may be used as a promising prognostic marker and a possible therapeutic target for human melanoma.

The E3 ubiquitin ligase Fbw7 (F-box and WD repeat domain-containing 7) is broadly considered as a tumor suppressor because of its role in the turnover of several well-known oncoproteins. However, the role of Fbw7 in melanomagenesis is not clear. To investigate the expression profile and biological functions of Fbw7 in melanoma, we examined Fbw7 expression level using melanoma tissue microarray and immunohistochemistry. Our data showed that Fbw7 expression is significantly reduced in primary melanoma compared with dysplastic nevi (P=0.020) and further reduced in metastatic melanoma compared with primary melanoma (P=0.011). Furthermore, we observed a strong correlation between negative Fbw7 expression and a worse 5-year survival of melanoma patients (P=0.015). We also found that both Fbw7 protein and mRNA expression was significantly reduced in nine melanoma cell lines compared with normal melanocytes. Moreover, our in vitro studies showed a remarkable increase of cell migration and stress fiber formation in Fbw7 knockdown cells, and treatment of selective MEK (MAPK/ERK kinase) inhibitor abrogated Fbw7α knockdown-induced melanoma cell migration. Taken together, our findings indicate that Fbw7 has an important role in melanoma progression, and it inhibits melanoma cell migration through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway and may serve as a prognostic marker.

Background: Distinguishing melanoma from dysplastic nevi can be challenging., Objective: To assess which putative molecular biomarkers can be optimally combined to aid in the clinical diagnosis of melanoma from dysplastic nevi., Methods: Immunohistochemical expressions of 12 promising biomarkers (pAkt, Bim, BRG1, BRMS1, CTHRC1, Cul1, ING4, MCL1, NQO1, SKP2, SNF5 and SOX4) were studied in 122 melanomas and 33 dysplastic nevi on tissue microarrays. The expression difference between melanoma and dysplastic nevi was performed by univariate and multiple logistic regression analysis, diagnostic accuracy of single marker and optimal combinations were performed by receiver operating characteristic (ROC) curve and artificial neural network (ANN) analysis. Classification and regression tree (CART) was used to examine markers simultaneous optimizing the accuracy of melanoma. Ten-fold cross-validation was analyzed for estimating generalization error for classification., Results: Four (Bim, BRG1, Cul1 and ING4) of 12 markers were significantly differentially expressed in melanoma compared with dysplastic nevi by both univariate and multiple logistic regression analysis (p < 0.01). These four combined markers achieved 94.3% sensitivity, 81.8% specificity and attained 84.3% area under the ROC curve (AUC) and the ANN classified accuracy with training of 83.2% and testing of 81.2% for distinguishing melanoma from dysplastic nevi. The classification trees identified ING4, Cul1 and BRG1 were the most important classification parameters in ranking top-performing biomarkers with cross-validation error of 0.03., Conclusions: The multiple biomarkers ING4, Cul1, BRG1 and Bim described here can aid in the discrimination of melanoma from dysplastic nevi and provide a new insight to help clinicians recognize melanoma.

Background: The cyclin-dependent kinase inhibitor p27 plays important roles in cell proliferation, cell motility, and apoptosis. Interestingly, the nuclear and cytoplasmic p27 exert opposite biological functions. In this study, we investigated the prognostic impact of subcellular p27 expression., Methods: We constructed melanoma tissue microarrays in a large series of melanoma patients, including 29 normal nevi, 52 dysplastic nevi, 270 primary melanomas, and 148 metastatic melanomas. The expression level of subcellular p27 in different stages of melanocytic lesions and its prognostic significance were evaluated., Results: Compared with dysplastic nevi, nuclear p27 expression was remarkably reduced in primary melanomas and further reduced in metastatic melanoma (P < 0.001 for both), whereas cytoplasmic p27 expression is significantly increased from dysplastic nevi to primary melanomas (P = 0.032) and further increased in melanoma metastases (P = 0.037). Although loss of nuclear p27 expression is correlated with a worse 5-year survival of primary melanoma patients in Kaplan-Meier analysis (P = 0.046), it is not a prognostic factor by multivariate Cox regression analysis. On the contrary, Kaplan-Meier analysis showed that gain of cytoplasmic p27 was associated with a poor 5-year survival of metastatic melanoma patients (P < 0.001). Multivariate Cox regression analysis revealed that positive cytoplasmic p27 expression is an independent prognostic factor to predict metastatic melanoma patient outcome., Conclusion: Cytoplasmic p27 may serve as a promising prognostic marker for metastatic melanoma., Impact: Because there is no reliable prognostic marker for metastatic melanoma, our finding may have important clinical implications using cytoplasmic p27 as a prognostic biomarker for advanced melanoma., (©2011 AACR)

Nodal, a potent embryonic morphogen in the transforming growth factor-beta family, is a proposed key regulator of melanoma tumorigenicity. However, there has been no systematic study of Nodal expression in melanocytic lesions. We investigated Nodal expression by immunohistochemistry in 269 melanocytic lesions, including compound nevi, dysplastic nevi, congenital nevi, Spitz nevi, melanoma in situ, malignant melanoma including the variant desmoplastic melanoma, and metastatic melanoma. We found that the Nodal expression was significantly increased in malignant lesions (including melanoma in situ, malignant melanoma, and metastatic melanoma) compared with compound nevi, Spitz nevi, and dysplastic nevi. Surprisingly, congenital nevi expressed a level of Nodal comparable with malignant lesions, whereas desmoplastic melanoma showed lower expression than nondesmoplastic malignant melanoma (P<0.05). Deep melanoma (Breslow depth >1 mm) displayed a higher percentage of Nodal-positive tumor cells than did superficial melanoma (Breslow depth < or =1 mm), although there was no statistical difference in the overall staining intensity (P=0.18). Melanomas in situ showed a lower level of Nodal expression than did deep melanomas and metastatic melanomas (P<0.05). The low expression of Nodal in normal and dysplastic nevi, and its increasing expression with the progression of malignant lesions, are suggestive of a role for Nodal in melanoma progression.

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Inhibitor of growth (ING) 4 has been reported as a tumor suppressor and shown to diminish colony-forming efficiency, induce p53-dependent apoptosis and arrest cell cycle at G(2)-M phase. In this study, we investigated the role of ING4 in human melanoma pathogenesis. Using the tissue microarray technology, we found that ING4 expression is significantly decreased in malignant melanoma compared with dysplastic nevi (P < 0.0001, chi(2) test) and reduced ING4 staining is associated with melanoma thickness, ulceration (P = 0.034 and 0.002, respectively, chi(2) test) as well as poor overall and disease-specific 5-year survival of primary melanoma patients (P = 0.0002 and 0.001, respectively, chi(2) test). Cox regression analysis revealed that reduced ING4 staining is an independent factor for the poor prognosis of patients with primary melanomas. Furthermore, we found that overexpression of ING4 suppressed cell migration by 63% and inhibited the activity of Ras homolog gene family member A (RhoA) small GTPase protein and Rho-associated kinase (ROCK)-mediated formation of stress fiber in melanoma cells. Moreover, our data showed that overexpression of ING4 inhibited melanoma cell invasion by 43% compared with the control (P = 0.006, t-test) and ING4-overexpressing melanoma cells showed significantly reduced activity of matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, this study highlights the importance of ING4 in melanoma pathogenesis and ING4 may serve as a promising prognostic marker and a potential therapeutic target for human melanoma.

The identification of molecular markers of melanoma progression is needed to more accurately stage and identify treatments for patients with malignant melanoma. Previously, we demonstrated that loss of the activator protein-2alpha (AP-2alpha) expression results in overexpression of the protease-activated receptor-1 (PAR-1) in human melanoma cell lines. Here, we used a tissue microarray platform that consisted of 64 melanocytic lesions, including dysplastic nevi (N=21), primary melanoma (N=20), and metastatic melanoma (N=23). We analyzed the expression of AP-2 and PAR-1 simultaneously by immunofluorescent microscopy with an automated quantification laser scanning cytometer. AP-2 was highly expressed in normal cutaneous melanocytes and dysplastic nevi but not in melanoma metastases. We observed a significantly higher number of AP-2-positive cells in the dysplastic nevi (P=0.0013) and primary melanoma (P=0.0023) compared to the metastatic melanoma. In contrast, we observed a significantly higher percentage of PAR-1-positive cells in the metastatic melanoma compared to dysplastic nevi (P=0.0072) and primary melanoma (P=0.0138). Increased expression of PAR-1 in metastatic melanomas contributes to tumor progression by modulating expression of genes, such as IL-8, matrix metalloproteinase-2, vascular endothelial growth factor, platelet-derived growth factor, and integrins. These findings support our hypothesis that loss of AP-2 is a crucial event in the progression of human melanoma and contributes to the acquisition of the metastatic phenotype via upregulation of PAR-1.

Cutaneous malignant melanoma is a severe and sometimes life-threatening cancer. The molecular mechanism of melanomagenesis is incompletely understood. Deregulation of apoptosis is probably one of the key factors contributing to the progression of melanoma. The inhibitor of growth (ING) family proteins are candidate tumour suppressors which play important roles in apoptosis. Downregulated expression of ING proteins have been reported in several tumour types, including the loss of nuclear expression of p33ING1b in melanoma. As ING2 exhibits 58.9% homology with p33ING1b, we hypothesized that the aberrant expression of ING2 may be involved in melanomagenesis. Here, we used tissue microarray technology and immunohistochemistry to examine ING2 expression in human nevi and melanoma biopsies. Our data showed that nuclear ING2 expression was significantly reduced in radial growth phase (RGP), vertical growth phase (VGP), and metastatic melanomas compared with dysplastic nevi (P < 0.05). Our data also revealed that nuclear ING2 expression was not associated with patient's gender, age or tumour thickness, ulceration, American Joint Committee on Cancer (AJCC) stage, tumour subtype, location and 5-year survival (P > 0.05). Taken together, our results suggest that nuclear ING2 expression is significantly reduced in human melanomas and that reduced ING2 may be an important molecular event in the initiation of melanoma development.

Melanocytic dysplastic nevi were first described in both patients and their relatives who had one or several cutaneous malignant melanomas. Most of these dysplastic lesions are biologically stable, but some of them have severe histological atypia and can progress further to melanomas. Although several studies have suggested the etiological importance of dysplastic nevi in the development of melanomas, comprehensive reviews of the molecular changes in these dysplastic lesions are still scarce. To remedy this issue, this article analyzes the available molecular information about dysplastic nevi and provides the current state of knowledge regarding the karyotypic abnormalities of the melanoma/dysplastic nevus trait and the involvement of allelic loss, tumor suppressor genes, mismatch repair proteins, microsatellite instability, oncogenes, extracellular matrix proteins, and growth factors in the genesis of these lesions. These studies suggest that although some of these lesions represent "genetic dead-ends," others represent intermediate lesional steps in the melanoma tumorigenesis pathway.

We investigated the role of alterations of HDM2, the human homologue of murine mdm2, in the tumorigenesis and progression of cutaneous melanoma. A well-characterized cohort of 172 cases representing different points in the spectrum of melanocyte transformation (16 dysplastic nevi, 11 melanomas in situ, 107 invasive primaries, and 38 metastatic lesions), as well as 11 human melanoma cell lines were examined by immunohistochemistry and Western blotting for HDM2 protein expression, and by either Southern blotting (SB) or fluorescence in situ hybridization for HDM2 gene amplification. HDM2 overexpression, defined as >20% tumor cells showing nuclear immunoreactivity, was observed in 1 of 16 (6%) dysplastic nevi, 3 of 11 (27%) melanomas in situ, and 81 of 145 (56%) invasive primary and metastatic melanomas. Comparable frequencies of HDM2 overexpression were observed among invasive primary cases with differing tumor thicknesses as well as among the metastatic cases: 21 of 40 (53%) at < or =1.5 mm; 31 of 50 (62%) at 1.6-3.9 mm; 10 of 17 (58%) at >4 mm; and 19 of 38 (50%) metastases. HDM2 amplification was observed in 1 of 88 (1%) primary cases using fluorescence in situ hybridization, and in 0 of 12 (0%) metastatic cases that overexpressed HDM2 using SB. Melanoma cell lines expressed HDM2 protein, but there was no evidence of amplification by SB. Our data suggest that HDM2 protein overexpression is common in invasive and metastatic melanoma. Observing HDM2 overexpression in noninvasive melanoma suggests that expression of this oncogene may play an early role in melanocyte transformation. HDM2 amplification occurs infrequently, and other mechanisms that up-regulate HDM2 expression are under investigation.

Objective: We previously surveyed cyclin D1 expression in common acquired nevi, Spitz nevi, and malignant melanomas and reported that benign nevi maintain a zonal pattern of cyclin D1 expression, in contrast with malignant melanomas. Our aim was to extend those observations by examining cyclin D1 expression in dysplastic nevi., Methods: Cyclin D1 overexpression in 23 dysplastic nevi was detected by an immunohistochemical technique. The extent of atypia of the nevi was graded as mild, moderate, or severe, using previously established criteria., Results: Cyclin D1 overexpression in dysplastic nevi maintained a zonal pattern, similar to Spitz nevi. Cyclin D1 overexpression was greatest in the region of the epidermal-dermal junction and was significantly less prominent in the papillary and reticular dermis, suggesting that cyclin D1 expression is under cell control and correlates with maturation of nevus cells. Cyclin D1 overexpression also correlated with cytologic atypia, as dysplastic nevi with moderate or severe cytologic atypia contained a greater percentage of cyclin D1-positive cells than did nevi with mild atypia. Six dysplastic nevi with many cyclin D1--positive cells were assessed by fluorescence in situ hybridization studies using cyclin D1--specific and chromosome 11 centromeric probes. In all cases, there was no evidence of 11q13 translocation, amplification, or trisomy of chromosome 11., Conclusions: Cyclin D1 may be involved in the pathogenesis of dysplastic nevi. Cyclin D1 overexpression does not appear to be explained by cyclin D1 locus amplification or translocation in most cases, and it may be a result of other cell abnormalities that up-regulate the protein level of cyclin D1.

Aims: Compelling evidence from cell culture studies implicates cadherins in the neoplastic progression of melanocytic tumours but few reports describe the expression of cadherins and the related transmembrane proteins, catenins, in a full range of benign and malignant excised melanocytic tumours., Methods: Using immunohistochemistry and western blotting after tissue fractionation, the pattern of expression of cadherins/catenins was studied in a range of surgically excised melanocytic tumours, from dysplastic naevi to stage III cutaneous metastatic malignant melanoma., Results: Appropriate membranous expression of E-cadherins and P-cadherins is seen in dysplastic naevocytes with an epithelioid phenotype and is largely maintained with malignant transformation to radial growth phase melanoma and primary vertical growth phase malignant melanoma. Loss of membranous E-cadherin is seen in a small number of vertical growth phase melanomas only when metastasis has occurred. However, there is a concomitant dramatic loss of membranous P-cadherin expression in all melanomas at the same stage. A minority of metastatic melanomas show de novo membranous N-cadherin expression in comparison with dysplastic naevi and primary melanoma. Membranous expression of the desmosomal cadherin, desmoglein, was not seen in any tumour studied. Frequently, beta catenin is aberrantly produced in the cytoplasm of cells in dysplastic naevi and metastatic malignant melanoma, with an implied compromise to adhesive function. Furthermore, membranous gamma catenin expression was not seen in any of the 70 melanocytic tumours studied, implying obligatory transmembrane binding of cadherins to beta catenin for maintenance of adhesive function., Conclusions: The most important alterations in membranous cadherin and catenin expression are seen late in the biological progression of melanocytic tumours at the stage of "in transit" or regional lymph node metastasis, with implications for tumour growth, invasion, and dissemination.

High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas--IIB-MEL-J, IIB-MEL-LES, and IIB-MEL-IAN--express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA. Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The overall data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.

Stimulation of epidermal growth factor (EGF) receptor by ligands such as transforming growth factor (TGF) alpha may be associated with cell proliferation or transformation in both nevocytes and keratinocytes. Previously, EGF receptors have been identified within a variety of pigmented lesions, suggesting a possible responsiveness to ligands such as TGF alpha. In the present study, we characterize the intralesional expression and distribution of immunoreactive TGF alpha protein by avidin-biotin immunoperoxidase localization in benign nevi, congenital nevi, dysplastic nevi, and malignant melanomas. In situ hybridization techniques with TGF alpha riboprobes confirmed the constitutive production of TGF alpha in all types of pigmented lesions. The localization of TGF alpha expression to nevocytes when coupled with the previous reports of expression in basal keratinocytes suggests the possibility of either an autocrine mechanism of action for TGF alpha or a paracrine interplay of TGF alpha between keratinocytes and nevocytes within melanocytic lesions. An increase in immunoreactive TGF alpha in nevocytes was noted in both benign and dysplastic nevi from dysplastic nevus patients, as compared to benign nevi from normal patients. Congenital nevi and malignant melanomas showed an intermediate and variable level of TGF alpha immunoreactivity. When coupled with previous studies the data suggest linkage of the TGF alpha/EGF receptor pathway in the evolution of melanocytic lesions.

Degradation of the extracellular matrix and other tissue barriers by proteases like plasminogen activators (PAs) is a prerequisite for neoplastic growth and metastasis. Recently, we reported that highly metastatic behavior of human melanoma cells in nude mice correlates with urokinase-type PA (u-PA) expression and activity and with PA inhibitor type 1 and 2 (PAI-1, PAI-2) expression. Here we report on the occurrence of components of the PA system in the various stages of human melanoma tumor progression in situ. We studied the protein distribution on freshly frozen lesions of common nevocellular nevi (n = 25), dysplastic (= atypical) nevi (n = 16), early primary melanomas (n = 8), advanced primary melanomas (n = 11), and melanoma metastases (n = 17). Tissue-type PA was present in endothelial cells in all lesions, whereas in metastases it could be detected in tumor cells in a minority of the lesions. u-PA, its receptor, PAI-1, and PAI-2 could not be detected in benign and in early stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. u-PA was detected in stromal cells and in tumor cells at the invasive front, the u-PA receptor and PAI-2 in tumor cells, and PAI-1 in the extracellular matrix surrounding tumor cells. Localization of the corresponding messenger RNAs and enzyme activities revealed a similar distribution. We conclude that plasminogen activation is a late event in melanoma tumor progression.

This study will review the clinical features of dysplastic melanocytic nevi (DMN) in Japanese cases, and then show the fine structure of melanosomes/melanogenesis in the dysplastic melanocytes and the immunohistochemical property of DMN as identified by monoclonal antibodies against human melanosome specific antigen (HMSA). The incidence of DMN is quite low in the Japanese. It is indicated that this fact may partly explain the low incidence of malignant melanoma in Japanese; in particular, low incidence in sun-exposed areas. The patients with DMN were largely males. They were different from ordinary Japanese in that they usually burn and tan poorly after sun exposure. In addition, the synthesis of melanosomes in the dysplastic melanocytes was found to be abnormal and characterized by a significant alteration in the fine structure of the melanosomal matrix and the pattern of melanization. These abnormal melanosomes reacted positively with MoAb HMSA-1 and HMSA-2, which identify the structural matrix protein of melanosomes unique to neoplastic melanocytes. Importantly, HMSA was present in both dysplastic melanocytes and melanoma cells, but not in epidermal melanocytes (nevus cells) of common melanocytic nevi.