Shedding light on PRAME expression in dysplastic nevi: a cohort study.

Dysplastic nevi represent one of the least agreed-upon entities in dermatopathology despite the existence of established criteria. This study explores preferentially expressed antigen in melanoma (PRAME) in dysplastic nevi, an uncharted area. We examined 22 common melanocytic nevi (CMN), 20 cutaneous melanomas (CM), 48 low-grade dysplastic nevi (LG-DN), and 40 high-grade dysplastic nevi (HG-DN). PRAME was immunohistochemically assessed using a five-tiered system (0 to 4 +). Among CMN, 59% scored 0, 32% scored 1 + , and 9% scored 2 + . CM had score 2 + and 4 + in 11% and 89% of cases, respectively. Among LG-DN, 38% presented score 0, 31% score 1 + , 17% score 2 + , 8% score 3 + , and 6% score 4 + . Thirty per cent of HG-DN demonstrated a score 0, 30% with score 1 + , 15% score 2 + , 10% score 3 + , and 15% score 4 + . Compared to CMN and CM, LG-DN and HG-DN showed heterogeneous expression profiles of PRAME. PRAME positivity effectively distinguished HG-DN from CM with 85% specificity and 80% sensitivity (p < 0.0001). Predictive values were 87% (negative) and 76% (positive). Furthermore, a trend of increased PRAME expression from LG-DN to HG-DN was observed. However, the applicability of PRAME in the differential diagnosis of dysplastic lesions remains unclear as can yield conflicting results with morphology, which remains the primary diagnostic tool for melanocytic lesions.

Transcriptome Analysis Identifies Oncogenic Tissue Remodeling during Progression from Common Nevi to Early Melanoma.

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.

Standardized Computer-Assisted Analysis of PRAME Immunoreactivity in Dysplastic Nevi and Superficial Spreading Melanomas.

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/mm2 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/mm2. 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.

Dysplastic nevi and melanoma: microRNAs tell a divergent story.

BACKGROUND: dysplastic nevi (DN) share some clinical and histological features with melanoma and have been considered intermediate lesions toward malignant transformation. However, scientific evidence of DN representing melanoma precursors is still incomplete, and many observations pointed toward their being a distinct biological entity. The current definition of DN is also confusing and the practical consequence of this uncertainty is the excessive excision of DN with severe atypia. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and whose global expression can classify normal and pathological tissues. OBJECTIVES: given these considerations, we decided to perform a small RNA profiling study in a group of DN and invasive melanomas obtained from the same patient, to assess tumor evolution according to the global microRNA expression. METHODS: we performed a small-RNA sequencing of 6 DN, 2 congenital nevi and 4 cutaneous melanomas obtained from 4 subjects and evaluated the global miRNA expression correlation between samples. RESULTS AND CONCLUSIONS: the hierarchical clustering and principal component analyses of global miRNA expression, independently grouped together DN and their matching congenital nevi and showed a divergence of DN miRNA profile from melanoma. Our study suggests that DN have a peculiar and different miRNA expression profile compared to melanomas developed in the same patient, thus supporting the hypothesis that DN are distinct biological entities and not melanoma precursors.

Molecular Proof of a Clinical Concept: Expression of Estrogen Alpha-, Beta-Receptors and G Protein-Coupled Estrogen Receptor 1 (GPER) in Histologically Assessed Common Nevi, Dysplastic Nevi and Melanomas.

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 (ERß), 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: ERß 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). ERß expression was significantly higher in margin melanocytes compared to melanoma melanocytes (p = 0.009). No difference in ERß 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 ERß and GPER in the pathogenesis of melanocytic lesions.

Preferentially Expressed Antigen in Melanoma Immunostaining in a Series of Melanocytic Neoplasms.

ABSTRACT: In their 2018 article, Lezcano et al [AJSP 2018(11):1456] show that diffuse tumor cell nuclear reactivity for Preferentially expressed Antigen in Melanoma (PRAME) is a feature of melanoma and that benign and atypical melanocytic tumors are PRAME negative or show only focal positivity for PRAME. We report our observations of PRAME staining in 253 melanocytic tumors. Tumors were classified by hematoxylin and eosin sections. The nuclear PRAME staining of neoplastic melanocytes in each case was categorized as absent, focally present, or diffusely present. The results were compared with those of Lezcano et al 105 of 134 (78%) melanocytic nevi were completely PRAME negative. Of the 29 PRAME-positive benign lesions, 28 exhibited focal but not diffuse positivity, including atypical (n = 11) and dysplastic nevi (n = 11). One of 11 Spitz nevi showed diffuse positivity (9%). Thirty-nine of 51 (76%) invasive melanomas, 41 of 50 (82%) melanoma in situ, and 15 of 18 (83%) metastatic melanomas were diffusely PRAME positive. Excluding desmoplastic melanomas, 39 of 49 (80%) primary melanomas were diffusely PRAME positive. Our findings of PRAME staining in melanocytic neoplasia are in general agreement with those of Lezcano et al. Diffuse PRAME reactivity in neoplastic melanocytes is a feature of malignancy and was only otherwise seen in 1 Spitz nevus. Caution is advised in interpretation of PRAME reactivity in melanocytic tumors of uncertain classification because melanoma arising in association with nevus and some atypical melanocytic tumors may show focal or incomplete PRAME staining. Routine histopathological findings, clinical information, PRAME staining, and judicious application of molecular studies are steps leading to accurate classification of melanocytic neoplasia.

Molecular analysis of atypical deep penetrating nevus progressing to melanoma.

Deep penetrating nevi (DPN) are dermal-based, heavily pigmented melanocytic proliferations primarily resulting from mutations in B-catenin and BRAF or, less commonly, NRAS. DPNs are considered to be intermediate grade tumors which are stable with low risk of malignant transformation. The precise risk for transformation is unknown. Only rare cases of DPN progressing to melanoma have been described. We present a case of a 53-year-old female with a blue-black thigh lesion, on histopathology illustrating a melanocytic proliferation with morphology most consistent with a DPN progressing to melanoma. Targeted next generation sequencing performed on both the atypical melanocytic proliferation and melanoma components showed NRAS and CTNNB1 mutations but no evidence of TERT promoter mutation or chromosomal copy number aberrations. The melanoma had additional mutations including a hotspot TERT promoter mutation as well as unbalanced chromosomal copy number aberrations. This report details the progression of DPN to melanoma through a prominent ultraviolet signature and acquisition of genetic aberrations. While the vast majority of DPNs are benign stable nevi, there are rare examples, which may progress to melanoma. This report documents a case and shows the molecular evolution by which the tumor transformed to melanoma.

The differential expression of micro-RNAs 21, 200c, 204, 205, and 211 in benign, dysplastic and malignant melanocytic lesions and critical evaluation of their role as diagnostic biomarkers.

Overlapping histological features between benign and malignant lesions and a lack of firm diagnostic criteria for malignancy result in high rates of inter-observer variation in the diagnosis of melanocytic lesions. We aimed to investigate the differential expression of five miRNAs (21, 200c, 204, 205, and 211) in benign naevi (n = 42), dysplastic naevi (n = 41), melanoma in situ (n = 42), and melanoma (n = 42) and evaluate their potential as diagnostic biomarkers of melanocytic lesions. Real-time PCR showed differential miRNA expression profiles between benign naevi; dysplastic naevi and melanoma in situ; and invasive melanoma. We applied a random forest machine learning algorithm to classify cases based on their miRNA expression profiles, which resulted in a ROC curve analysis of 0.99 for malignant melanoma and greater than 0.9 for all other groups. This indicates an overall very high accuracy of our panel of miRNAs as a diagnostic biomarker of benign, dysplastic, and malignant melanocytic lesions. However, the impact of variable lesion percentage and spatial expression patterns of miRNAs on these real-time PCR results was also considered. In situ hybridisation confirmed the expression of miRNA 21 and 211 in melanocytes, while demonstrating expression of miRNA 205 only in keratinocytes, thus calling into question its value as a biomarker of melanocytic lesions. In conclusion, we have validated some miRNAs, including miRNA 21 and 211, as potential diagnostic biomarkers of benign, dysplastic, and malignant melanocytic lesions. However, we also highlight the crucial importance of considering tissue morphology and spatial expression patterns when using molecular techniques for the discovery and validation of new biomarkers.

Novel immune signatures associated with dysplastic naevi and primary cutaneous melanoma in human skin.

Dysplastic naevi (DN) are benign lesions with atypical features intermediate between that of common melanocytic naevi (CMN) and malignant melanoma (MM). Debate remains over whether DN represent progressive lesions from CMN. Through gene expression profiling and analysis of molecular gene signatures, our study revealed progressive increases in immune activation and regulation, along with pathways implicated in melanomagenesis, from CMN to DN to MM. Using criteria of 1.5-fold change and false discovery rate ≤0.05, we found differential expression of 7186 probes (6370 unique genes) with the largest difference detected between DN and MM from the standpoint of genomic melanoma progression. Despite progressive increases in the T-helper type 1 (Th1)-inducing gene (IL-12), RT-PCR indicated impaired Th1 or cytotoxic T-cell response (decreased IFN-γ) in MM. Concordantly, our results indicated progressive increases in molecular markers associated with regulatory T cells, exhausted T cells and tolerogenic dendritic cells, including detection of increased expression of suppressor of cytokine signalling 3 (SOCS3) in dendritic cells associated with MM. All together, our findings suggest that the increased immunosuppressive microenvironment of melanoma may contribute to unhampered proliferation of neoplastic cells. In addition, the detection of increased markers associated with tolerogenic dendritic cells in MM suggests that targeting these suppressive immune cell types may represent an alternative avenue for future immunotherapy.

Melanomas and Dysplastic Nevi Differ in Epidermal CD1c+ Dendritic Cell Count.

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/mm2 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.

Association of vascular endothelial growth factor expression with patohistological parameters of cutaneous melanoma.

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.

IMP-3 EXPRESSION IN BENIGN MELANOCYTIC NEVI, DYSPLASTIC NEVI AND MALIGNANT MELANOMA: PRELIMINARY FINDINGS IN BULGARIAN PATIENTS.

IMP-3 is generally considered as an oncofetal protein, which plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells. IMP-3 expression has been detected in malignancies with various origins, while its appearance in adult tissue is generally considered abnormal, with some exceptions. IMP3 is also considered a prognostic biomarker in patients with renal cell carcinoma and clear-cell type ovarian carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma and in patients with poorly differentiated thyroid carcinoma and uterine cervical carcinomas, testicular cancer and malignant melanoma. To our knowledge, no more than 4 PubMed-indexed studies have investigated the expression of IMP-3 in melanocytic lesions, namely its role in the differentiation between benign and malignant neoplasms. We investigated the expression of IMP-3 in a small series of benign melanocytic lesions, dysplastic nevi and melanomas, aiming to establish its significance as a marker for their distinction, comparing the results with those from the literature. IMP- 3 immunostaining was performed in 30 melanocytic lesions: 10 malignant melanomas, 10 dysplastic nevi and 10 benign melanocytic nevi. Our results revealed expression in 20% of dysplastic lesions and 40% of melanoma cases, while none of the benign nevi showed positive expression. These data contradict some of the results from other studies and raise some questions regarding the correlation between IMP- 3 and the degree of dysplasia of melanocytic nevi, as well as its potential relationship with prognostic parameters in melanoma, including tumor thickness and mitotic rate. Our results suggest that IMP-3 expression could be only an auxiliary marker for differentiation between dysplastic nevi and benign nevi, since although it is not expressed in all dysplastic lesions, staining correlates with the degree of dysplasia/atypia. It seems that IMP-3 expression is not a useful discriminator between dysplastic nevi and melanoma nor a good prognostic marker in melanoma.

α-Methylacyl-coenzyme A racemase (AMACR, p504s) is a marker to distinguish malignant melanomas from dysplastic nevi and melanocytic nevi.

Routinely processed skin biopsies are still the mainstay for the diagnosis of melanocytic skin neoplasms (MSNs) and are considered the "gold standard" for individual patient management and clinical trials. The diagnostic challenge of melanocytic lesions of the skin prompts histopathologists to consider new diagnostic tools; among these, immunohistochemistry. We aimed to find putative new immunohistochemical markers, which can supplement the histological criteria used to detect dysplasia. In this immunohistochemical study, we chose a panel of promising biomarkers which could potentially differentiate between different MSN entities. These included α-methylacyl-coenzyme A racemase (AMACR; p504s), which is involved in the degradation of branched chained fatty acid derivates. We analysed a cohort of benign nevi and malignant melanomas. The design of the study included 78 melanocytic skin neoplasms (26 malignant melanomas and 52 benign nevi) in a tissue microarray. Immunohistochemistry of cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1, and E-cadherin was performed and assessed. We have observed that the p16Ink4a, AMACR, cyclin D1, and E-cadherin showed no exclusive staining for nevi or melanomas. However, a significant overexpression of AMACR was found in malignant melanomas compared to benign nevi. AMACR overexpression was also associated with an increased p16Ink4a staining. Our results suggest AMACR as an immunohistochemical marker for distinguishing malignant melanomas and dysplastic nevi from conventional melanocytic nevi.

Suprabasal spread of melanocytes in dysplastic nevi and melanoma in situ: Ki-67-labeling rate of junctional melanocytes and suprabasal cells may be a helpful clue to the diagnosis.

Multiple criteria on routinely stained sections allow one to make a diagnosis of a dysplastic or "Clark" nevus (CN) versus melanoma in situ (MIS), and one of these is suprabasal spread of melanocytes. The extent of suprabasal spread of melanocytes in otherwise conventional CN and the combination of a sensitive marker of melanocytes combined with Ki-67 to assess the differences between the proliferation of melanocytes at the junction and those above it have not yet been studied. Fifty classic examples of CN and 27 cases of MIS were culled from the files of a university-based dermatopathology practice. All cases were stained with a 2-color method (MART-1/tyrosinase red, Ki-67 brown) to evaluate morphologic and immunohistochemical differences in these lesions. Fifteen of 50 cases of benign CN demonstrated suprabasal spread compared with 27 of 27 cases of MIS. The majority of CNs with suprabasal spread (13 of 15) showed a 0% Ki-67-labeling rate among the suprabasal melanocytes, and the majority of MISs (23 of 27) showed a 20% or greater Ki-67-labeling rate in suprabasal cells. Suprabasal melanocytes can be seen by immunostaining in otherwise unremarkable CN, wherein they are not notable in routinely stained sections, but their proliferation rate is much less than in MIS.

Cell cycle analysis can differentiate thin melanomas from dysplastic nevi and reveals accelerated replication in thick melanomas.

Cell replication integrates aberrations of cell cycle regulation and diverse upstream pathways which all can contribute to melanoma development and progression. In this study, cell cycle regulatory proteins were detected in situ in benign and malignant melanocytic tumors to allow correlation of major cell cycle fractions (G1, S-G2, and G2-M) with melanoma evolution. Dysplastic nevi expressed early cell cycle markers (cyclin D1 and cyclin-dependent kinase 2; Cdk2) significantly more (p < 0.05) than common nevi. Post-G1 phase markers such as cyclin A, geminin, topoisomerase IIα (peaking at S-G2) and aurora kinase B (peaking at G2-M) were expressed in thin (≤1 mm) melanomas but not in dysplastic nevi, suggesting that dysplastic melanocytes engaged in the cell cycle do not complete replication and remain arrested in G1 phase. In malignant melanomas, the expression of general and post-G1 phase markers correlated well with each other implying negligible cell cycle arrest. Post-G1 phase markers and Ki67 but none of the early markers cyclin D1, Cdk2 or minichromosome maintenance protein 6 (Mcm6) were expressed significantly more often in thick (>1 mm) than in thin melanomas. Marker expression did not differ between metastatic melanomas and thick melanomas, with the exception of aurora kinase A of which the expression was higher in metastatic melanomas. Combined detection of cyclin A (post-G1 phase) with Mcm6 (replication licensing) and Ki67 correctly classified thin melanomas and dysplastic nevi in 95.9 % of the original samples and in 93.2 % of cross-validated grouped cases at 89.5 % sensitivity and 92.6 % specificity. Therefore, cell cycle phase marker detection can indicate malignancy in early melanocytic lesions and accelerated cell cycle progression during vertical melanoma growth.

The significance of Melan-A-positive pagetoid melanocytosis in dysplastic nevi.

Dysplastic nevi may occasionally display alarming histological features. One of these features is the presence of upward spread of melanocytes (pagetoid melanocytosis), identified either on routine histologic sections or after immunohistochemistry using one of the melanocytic markers. Forty-three cases of dysplastic nevi with mild to moderate atypia were selected and retrieved, and Melan-A staining was performed. Melan-A-positive cells with pagetoid architecture were present in 27 cases (63%). Of these, only 5 cases demonstrated pagetoid architecture on routine staining. It is concluded that Melan-A staining should be used only with caution as an adjunct to routine histology in the evaluation of dysplastic nevi with mild to moderate atypia because the identification of pagetoid melanocytosis using this technique has the potential to lead to an erroneous diagnosis of melanoma.

Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4.

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.

Prognostic significance of Fbw7 in human melanoma and its role in cell migration.

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.

Novel multiple markers to distinguish melanoma from dysplastic nevi.

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.