Immunohistochemical analysis of Bcl-2 protein regulation in cutaneous melanoma.

Cutaneous melanoma is becoming increasingly common. Genetic and environmental factors are thought to play a role in its pathogenesis. We have previously shown that normal human melanocytes strongly express the oncoprotein, Bcl-2. To determine the role of Bcl-2 in melanocytic tumors, we studied human benign nevi and melanomas for expression of Bcl-2 protein using immunohistochemistry. Our results show that benign melanocytes from 3 of 4 normal skin biopsies and 5 of 7 common acquired nevi strongly express Bcl-2. Conversely, only 3 of 23 primary cutaneous melanomas and 3 of 9 metastatic melanomas showed strong staining in comparison with melanocytes from normal skin and common acquired nevi (chi 2, P = 0.0021). Interestingly, 0 of 6 dysplastic nevi, a precursor of melanoma, demonstrated strong staining as compared with melanocytes and nevi (8 of 11; chi 2, P = 0.02), but similar expression to that of melanoma (6 of 32; chi 2, P = 0.6). We conclude that Bcl-2 expression decreases in malignant melanoma and suggest that this may be related to the autonomous growth characteristics of malignant melanoma.     

Increased expression of stem cell markers in malignant melanoma.

The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P=0.005), CD133 (P=0.003), and nestin (P=0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P=0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P=0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.     

CD10 protein expression in tumor and stromal cells of malignant melanoma is associated with tumor progression.

CD10 antigen is a 100-kDa-cell surface zinc metalloendopeptidase expressed in a variety of normal and neoplastic lymphoid and nonlymphoid tissues including melanomas. It was recently shown that metastatic melanomas express more CD10 than primary tumors. We evaluated CD10 expression in tumor and stromal cells in 70 biopsies with primary and 28 with metastatic malignant melanomas. Ki-67, Bcl-2, and Bax were also examined to investigate whether CD10 expression is associated with tumor proliferation index or factors of apoptosis. Formalin-fixed/paraffin-embedded tissues were studied by immunohistochemistry. More advanced primary tumors had higher CD10 expression in the tumor cells (r = 0.27, P = 0.03 for Clark levels and r = 0.29, P = 0.02 for Breslow) and higher Ki-67 proliferation fraction (r = 0.32, P = 0.007 for Clark levels and r = 0.32, P = 0.001 for Breslow). Similarly, CD10 expression in the intratumoral stromal cells was also higher in primary tumors with higher Clark level (P = 0.04, linear-by-linear association) and tumor thickness according to Breslow (r = 0.33, P = 0.01). The presence of CD10+ peritumoral stromal cell cuffs was also positively associated with tumor thickness according to Breslow (r = 0.27, P = 0.05). Also, expression of CD10 and Ki-67 were significantly higher in metastatic than in primary tumors (P = 0.01 and 0.02 respectively), but Bcl-2 expression was higher in primary melanomas (P = 0.02). We conclude that CD10 expression in malignant melanoma is associated with tumor progression.     

Versican is differentially expressed in human melanoma and may play a role in tumor development

Undifferentiated human melanoma cell lines produce a large chondroitin sulfate proteoglycan, different from the well-known melanoma-specific proteoglycan mel-PG (Heredia and colleagues, Arch Biochem Biophys, 333: 198-206, 1996). We have identified this proteoglycan as versican and analyzed the expression of versican in several human melanoma cell lines. Versican isoforms are expressed in undifferentiated cell lines but not in differentiated cells, and the isoform expression pattern depends on the degree of cell differentiation. The V0 and V1 isoforms are found on cells with an early degree of differentiation, whereas the V1 isoform is present in cells with an intermediate degree of differentiation. We have also characterized some functional properties of versican on human melanoma cells: the purified proteoglycan stimulates cell growth and inhibits cell adhesion when cells are grown on fibronectin or collagen type I as substrates, and thus may facilitate tumor cell detachment and proliferation. Furthermore, we have analyzed the expression of versican in human melanocytic nevi and melanoma: 10 benign melanocytic nevi, 10 dysplastic nevi, 11 primary malignant melanomas, and 8 metastatic melanomas were tested. Immunoreactivity for versican was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas. Our results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis.     

Differential gene expression profiling of primary cutaneous melanoma and sentinel lymph node metastases

Limited understanding of molecular mechanisms of metastasis in melanoma contributes to the absence of effective treatments. Increased knowledge of alterations in genes that underpin critical molecular events that lead to metastasis is essential. We have investigated the gene expression profiles of primary melanomas and melanoma metastases in sentinel lymph nodes. A total of 19 samples (10 primary melanomas and 9 sentinel lymph node metastases) were evaluated. Melanoma cells were dissected from tissue blocks. Total mRNA was isolated, amplified, and labeled using an Ambion Recover All Total Nucleic Acid Isolation kit, Nu-GEN WT-Ovation formalin-fixed, paraffin-embedded RNA Amplification System, and FL-Ovation cDNA Biotin Module V2, respectively. Samples were hybridized to the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data were analyzed using Partek Genomics Suite Version 6.4. Genes selected showed ≥2-fold difference in expression and P<5.00E-2. Validation studies used standard immunohistochemical assays. Hierarchical clustering disclosed two distinct groups: 10 primary melanomas and 9 sentinel lymph node metastases. Gene expression analysis identified 576 genes that showed significant differential expression. Most differences reflected decreased gene expression in metastases relative to primaries. Reduced gene expression in primaries was less frequent and less dramatic. Genes significantly increased or decreased in sentinel lymph node metastases were active in cell adhesion/structural integrity, tumor suppression, cell cycle regulation, and apoptosis. Validation studies indicate that MAGEC1 (melanoma antigen family C1) and FCRL1 (Fc receptor-like 1) are involved in melanoma progression. There are striking differential gene expression patterns between primary and nodally metastatic melanomas. Similar findings were seen with autologous paired primary melanomas and sentinel lymph node metastases, supporting involvement of these gene alterations in evolution of metastases. With further study, it may be possible to determine the exact sequence of molecular events that underlie melanoma metastases.     

Malignant melanoma: analysis by DNA flow cytometry

The DNA content of 14 primary and 111 secondary melanomas was determined by flow cytometry. Aneuploidy was detected in 67% of samples. The frequency with which aneuploid cells were found was similar in primary and metastatic melanomas and, in the metastatic group, for melanotic and amelanotic tumours. Aneuploid diversity was marked with a wide variation in DNA content between tumours. Serial biopsies were performed in 14 patients, and in 10 there was discordance in DNA profiles between first and subsequent biopsies. Tumour biopsies taken from different sites at the same time also showed discordance in 3 of 5 cases. These features highlight the degree of cellular heterogeneity in malignant melanoma.     

Microtubule-associated protein 2, a marker of neuronal differentiation, induces mitotic defects, inhibits growth of melanoma cells, and predicts metastatic potential of cutaneous melanoma

Dynamic instability of microtubules is critical for mitotic spindle assembly and disassembly during cell division, especially in rapidly dividing tumor cells. Microtubule-associated proteins (MAPs) are a family of proteins that influence this property. We showed previously that MAP2, a neuron-specific protein that stabilizes microtubules in the dendrites of postmitotic neurons, is induced in primary cutaneous melanoma but is absent in metastatic melanomas. We proposed that induction of a microtubule-stabilizing protein in primary melanoma could disrupt the dynamic instability of microtubules, inhibit cell division and prevent or delay tumor progression. Here we show, by Kaplan-Meier survival and multivariate Cox regression analysis, that patients diagnosed with MAP2+ primary melanomas have significantly better metastatic disease-free survival than those with MAP2- disease. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Disruption of microtubule dynamics by MAP2 resulted in multipolar mitotic spindles, defects in cytokinesis and accumulation of cells with large nuclei, similar to those seen in vivo in MAP2+ primary melanomas cells. These data suggest that ectopic activation of a neuronal differentiation gene in melanoma during early tumor progression inhibits cell division and correlates with inhibition or delay of metastasis.     

Ocular melanoma: an overview of the current status

Ocular melanoma is the second most common type of melanoma after cutaneous and the most common primary intraocular malignant tumor in adults. Large majority of ocular melanomas originate from uvea, while conjunctival melanomas are far less frequent. Incidence of uveal melanoma has remained stable over last three decades. Diagnosis is in most cases established by clinical examination with great accuracy. Local treatment of uveal melanoma has improved, with increased use of conservative methods and preservation of the eye, but survival rates have remained unchanged. Recent advances in cytogenetics and genetics enhanced prognostication and enabled to determine tumors with high metastatic potential. However, due to lack of effective systemic therapy, prognosis of patients with metastasis remains poor and metastatic disease remains the leading cause of death among patients with uveal melanoma. Conjunctival melanoma is rare, but its incidence is increasing. It mostly occurs among white adults. In majority of cases it originates from preceding primary acquired melanosis. Current standard treatment for conjunctival melanoma is wide local excision with adjuvant therapy, including brachytherapy, cryotherapy and topical application of chemotherapeutic agent. Rarity of this tumor limits conduction of controlled trials to define the best treatment modality. As well as for uveal melanoma, prognosis of patients with metastasis is poor because there is no effective systemic therapy. Better understanding of underlying genetic and molecular abnormalities implicated in development and progression of ocular melanomas provides a great opportunity for development of targeted therapy, which will hopefully improve prognosis of patients with metastatic disease.     

Cell growth and p53 expression in primary acquired melanosis and conjunctival melanoma.

AIMS: To evaluate cell growth and the pattern of p53 suppressor gene expression in atypical primary acquired melanosis (PAM) and in recurrent conjunctival melanoma. METHODS: Eighteen specimens of PAM with atypia and 24 specimens, comprising early and late lesions, from 12 patients with conjunctival melanoma were stained for the proliferating cell nuclear antigen using the PC10 antibody, and for the p53 gene product using the BP53-12-1, 1801 and DO7 clones. The immunoreactive cells were counted manually and the data evaluated statistically. RESULTS: Seven of nine PAM specimens progressing to melanoma expressed PC10. None of these lesions expressed the p53 gene product. The number of proliferating cells was higher in the late than in the early lesions of conjunctival melanoma. Four of the 12 recurrent melanomas displayed focal, but minimal, p53 expression. The proliferating cell count in the p53 positive tumours was very similar to that of the p53 negative conjunctival melanomas. CONCLUSION: Examination of the expression of proliferating cells in atypical PAM may be used as an adjunct to predict which lesions will progress to melanoma. The increase in the number of proliferating cells over time in recurrent conjunctival melanomas probably reflects more aggressive behaviour and may be used to monitor recurrence. The absence of p53 expression in PAM and minimal staining of conjunctival melanomas did not correlate with cell growth, suggesting that alterations in the p53 tumour suppressor gene are uncommon and late events in conjunctival melanoma, and that p53 expression is unlikely to be a useful prognostic indicator.     

Monoclonal antibody to a highly glycosylated protein reacts in fixed tissue with melanoma and other tumors

A highly glycosylated protein with a molecular weight of 30,000 to 60,000 and a protein core of 20,000 daltons has been identified by antimelanoma monoclonal antibodies. The antigenicity of this melanoma-associated glycoprotein (MAG) was not destroyed in fixed paraffin-embedded melanoma tissue, and was present in malignant cells of cutaneous superficial spreading melanomas in skin (31/33) and in half of all metastatic melanomas examined (5/10). The antigen was not expressed by normal melanocytes. The strong reactivity of dysplastic nevi with the anti-MAG antibodies was comparable to that seen in radial growth phase melanoma. Antigen expression was much weaker in compound nevi where reactivity ranged from moderate in the junctional component and the upper dermis to absent at the base of the nevus.     

KP1 (CD 68) staining of malignant melanomas

The monoclonal antibody KP1, which recognizes the CD 68 antigen on macrophages and myeloid precursors, was tested on 28 malignant (primary and metastatic) melanomas, 28 naevi, and 17 skin biopsies showing either normal (10) or hyperplastic melanocytes (7). Sixteen of 20 primary melanomas and six of eight metastatic melanomas showed variable numbers of KP1 positive tumour cells. All but five benign melanocytic proliferations (two Spitz naevi and three intradermal naevi), as well as normal and hyperplastic melanocytes were negative. These results indicate that difficulties may occur with the use of KP1 in the differential diagnosis between melanomas and neoplasms derived from histiocytes-macrophages, and that the expression of CD 68 antigen might be related to tumour progression in melanocytic cells.     

Downregulation of the retinoblastoma gene expression in the progression of malignant melanoma.

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.     

A study of adhesion molecules as markers of progression in malignant melanoma.

Adhesion molecules are substances which are involved in the interactions between cells, and between cells and the extracellular matrix in both benign and malignant tissues. Two members of this group--intercellular adhesion molecule-1 (ICAM-1) and MUC18--have previously been found to be expressed on melanoma; however, studies seeking a correlation between expression and metastatic behaviour have yielded conflicting results. In this study we investigated the expression of these two antigens and that of a number of other adhesion molecules [VCAM-1, ELAM, and the neural cell adhesion molecule (NCAM)] on a range of benign and malignant melanocytic lesions. Both ICAM-1 and MUC18 were found on a high percentage of all melanocytic lesions including benign naevi. VCAM-1 was found to be expressed on 79 per cent of benign naevi, 62 per cent of primary melanomas less than 1.5 mm in depth, and 6 per cent of thick primaries. The antigen was present on 14 per cent of lymph node metastases and on no extranodal deposits. This suggests that loss of melanoma cell adhesion mediated by VCAM-1 may be important in the development of metastatic melanoma.     

Expression of interleukin-8 detected by in situ hybridization correlates with worse prognosis in primary cutaneous melanoma

Previous in vitro studies have demonstrated that endogenously produced human interleukin-8 (IL-8) can act as an important growth factor for human melanoma cells in vitro. The present study, has investigated whether IL-8 mRNA expression in primary melanomas may be of prognostic relevance with regard to melanoma progression and metastatic spread. In order to evaluate the clinical significance of IL-8 mRNA expression of melanoma cells in vivo, 59 melanocytic tissue specimens (37 primary melanomas and 22 melanocytic naevi) were studied using a semiquantitative in situ hybridization technique. Significant mRNA expression of IL-8 was found in 59 per cent (22/37) of melanomas. In 19 per cent (7/37) of the malignant melanomas, additional hybridization signals were noted within keratinocytes of the overlying epidermis. In contrast, paralesional normal-appearing epidermis and melanocytes in non-malignant lesions (melanocytic naevi) showed no IL-8 mRNA. Analysis of the relationship between IL-8 expression and clinico-histopathological features showed a significant association between IL-8 mRNA expression and the histological melanoma subtype (IL-8 mRNA: 14/19 in superficial spreading melanoma versus 4/12 in nodular melanoma, p< 0.05). Furthermore, IL-8 expression in primary tumours could be correlated with the patients' clinical course, with time to progression being significantly reduced in primary tumours expressing IL-8 in either the tumour cells or keratinocytes of the overlying epidermis. These results demonstrate for the first time that IL-8 expression, as detected by in situ hybridization in primary tumours, may serve as a significant prognostic factor for tumour progression in human malignant melanoma.     

Development of a tridimensional microvascularized human skin substitute to study melanoma biology

Cutaneous malignant melanomas represent an important clinical problem because they are highly invasive, they can metastasize to distant sites and are typically resistant to available therapy. The precise molecular determinants responsible for melanoma progression and chemo-resistance are not yet known, in part due to lack of pertinent experimental models that mimic human melanoma progression. Accordingly, we developed a complex human microvascularized reconstructed skin substitute in which the organized three-dimensional (3D) architecture of the native skin is reproduced. Human melanoma cell lines derived from primary and metastatic sites were added to this 3D model. Our results demonstrate that histological features and behavior of melanoma cells applied in our skin substitute model are specific to their site of origin. In particular, the ability of melanoma cells to cross the dermal–epidermal junction correlates with their metastatic potential. In addition, a potent angiogenic effect was detected for an aggressive metastatic cell line that produces VEGF. The presence of a microvascular network within this model will allow studying a crucial step of the metastatic process. We conclude that such an in vitro human tumor microvascularized reconstructed skin substitute promises to be a versatile and efficient model to investigate skin cancer progression and to screen new anticancer drugs to improve currents clinical treatments.