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Identification of antigens by monoclonal antibodies (MAbs) on sections of human melanoma by immunoperoxidase techniques was used to determine whether certain adhesion molecules and "selectin-like" molecules may be related to the metastatic potential of primary melanoma. The adhesion molecules examined were the leukocyte function antigen (LFA-1) and its ligand--intercellular adhesion molecule-1 (ICAM-1), the receptor alpha V beta 3 for vitronectin, its subunits alpha V and beta 3, and the CD36 receptor for thrombospondin (TSP). The criteria used to establish metastatic potential were relation of the molecules to tumor thickness and differences in expression: (i) between radial and vertical growth phases of the primary tumors and (ii) between 34 primary and 21 unrelated metastases. By these criteria ICAM-1, alpha V beta 3 and its subunit were associated with the malignant potential of primary melanoma. These molecules were not expressed on nevi or other skin cancers with low metastatic potential such as squamous (SCC) and basal cell carcinomas (BCC). In contrast, expression of TSP and the CD36 receptor for TSP were not related to metastatic potential. CD36 was expressed widely not only on melanoma but also on BCC, SCC and nevi. Similarly, the selectin-like molecule, CD44, was widely expressed on melanoma and non-melanoma carcinomas. The lymph node homing receptor, Leu 8, and the cutaneous lymphocyte antigen (CLA) were not detected on melanoma. Leu 8 was present on normal epithelium and SCCs, and common leucocyte antigen (CLA) was detected on lymphocytes in the epithelium and near melanoma. These results support previous suggestions that expression of ICAM-1 and V beta 3 integrin or its subunit beta 3 on melanoma may be a useful prognostic marker in primary melanoma. They do not support a role for CD44, Leu 8, CLA and TSP or its receptor CD36 in the metastatic process in melanoma.

MicroRNAs are small non-protein-coding RNAs which repress gene expression, through base pair matching with messenger RNA (mRNA). A single microRNA is capable of regulating hundreds of mRNA sequences. Only a small fraction of the over 1000 discovered microRNAs have currently known functions; many are crucial in the regulation of genetic signalling, including cellular processes such as cellular differentiation, growth, proliferation and death. Dysfunction in microRNA signalling is present in all cancers studied thus far, leading to overactive oncogenic and underactive tumour suppressor gene signalling. Current research is actively pursuing the potential to use microRNAs as diagnostic tools and novel therapies in a variety of diseases. This review summarises normal and abnormal maturation and function of microRNAs and their role in the pathogenesis of various human tumours and highlights how microRNAs may be used as diagnostic and treatment tools in human cancers in the future.

The generation of anti-apoptotic factors is complex. Previous studies have demonstrated that endoplasmic reticulum (ER) stress results in both apoptosis and anti-apoptosis in some tumours. ER stress may induce apoptosis via multiple pathways. Adaptation of melanoma to ER stress may result in anti-apoptosis and resistance to therapy. The anti-apoptosis mechanisms include RAS/RAF/MEK/ERK activation, increased GRP78 and Mcl-1 protein. In the present study, we sought to better characterise the correlation of the ER stress protein GRP78 expression with ERK activation and Bcl-2 family proteins. The expression of GRP78, Bcl-2 and Mcl-1 in formalin-fixed sections of 135 naevi, primary and metastatic melanoma by immunohistochemistry was examined. p-ERK expression was assessed in 82 of the 135 cases. The correlations of GRP78 with p-ERK, Bcl-2 and Mcl-1 were analysed. The immunoreactive score (IRS) of GRP78 and Mcl-1 were related to melanocytic tumour progression. The intensity of GRP78 and Mcl-1 increased with the progression of melanocytic tumours. In contrast, Bcl-2 showed high expression in melanoma ≤1 mm and less expression in melanoma metastases. GRP78 expression increased with increased p-ERK expression ( p p The findings of this study suggest that activated ERK may enhance GRP78 levels and are consistent with previous results from melanoma cell lines. GRP78 and Mcl-1 expression may play a role in protecting cells from ER stress-induced apoptosis. Bcl-2 is increased in thin melanoma and this suggests that Bcl-2 plays its anti-apoptotic role in the early stage of melanoma progression.

E-cadherin mediated intercellular adhesion is regulated by a family of cytoplasmic proteins that include alpha-, beta- and gamma-catenin and p120cas. Changes in expression of E-cadherin are believed to be an early event in melanoma development. Recent studies have also drawn attention to the over-expression of beta-catenin and its possible indirect role as an oncogene in melanoma. In view of these studies, we have examined the expression of cytoplasmic proteins immunohistochemically in 13 melanocytic nevi, 34 primary cutaneous melanomas and 20 metastatic melanomas. alpha-, gamma-catenin and p120cas were heterogeneously expressed in melanocytic nevi and melanomas and were frequently absent, whereas beta-catenin expression was observed in all lesions. The pattern of expression of alpha-, beta- and gamma-catenin and p120cas was characterised by cytoplasmic and membranous immunoreactivity of varying intensity. No significant difference was found in expression of these proteins between melanocytic nevi and primary melanoma. In contrast, there was an inverse correlation between alpha-catenin expression and tumor thickness and alpha-catenin was more frequently expressed in radial compared to vertical growth phase in primary melanoma. Loss of alpha-catenin expression was observed in ten of 20 metastases compared to six of 34 primaries and the expression was more marked in primaries than in metastases. These results indicated that alterations in alpha-, beta- and gamma-catenin and p120cas expression were common in melanocytic nevi and melanomas, and that loss of alpha-catenin expression was associated with melanoma invasiveness and metastasis.

Background In melanoma, glycogen synthase kinases 3 (GSK-3) has been implicated in many critical signalling pathways, including the PI3K-AKT and Wnt-β-catenin pathways and also in apoptosis. This study sought to determine the role of the GSK-3α, p-GSK-3α, GSK-3 β, p-GSK-3β and other related factors McL-1 and p53 in melanoma progression. Methods Immunohistological staining for the aforementioned proteins was performed in formalin-fixed, paraffin-embedded sections of 250 excised human melanocytic tumours including primary melanomas, metastatic melanomas and benign naevi. Results The expression of GSK-3α, GSK-3b, MCL-1 and p53 expression was significantly greater in primary melanomas > 1 mm in thickness (each p p 1 mm in thickness ( p = 0.001) and soft tissue metastases ( p = 0.027). Conclusions These results show GSK-3α/β expression increases with tumour progression in melanocytic tumours. The findings support functional studies which implicate GSK-3 in melanoma proliferation and survival.

Summary DNA ploidy in benign nevi (BN), thin non-metastasizing melanomas (TNM) and thin metastasizing melanomas (TMM) was investigated using an image analyser to determine whether characteristics such as nuclear area (NA) and nuclear integrated optical density (IOD) could be used to distinguish between these lesions. NA measurements showed significant differences between samples of nevus cells and melanoma cells and nuclear IOD differences were significant between TNM and TMM samples. Differences in NA and nuclear IOD were detected across the three groups (BN, TNM and TMM) but the large variability within samples and within groups indicate further studies would be necessary to determine the usefulness of these results in terms of the rate of correct group classification of a single sample for diagnostic purposes.

MHC antigen expression on 20 nevi, and 35 primary and 95 metastatic melanomas was studied by immunoperoxidase techniques using monoclonal antibodies to identify the antigens on frozen tissue sections. DR antigens were not detected on nevi but were detected on 71% of primary melanomas and 56% of metastases, suggesting that this antigen may be a useful marker of malignant transformation of nevi. Expression of class II antigen could not be related to other prognostic histological features of primary melanoma such as tumour thickness, but comparison of the common phenotypes of primary and metastatic melanoma suggested that expression of DR antigens alone in the absence of DP, DQ and ABC antigens may be an indicator of metastatic potential. Class I (HLA-A,B,C) antigens were also expressed infrequently on nevi but were detected on 43% of primary melanomas and 34% of metastases. HLA-A,B,C expression was inversely related to thickness of the primary melanoma. This as well as the lower expression of class I antigens on metastases, may indicate that growth and spread of melanoma may be inhibited by MHC (class I) dependent cytotoxic T cell responses. Expression of class I MHC antigens was unrelated to class II antigens. Expression of DR was more common than DP or DQ, but the latter with one exception, were not expressed in the absence of DR antigens. Significant differences were not found in MHC antigen expression on metastases in lymph nodes compared to those in subcutaneous sites, but further studies are needed to determine whether such differences may exist between metastases in other visceral sites.

Immunoperoxidase localization of monoclonal antibodies in sections of melanoma has been used to identify histological features which may be of prognostic importance in melanoma, in particular whether certain structures on melanoma cells may determine the degree and nature of lymphoid infiltrates and whether these may be related to prognosis. Monoclonal antibodies to lymphocyte subpopulations were used to identify and quantitate lymphoid infiltrates in 15 primary melanomas and 8 cutaneous metastases. These were correlated with histological features identified in routine sections. There was a wide variation in the numbers of mononuclear cells associated with both primary and metastatic melanoma. T cells and to a lesser extent macrophages accounted for the majority of the cells, B cells and natural killer (NK) cells for only 2% of the infiltrates. The Leu 3a (helper) T cell subpopulation predominated in primary tumours, OKT8 positive (suppressor/cytotoxic) cells in metastases. Infiltrates in which OKT8 cells predominated were associated with ulceration, a high mitotic rate and thick primary tumours. The converse applied to Leu 3a infiltrates. Infiltrates with a high proportion of Leu 3a positive cells tended to be associated with Thy-1 positive, DR antigen negative tumours. Thy-1 antigens were predominantly expressed on primary tumours and rarely on metastases whereas the converse applied to expression of the acute lymphoblastic leukemia antigen (CALLA) on melanoma cells. These findings suggest that certain melanoma antigens may be related to the nature of the lymphoid infiltrate associated with melanoma and possibly with the behaviour of the tumour in the host. They further suggest that identification of cell surface structures and lymphoid infiltrates by these techniques may be a valuable extension of routine histopathological assessment of prognosis in melanoma.

Summary A number of previous studies have suggested that certain C-type viruses from non-human primates are associated with acute leukaemia in humans. The present study sought to examine this association further by detection of antigens related to these viruses on the surface of acute leukaemia cells. Antisera against the whole viruses or the major internal protein of simian sarcoma, endogenous baboon and Rauscher viruses were tested against mononuclear cells from patients with acute leukaemia at various stages of their disease in 51 Cr release leucocyte dependent cytotoxic assays. A high proportion of the patients had antigens related to these viruses detectable on their blood leucocytes at some stage in their disease whereas with one exception blood leucocytes from non-leukaemic subjects did not. The expression of these antigens did not show a close relation to the number of blast cells in blood detected on morphological grounds. Antigens related to the non-human primate viruses were detected on mononuclear cells from bone marrow samples of several non-leukaemic patients which indicated they were not specifically associated with leukaemia. Antigens related to the Rauscher virus were detected on cells of practically all bone marrow samples tested. These results suggest that assays to detect these antigens would be of limited usefulness to monitor disease activity in patients with acute leukaemia. Their expression on blood leucocytes however appeared strongly associated with acute leukaemia and further studies to assess the prognostic and epidemiological significance of detection of these antigens appears warranted.

Previous studies in vitro have shown that monoclonal antibodies (MAbs) against gangliosides GD3 and GD2 potentiate lymphocyte responses to a variety of stimuli. The purpose of the present study was to determine by immunohistological techniques whether GD3 and GD2 was expressed on lymphoid cells in vivo around melanoma cells. Studies on metastases in lymph nodes indicated that the lymphoid infiltrate around the margins of the metastases was predominantly CD4+ T cells, which were shown by dual labelling techniques to express mainly GD2 and to a lesser extent GD3. CD4+GD3+ T cells were detected more frequently in cortical regions of the lymph nodes. CD8+ T cells were less numerous than CD4+ T cells and expressed both GD3 and GD2. Expression of GD2 was also prominent on CD4+ T cells, B lymphocytes and dendritic reticular cells in germinal centres, whereas GD3 was mainly expressed on T cells in the margins of the follicles. In contrast to the predominance of CD4+ T cells in lymph nodes, CD4+ and CD8+ T cells were in approximately equal proportions about primary melanoma and metastases in skin. GD2 was largely undetectable on lymphocytes at these sites. In contrast, GD3 was detected on both CD8+ and CD4+ lymphocytes but not on B lymphocytes. The absence of GD2 on CD4+ T cells in skin suggested the latter were a different subpopulation to those in lymph nodes. There appeared to be no clear correlation, however, with subsets of CD4 T cells defined by the 2H4 and Leu 8 MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

The introduction of monoclonal antibody techniques has led to a rapid advance in information concerning antigenic structures in melanoma cell membranes. These have been classified according to the extent of their expression on cells of other tissues, but it is evident that a more precise classification based on their biochemical nature is possible. Several monoclonal antibodies appear to define antigens restricted to melanoma cells and fetal tissues. Many antibodies recognize antigens shared with gliomas and nevi, whereas other groups can be defined which recognize antigens on melanocytes or other carcinomas. One of the commonly detected antigens was shown to be a high molecular weight (MW) proteoglycan which may be involved in reactions with other cells and the intercellular matrix. A second antigen was shown to be a ganglioside which may have receptor functions in cells. A third was shown to be a glycoprotein with iron transport functions. The latter antigen and the large MW proteoglycan have been a focus of attention for in vivo targeting studies in treatment and diagnosis. The ganglioside, large MW proteoglycan and a melanocarcinoma antigen may be detected in the circulation of patients and are being evaluated for monitoring of disease activity in patients with melanoma. Several monoclonals may be of value in histological evaluation of melanoma, e.g. diagnosis of preneoplastic lesions, metastatic lesions of unknown origin and identification of cell structures related to metastatic behaviour in the host. Further studies should help to define cellular structures recognized by the immune system in humans.