The clinical significance of antityrosinase antibodies in melanoma and related hypopigmentary lesions

Summary Antityrosinase antibody is a newly detected antibody in the sera of patients with melanoma or vitiligo. The serum level of the antibody is measured by enzyme-linked immunosorbent assay (ELISA). The autoantigen is tyrosinase itself, the enzyme that participates in pigment (melanin) formation by both melanocytes and melanoma cells Antityrosinase IgG antibodies were found to be present in high titers in sera of patients with vitiligo in comparison to patients with melanoma or healthy volunteers. The level of antityrosinase antibodies in patients with metastatic melanoma was significantly higher than the level in healthy subjects, but insignificantly higher than the level in patients with no evidence of disease. Patients with melanoma and MAH (melanoma-associated hypopigmentation; vitiligo-like) had the same level of antityrosinase antibodies as the controls or the patients with metastatic melanoma. This observation reflected the possible absorption of antityrosinase antibodies by melanoma antigens, and pointed to the participation of the antibodies in the destruction of normal melanocytes in patients with melanoma, as part of the immune reaction towards this disease. The most interesting observation was the high level of antityrosinase antibodies in patients with vitiligo in comparison with the low level in patients with melanoma, patients with MAH, and patients with NED. Although the cutaneous manifestations of vitiligo and MAH are similar and result from destruction of melanocytes by specific antibodies, the two situations are immunologically different. The serum level of free antityrosinase antibodies could not serve as marker for the state of the disease or disease progression or relapse, as no significant difference could be detected between the levels in patients without evidence of disease to those with metastatic melanoma; nor could the levels of antityrosinase antibodies differentiate between the different sites of the primary lesion. However, we have shown that antityrosinase antibodies could be used for monitoring the response to active specific immunotherapy by injection of anti-idiotypic antibodies mimicking the HMW-MAA. In the future, antityrosinase antibodies may be incorporated into immunotherapy for malignant melanoma.     

The extracellular matrix in pigmented skin lesions: an immunohistochemical study

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.     

ESR study of plasmatic membrane of the transplantable melanoma cells in relation to their biological properties.

Using the spin labelling method we studied changes in the structure and dynamics of molecular mobility in the plasmatic membrane accompanied by a spontaneous alteration of a melanotic melanoma line into an amelanotic form with a higher growth rate, changed antigenicity and immunogenicity. The calculated ratio of the low-field line (A) intensity to the central line (C) intensity of the spectrum showed statistically significant differences in the order of parameters in the plasmatic membranes of both forms of melanocytes. The significantly broader central line (deltaW0) in the spectra of labelled amelanotic melanoma cells than in the original cell line indicated changes in the membrane structure leading to a lowering of the degree of order in the phospholipid bilayer. It has been suggested that a progression of transplantable melanomas is accompanied by an increase in membrane fluidity and reduction in molecular mobility dynamics within it.     

Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance

The lack of effective conventional therapies for the treatment of advanced stage melanoma has stimulated interest in the application of novel strategies for the treatment of patients with malignant melanoma. Because of its expression in a large percentage of melanoma lesions and its restricted distribution in normal tissues, the high molecular weight-melanoma-associated antigen (HMW-MAA), also known as the melanoma chondroitin sulfate proteoglycan (MCSP), has been used to implement immunotherapy of melanoma. The potential clinical relevance of HMW-MAA/MCSP has stimulated investigations to characterize its structural properties and biological function in melanoma cells. Over the last 10 years, the field of HMW-MAA/MCSP research has seen tremendous growth. Specifically, a significant amount of information has been accumulated regarding (1) the structural characteristics of the HMW-MAA/MCSP, (2) its role in the biology of melanoma cells, and (3) the potential molecular mechanisms underlying the association between HMW-MAA/MCSP-specific immunity and survival prolongation in melanoma patients immunized with HMW-MAA/MCSP mimics. In this review, we summarize the characteristics of the HMW-MAA/MCSP in terms of its structure, antigenic profile, tissue distribution, and similarities with its counterparts in other animal species. Additionally, we discuss the role the HMW-MAA/MCSP plays in melanoma cell biology with emphasis on the recently identified signal transduction pathways triggered by the HMW-MAA/MCSP. Finally, we discuss the potential molecular mechanisms underlying the beneficial effect of anti-HMW-MAA/MCSP antibodies on the clinical course of the disease in patients with melanoma.     

Apurinic/apyrimidinic endonuclease/redox effector factor-1(APE/Ref-1): a unique target for the prevention and treatment of human melanoma

Management of melanoma is a growing and challenging public health issue requiring novel and multidisciplinary approaches to achieve more efficient prevention and therapeutic benefits. The aim of this article is to show the critical role of APE/Ref-1 on melanomagenesis and progression. APE/Ref-1 serves as a redox-sensitive node of convergence of various signals as well as a DNA-repair enzyme, and its activation protects melanocytes and melanoma cells from chronic oxidative stress and promotes cell survival via mediation of downstream pathways. APE/Ref-1 is a strong candidate as a potential drug-treatable target for the prevention and treatment of human melanoma. Lead compounds exhibiting inhibitory effects on APE/Ref-1 are also reviewed. We anticipate potential clinical benefit in the future through inhibition of APE/Ref-1 and/or Ref-1-mediated signaling.     

Raf kinase inhibitor protein (RKIP) and phospho-RKIP expression in melanomas

Melanoma, a cancer notorious for its high potential to metastasize, arises from melanocytes, cells dedicated to melanin production and located in the basal layer of the epidermis. Raf-1 kinase inhibitor protein (RKIP) is an inhibitory molecule that down-regulates the effects of the Ras/Raf/MEK/ERK signaling pathway. The aim of this study was to examine the expression of RKIP and pRKIP in melanomas at different stages. We evaluated the RKIP and pRKIP protein by immunohistochemistry in control skin, pigmented nevi and melanomas, and through Western blotting in human normal melanocytes and in four different melanoma-derived cell lines (WM35, A375, M14, and A2058). Our results demonstrated a correlation between the expression of RKIP and pRKIP, and metastatic ability in melanoma cells. This raises the possibility to analyze both RKIP and pRKIP in all melanomas. Down-regulation of both RKIP and pRKIP expression could represent a useful marker of metastatic melanoma. On the contrary for non-metastatic melanoma, especially in Clark I and II, low RKIP and high pRKIP expression could be indicative. In conclusion, the observed negative correlation of the RKIP and pRKIP expression in metastatic melanomas indicates that expression of these proteins may become a prognostic marker for the progression of human cutaneous melanoma. We propose that the investigation of both RKIP and pRKIP may provide a useful tool indicative for metastatic or non-metastatic melanoma in different Clark's level melanomas. Further studies are required to verify the molecular background of the observed RKIP and pRKIP variations.     

Atypically low spontaneous sister chromatid exchange formation in uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular cancer of adults and is characterized by several well-established chromosomal changes. More recently, a specific mutation of guanine nucleotide binding protein Gq alpha subunit (GNAQ) has also been identified in a proportion of UM. Although some of these alterations have been suggested to be early changes, the genetic alterations responsible for the development of UM have yet to be clearly determined. Cancers are characterized by increased genetic instability, and analysis of established cancer cell lines and blood from cancer patients has universally been associated with an increased level of sister chromatid exchange (SCE). We have observed that the spontaneous frequency of SCE in primary cultures of UM and UM-derived cell lines is decreased below normal baseline levels, a phenomenon unique to UM when compared with multiple other cancers. This finding was specific to the tumor and not found in lymphocytes from the patients. Although we cannot exclude the possibility that low SCE (LSCE) is peculiar to the uveal melanocytes lineage, as it was consistently observed in all UM studied, regardless of other genetic defects, we propose that this phenomenon contributes to the molecular pathogenesis of UM.     

Nuclear to cytoplasmic compartment shift of the p33ING1b tumour suppressor protein is associated with malignancy in melanocytic lesions

AIMS: Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation-associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co-operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia. METHODS AND RESULTS: Sixty-seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma. CONCLUSIONS: Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with 'activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.     

Distinct MHC gene expression patterns during progression of melanoma

Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas. © 2009 Wiley‐Liss, Inc.     

Attempts to optimize active specific immunotherapy for melanoma.

During the past 5 years, we have been conducting clinical trials with a therapeutic melanoma vaccine (melanoma "theraccine"). Mechanical lysates of two melanoma cell lines chosen for their complementary characteristics were combined with the adjuvant DETOX and injected subcutaneously on weeks 1, 2, 3, 4 and 6 for one or two courses, and then monthly in patients with objective clinical responses. Of 109 patients, 22 (20%) have had objective clinical regression of tumor masses, with 5% complete responses. Ten patients have lived more than a year. Eight of the 10 are still alive, five of whom have lived more than 3 years. It was not necessary to achieve complete remissions to cause an increase in survival, and most of the long-surviving patients have one or more (stable) residual nodules. The pace of the disease process has clearly been slowed in those individuals. A rise in the level of cytotoxic T lymphocyte precursors in the blood (pCTL) has correlated with clinical response. Only one patient without such a rise in pCTL has had a response, and assays in that patient were considered unreliable. Both CD4+ and CD8+ CTL have been cloned from the blood of immunized patients. Both types of CTL killed a number of melanoma cell lines, but not other types of tumor or normal cells (lymphoblasts and melanocytes). CD8+ CTL have not been restricted to killing the autologous melanoma. MHC restriction by the HLA-A2 locus was identified. CD4+ CTL were not restricted only by Class II HLA antigens. Many CD4+ clones killed HLA Class II-negative melanomas, and we were able to block cytotoxicity of a particular clone with either anti-HLA Class I or anti-Class II MHC monoclonal antibodies, or both. An association of clinical response to the theraccine with certain HLA phenotypes, notably HLA-C3, -A2 (and the cross-reactive HLA-A28), B12 (and the related alleles (HLA-B44 and -B45) and perhaps DR4, particularly when combinations of those alleles were present, was suggested by our analysis of 70 patients. It is possible that this simply indicates the sharing of MHC antigens between the immunizing melanomas and the patient's melanoma. However, these MHC molecules may be important in their own right in presenting melanoma-associated antigens in CTL in vivo. Subtractive hybridization of mRNA from lung squamous carcinoma cells from cDNA of the M-1 melanoma cell line has yielded several DNA sequences unique to melanoma. Those are now being analyzed for possible immunogenicity, with cytotoxicity by CTL from immunized patients as the major criterion.(ABSTRACT TRUNCATED AT 400 WORDS)     

The genome and epigenome of malignant melanoma

Malignant melanoma originates in melanocytes, the pigment-producing cells of the skin and eye, and is one of the most deadly human cancers with no effective cure for metastatic disease. Like many other cancers, melanoma has both environmental and genetic components. For more than 20 years, the melanoma genome has been subject to extensive scrutiny, which has led to the identification of several genes that contribute to melanoma genesis and progression. Three molecular pathways have been found to be nearly invariably dysregulated in melanocytic tumors, including the RAS-RAF-MEK-ERK pathway (through mutation of BRAF, NRAS or KIT), the p16 INK4A-CDK4-RB pathway (through mutation of INK4A or CDK4) and the ARF-p53 pathway (through mutation of ARF or TP53). Less frequently targeted pathways include the PI3K-AKT pathway (through mutation of NRAS, PTEN or PIK3CA) and the canonical Wnt signaling pathway (through mutation of CTNNB1 or APC). Beyond the specific and well-characterized genetic events leading to activation of proto-oncogenes or inactivation of tumor suppressor genes in these pathways, systematic high-resolution genomic analysis of melanoma specimens has revealed recurrent DNA copy number aberrations as well as perturbations of DNA methylation patterns. Melanoma provides one of the best examples of how genomic analysis can lead to a better understanding of tumor biology. We review current knowledge of the genes involved in the development of melanoma and the molecular pathways in which these genes operate.     

A putative marker for human melanoma. A monoclonal antibody derived from the melanoma gene in the Xiphophorus melanoma model.

A MoAb was raised against a peptide corresponding to an exposed domain of the putative tyrosine kinase receptor protein encoded by Xmrk, a gene involved in melanoma formation and/or progression in the Xiphophorus fish melanoma model. The antibody reacts specifically with cells from human melanocytic lesions, ie, common acquired nevi, primary and metastatic melanoma biopsies. No reactivity with other cells, including normal melanocytes, was observed in the biopsies or with cells in biopsies from normal tissue (skin, liver, lung, spleen) and from other malignancies including those of neuroectodermal origin. The reactivity was very weak and variable in metastatic melanomas but very strong and characteristic of a receptor-type antigen in primary melanomas, a stage in melanoma progression in which cells have acquired metastasizing potential. It is suggested that the antigen recognized may be involved in growth promotion and represents the human equivalent of the fish melanoma gene product.     

E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors

In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation.     

Primary acquired melanosis of the conjunctiva

Forty-one cases of conjunctival primary acquired melanosis (PAM) were studied to determine the frequency of progression to malignant melanoma and to establish prognostic parameters for progression to melanoma. Two subdivisions were identified: lesions with cytologically atypical melanocytes (PAM with atypia, 28 lesions, 68.3 per cent) and those lacking cytologically atypical melanocytes (PAM without atypia, 13 lesions, 31.7 per cent). None of the lesions of PAM without atypia progressed to melanoma. Thirteen of the 28 lesions of PAM with atypia (46.4 per cent) progressed to melanoma. Progression to melanoma was more frequent in the lesions of PAM with atypia if basilar hyperplasia was not the dominant histologic pattern (90 per cent progression, P = 0.02) or if any epithelioid cells were present (75 per cent progression, P = 0.02). It was not possible to determine which lesions were atypical on the basis of clinical appearance. Lesions at risk for the development of melanoma should be totally extirpated.     

Attempts to Optimize Active Specific Immunotherapy for Melanoma

During the past 5 years, we have been conducting clinical trials with a therapeutic melanoma vaccine (melanoma“theraccine”). Mechanical lysates of two melanoma cell lines chosen for their complementary characteristics were combined with the adjuvant DETOX and injected subcutaneously on weeks 1, 2, 3, 4 and 6 for one or two courses, and then monthly in patients with objective clinical responses. Of 109 patients, 22 (20%) have had objective clinical regression of tumor masses, with 5% complete responses. Ten patients have lived more than a year. Eight of the 10 are still alive, five of whom have lived more than 3 years. It was not necessary to achieve complete remissions to cause an increase in survival, and most of the long-surviving patients have one or more (stable) residual nodules. The pace of the disease process has clearly been slowed in those individuals.

A rise in the level of cytotoxic T lymphocyte precursors in the blood (pCTL) has correlated with clinical response. Only one patient without such a rise in pCTL has had a response, and assays in that patient were considered unreliable. Both CD4+ and CD8+ CTL have been cloned from the blood of immunized patients. Both types of CTL killed a number of melanoma cell lines, but not other types of tumor or normal cells (lymphoblasts and melanocytes). CD8+ CTL have not been restricted to killing the autologous melanoma. MHC restriction by the HLA-A2 locus was identified. CD4+ CTL were not restricted only by Class IIHLA antigens. Many CD4+ clones killed HLA Class H-negative melanomas, and we were able to block cytotoxicity of a particular clone with either anti-HLA Class I or anti-Class II MHC monoclonal antibodies, or both.

An association of clinical response to the theraccine with certain HLA phenotypes, notably HLA-C3, -A2 (and the cross-reactive HLA-A28), B12 (and the related alleles (HLA-B44 and -B45) and perhaps DR4, particularly when combinations of those alleles were present, was suggested by our analysis of 70 patients. It is possible that this simply indicates the sharing of MHC antigens between the immunizing melanomas and the patient's melanoma. However, these MHC molecules may be important in their own right in presenting melanoma-associated antigens in CTL in vivo.

Subtractive hybridization of mRNA from lung squamous carcinoma cells from cDNA of the M-l melanoma cell line has yielded several DNA sequences unique to melanoma. Those are now being analyzed for possible immunogenicity, with cytotoxicity by CTL from immunized patients as the major criterion. Our hope is eventually to produce a wholly synthetic melanoma theraccine incorporating a group of critical antigens proved to be immunogenic to humans.     

Associated expression of HLA class I and class II antigens on melanoma cells in surgically removed metastases

Malignant transformation of melanocytes and further neoplastic progression may be associated with qualitative and/or quantitative changes in expression of HLA class I and class II antigens. Since previous immunohistochemical studies of surgically removed melanoma lesions have suggested a relationship in the expression of HLA class I and class II antigens, we have investigated the expression of these antigens at the single cell level. Double immunofluorescence staining of frozen sections of melanoma metastases and immunoelectron microscopic double labelling of melanoma cell suspensions prepared from three of these lesions has detected three HLA phenotypes on the large majority of melanoma cells: either both HLA class I and class II antigens, neither HLA antigen or only HLA class I antigens. In four out of the 11 lesions a few melanoma cells were found to express HLA class II antigens and to lack HLA class I antigens. A relationship was also found in the level of expression of HLA class I and class II antigens, as estimated by the intensity of staining with monoclonal antibodies. The level of expression of HLA class II antigens appeared to be similar to or lower than that of HLA class I antigens on the large majority of melanoma cells. This coordinated heterogeneity in the expression of HLA class I and class II antigens by melanoma cells may have implications in the interactions of tumour cells with the host's immune system.