Glycoconjugate profiling of primary melanoma and its sentinel node and distant metastases: implications for diagnosis and pathophysiology of metastases.

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

The proteasome inhibitor bortezomib augments anti-proliferative effects of mistletoe lectin-I and the PPAR-gamma agonist rosiglitazone in human melanoma cells.

BACKGROUND: The NFkappaB signalling pathway plays an important role in chemoresistance and decreased apoptosis. One indirect way to inhibit the NFkappaB pathway is to slow down the proteasomal degradation of its inhibitor IkappaB, thus preventing NFkappaB from translocation into the nucleus. Hence, the effect of the proteasome inhibitor bortezomib (Velcade) on the cell proliferation of the MV3, FemX-1 and G361 human melanoma cell lines and its action in combination with the PPAR-gamma agonist rosiglitazone or the mistletoe lectin ML-I, both having anti-proliferative effects on melanoma cells in single agent use, was investigated. MATERIALS AND METHODS: Proliferation of melanoma cells under the different treatment regimes over a broad concentration range (0.0001-100 microg/ml) was assessed by means of the XTT cell proliferation assay. RESULTS: At a concentration of 0.1 microg/ml bortezomib significantly reduced the proliferation rate of all melanoma cells to 1-13% of the control, which was mediated through increased apoptosis and inhibition of NFkappaB expression. Furthermore, the combination of bortezomib and rosiglitazone was the most potent and increased the effectiveness against melanoma cell growth by 63-71% (compared to single use of rosiglitazone) and by 27-39% (compared to single use of bortezomib), respectively. CONCLUSION: This combination strategy might be a promising approach for future melanoma therapy.

CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease.

PURPOSE: The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis. PATIENTS AND METHODS: CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression. RESULTS: A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P <.0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness. CONCLUSION: Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.

Influence of mistletoe lectins and cytokines induced by them on cell proliferation of human melanoma cells in vitro.

Although aqueous mistletoe extracts are widely used in complementary cancer therapy, the precise mode of action of their main therapeutic agents, the three mistletoe lectins (MLs), is poorly understood as they act both as cytotoxic agents and as immunomodulators due to their cytokine release by mononuclear cells. Thus, this study aims to investigate both the direct and the indirect effects of MLs on the growth of human melanoma cells in vitro. Proliferation of six human melanoma cell lines under ML treatment and additionally under the influence of cytokines induced by them (TNF-alpha, IL-1, IL-6) was assessed by means of the tetrazolium derived reduction (XTT) assay. Furthermore, ML binding patterns were analysed and correlated with the biological effects. All three MLs inhibited melanoma cell proliferation in a dose-dependent manner starting at very low ML concentrations (0.001-100 ng/ml) with ML-I being the most cytotoxic lectin (significant inhibition of ultra-sensitive cell line MV3 at 1 x 10(-13) ng ML-I/ml). Even if applied in a broad concentration range (0.0001-100 ng/ml) cytokines had no influence on cell proliferation at all. For ML-I, no association between binding intensity and cytotoxicity was observed, while for ML-II and -III an association between binding and toxicity was established. In conclusion, this study emphasises the direct anti-proliferative effect of the mistletoe lectins on melanoma cells with ML-I being superior to MLs-II and -III. The observation of an ultra-sensitivity of one cell line towards ML-I toxicity may serve as an explanation for the therapeutic success in anecdotal case reports and needs further investigations.

Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma.

Modulation of cell adhesion molecule expression plays a key role in melanoma metastasis. In particular, the expression of the cell adhesion molecule L1 has been associated with the metastatic phenotype in a murine model of malignant melanoma. However, no such association between L1 expression and metastasis has been investigated in a clinical study. Therefore, L1 expression was determined immunohistochemically in 100 cases of malignant melanoma and correlated with metastasis in a 10-year retrospective study. Furthermore, nine distant metastases and five sentinel lymph node metastases were analysed for their L1 expression. Additionally, the expression of alpha2,3 sialic acid residues, which are recognised by the siglec domain of L1, was determined by Maackia amurensis agglutinin (MAA) lectin histochemistry. The log-rank test between Kaplan-Meier curves revealed a positive association between L1 expression and metastasis (P<0.0001) and multivariate Cox regression analysis adjusted for tumour thickness, ulceration and mitotic rate confirmed the prognostic power of L1 in malignant melanoma. As alpha2,3 sialic acid residues were absent in melanoma cells, homotypic adhesion between melanoma cells via their siglec domain can be excluded, suggesting a different adhesive function of L1 during melanoma metastasis. The functional role of L1 was further stressed by the fact that its expression was preserved in metastatic lesions.

Magnetic resonance imaging of melanoma metastases in a clinical relevant human melanoma xenograft scid mouse model.

This study aimed to analyse (i) the metastatic behaviour of human melanoma FEMX-1 cells in scid mice after surgical excision of the PT and (ii) to evaluate the feasibility of magnetic resonance imaging (MRI) for the detection of melanoma metastases. Histology proved both high specificity (95%), and high sensitivity of MRI detection of melanoma metastasis. CEACAM1, L1, and HPA-binding site expression, all markers predicting metastasis in clinical studies, were preserved in the metastatic nodules. Thus, our xenograft model closely resembles the clinical situation of post-operative development of distant organ metastasis and demonstrates that MRI is a sensitive and highly qualified technology for intra-vital monitoring of melanoma progression.

Anti-proliferative effect of peroxisome proliferator-activated receptor gamma agonists on human malignant melanoma cells in vitro.

Malignant melanoma has a poor reputation for early spread and no curative treatment is yet available. As peroxisome proliferator-activated receptor gamma (PPARgamma) agonists (glitazones) have recently been shown to have growth-inhibiting effects on different cancer lineages, the aim of this study was to analyze the effects of four glitazones (rosiglitazone, ciglitazone, pioglitazone and troglitazone) on the growth of six human malignant melanoma cells in vitro. Proliferation of six human melanoma cell lines under glitazone treatment over a broad concentration range (0.15-300 micromol/l) was assessed by means of the XTT cell proliferation assay, and expression of PPARgamma in these cell lines was analyzed using both immunohistochemical and molecular biological techniques. All four glitazones showed a significant dose-dependent anti-proliferative effect on all six cell lines starting at a concentration of 0.3 micromol/l, with ciglitazone being the most potent inhibitor of cell growth, followed by troglitazone, rosiglitazone and pioglitazone. PPARgamma was predominantly localized in the cytoplasm; however, there were quantitative differences in PPARgamma expression between the different cell lines as demonstrated by quantification of Western blots. As an already approved class of drugs, glitazones have been found to significantly inhibit growth of human malignant melanoma cells in vitro and might be a promising tool for further therapeutic studies.

The developmentally regulated neural crest-associated glycotope HNK-1 predicts metastasis in cutaneous malignant melanoma.

Aberrant glycosylation is a common feature of metastatic sub-clones of malignant tumours and in uveal melanoma in particular, the HNK-1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK-1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10-years' follow-up. Furthermore, HNK-1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan-Meier analysis revealed a positive association between HNK-1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK-1 expression as an independent prognostic marker. HNK-1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK-1 immunoreactivity. None of the benign pigmented lesions exhibited HNK-1 immunoreactivity. Expression of the HNK-1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK-1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK-1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma.