Comparison of two prognostic markers for malignant melanoma: MIA and S100 beta.

It has recently been shown that the serum level of melanoma-inhibitory protein (MIA) provides useful information for the therapy and follow-up of patients with malignant melanoma. Previously, S100 beta has been described as a useful tumor marker for malignant melanoma. In this study, we compare the significance of the two markers in follow-up, therapy outcome and prognosis by measuring MIA and S100 beta serum levels in 50 melanoma patients. Serum levels were measured in patients with malignant melanomas of stages I-IV with at least 3 time points of measurement. Serial MIA and S100 beta measurements were obtained from 32 patients with stage IV disease in parallel to chemotherapy and from 18 patients with a history of stage I and stage II disease during follow-up. The response to chemotherapy in stage IV disease and relapse of melanoma during follow-up correlated with changes in MIA and S100 beta serum levels. In comparison, MIA revealed slightly higher specificity and sensitivity. In conclusion, both markers are useful for detection of progression from localized to metastatic disease during follow-up and for monitoring therapy of advanced melanomas.     

Ultraviolet radiation induces p16CDKN2A expression in human skin.

The loss of the tumor suppressor gene product p16 in melanoma is well documented, although the normal physiological function of p16 in skin melanocytes is unknown. In this report, we demonstrate that when human skin was irradiated with suberythemal doses of UV radiation, levels of p16 were dramatically increased by 16 h postirradiation, peaking at 24 h, and declining by 72 h. p16 was expressed in the nucleus and cytoplasm of melanocytes and keratinocytes within the epidermis, and the pattern of p16 expression within the epidermis was dependent on the penetrative ability of the different UV wavebands. The existence of a UV-induced response pathway involving up-regulated p16 expression may provide a mechanism linking the loss of p16 and UV exposure with the development of melanoma.     

Allelotype of posterior uveal melanoma: implications for a bifurcated tumor progression pathway.

To further elucidate the somatic genetic alterations leading to acquired choroidal and ciliochoroidal melanoma, we screened every autosomal arm and the X chromosome of 50 primary posterior melanomas (31 choroidal tumors and 19 ciliochoroidal tumors). A minimum of two microsatellite markers were used to achieve at least 90% informativity (excluding X). Twenty-eight of 47 informative tumors (59%) showed allelic loss of all informative markers on chromosome 3, consistent with monosomy 3 (M3). Allelic imbalance of 8q was observed in 60% of tumors. A total of 28% of tumors displayed allelic loss of 6p. We then compared these genetic alterations with the status of chromosome 3 and found a relative absence of 6p alteration in tumors with M3 (P = 0.0005). Additionally, all observed 8q imbalance was associated with either M3 or alteration of 6p, suggesting that 8q alterations occur later in tumor progression. The mutual exclusivity of M3 and 6p alterations suggests a bifurcated tumor progression model. In this model, M3 or 6p loss identify distinct pathways, both followed by 8q loss in tumor progression.     

Pyrimethamine induces apoptosis of melanoma cells via a caspase and cathepsin double-edged mechanism

The unresponsiveness of metastatic melanoma to conventional chemotherapeutic and biological agents is largely due to the development of resistance to apoptosis. Pyrimethamine belongs to the group of antifolate drugs, and in addition to antiprotozoan effects, it exerts a strong proapoptotic activity, which we recently characterized in human T lymphocytes. However, no data regarding pyrimethamine anticancer activity are available thus far. To this end, we examined the in vitro effects of pyrimethamine on apoptosis, cell cycle distribution, and cell proliferation of human metastatic melanoma cell lines. The in vivo antitumor potential of pyrimethamine was evaluated in a severe combined immunodeficiency (SCID) mouse xenotransplantation model. Our data indicate that pyrimethamine, when used at a clinically relevant concentration, induced apoptosis in metastatic melanoma cells via the activation of the cathepsin B and the caspase cascade (i.e., caspase-8 and caspase-9) and subsequent mitochondrial depolarization. This occurred independently from CD95/Fas engagement. Moreover, pyrimethamine induced a marked inhibition of cell growth and an S-phase cell cycle arrest. Results obtained in SCID mice, injected s.c. with metastatic melanoma cells and treated with pyrimethamine, indicated a significant inhibitory effect on tumor growth. In conclusion, our results suggest that pyrimethamine-induced apoptosis may be considered as a multifaceted process, in which different inducers or regulators of apoptosis are simultaneously implicated, thus permitting death defects of melanoma cells to be bypassed or overcome. On these bases, we hypothesize that pyrimethamine could represent an interesting candidate for the treatment of metastatic melanoma.     

HSP90 as a marker of progression in melanoma.

BACKGROUND: HSP90 chaperones molecules critical for cell survival and malignant progression, including mutated B-raf. HSP90-targeting agents are in clinical trials. No large studies have been conducted on expression of HSP90 in melanomas. MATERIALS AND METHODS: Tissue microarrays containing 414 nevi, 198 primary and 270 metastatic melanomas were assessed using our automated quantitative analysis (AQUA) method of in situ protein measurement; we use S-100 to define pixels as melanocytes (tumor mask) within the array spot, and measure HSP90 expression within the mask using Cy5-conjugated antibodies. RESULTS: HSP90 expression was higher in melanomas than nevi (P < 0.0001) and higher in metastatic than primary specimens (P < 0.0001). No association was seen between high HSP90 expression and survival in the primary or metastatic patient subsets. In primary melanomas, high HSP90 expression was associated with higher Clark level (P = 0.0167) and increased Breslow depth (P < 0.0001). CONCLUSIONS: HSP90 expression was significantly higher in tumors than nevi and was associated with disease progression, indicating that it might be a valuable drug target in melanoma, as well as a useful diagnostic marker. Prospective studies are needed to confirm the diagnostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.     

Adjuvant immunotherapy in melanoma: a new approach.

Patients with metastatic cutaneous melanoma to two or more regional lymph nodes have an extremely poor prognosis despite radical lymphadenectomy. In an attempt to improve the survival and to determine the safety of a new method of tumor specific adjuvant immunotherapy in such a high risk group of patients, nine patients were studied. Three to four weeks after regional lymphadenectomy, each of them received a single intradermal injection of Bacillus Calmette-Guérin. Three weeks later, they were immunized by allogenic melanoma cells obtained from live donors with distant metastases. Each patient received three vaccinations, each from a different donor (except in one), to avoid development of HLA response, but maintaining exposure to melanoma antigens. No cultured melanoma cells were used. Each vaccine consisted of mitomycin-C treated tumor cells mixed with purified protein derivative (PPD) of tuberculin given intradermally once per month for 3 months. The patients were then observed with no further treatment. Utilizing the leukocyte migration inhibition test, there was some in vitro evidence of tumor specific cell mediated response which seemed to disappear 1-2 months postimmunization. At 5 years, five of the nine patients (55%) were alive free of disease. No autoimmune diseases were detected in any of the immunized patients. A major hindering factor for such an approach was the limited availability of the allogenic melanoma cells.     

A new Mr 55,000 surface protein implicated in melanoma progression: association with a metastatic phenotype

Emergence of the invasive phenotype is a key event in the progression of human melanoma from benign proliferative lesions to malignant lesions. Recently we successfully selected in vivo from a poorly metastatic M4Beu. human melanoma cell line two variants (7GP and T1P26) that generate a higher frequency of spontaneous metastases to the lungs into immune-suppressed neonatal rats. Both cell lines showed no significant differences in the integrin profile of the subunits analyzed except for beta3, which was reduced to a background level in metastatic variants. To investigate how these variant sublines of human melanomas manage to sustain growth in the absence of alpha(v)beta3, a subtractive immunization approach was used to elicit host antibody response against cell surface proteins expressed on metastatic variants. In this study, a new monoclonal antibody (MoAb), LY1, that is highly specific for the 7GP and T1P26 variants, was isolated. LY1 identifies a membrane protein of Mr 55,000 on melanoma variants with epitopes that were resistant to sugar-cleaving enzymes. Immunostaining cells from variants by LY1 showed that staining is distributed to the cell periphery with high labeling intensity at the cell-to-cell contact points. This MoAb significantly inhibited invasion of metastatic variants through a reconstituted basement membrane (Matrigel) in vitro. Moreover, tumor growth of melanoma variants was dramatically affected in vivo with this MoAb. In vitro studies indicate that the LY1 MoAb does not inhibit chemotactic migration of the metastatic variants, the adhesion of tumor cells to vitronectin, collagen IV, fibronectin, and laminin, or cell proliferation. Expression of this antigen is high in human striated muscle, heart, spleen, brain, and lung and absent in kidney, liver, and pancreas. Using 59 fixed, paraffin-embedded archival tissues of human melanomas and nevi, LY1-reactive cells were not observed in melanocytes, nevi, or radial growth phase primary melanomas. In sharp contrast, LY1 selectively stained melanocytes derived from the vertical growth phase of many primary melanomas and metastatic melanomas. These results provide evidence that the Mr 55,000 protein expressed by selected variants with increased metastatic properties in vivo plays a functionally important role in determining metastasis. This molecule may represent a new metastatic risk marker in human melanoma and may be of biological importance in the identification of fatal metastatic subpopulations that have acquired competence for metastasis production.     

Up-regulation of ephrin-A1 during melanoma progression.

Ephrin-A1, formerly called B61, is a new melanoma growth factor; it is angiogenic and chemoattractant for endothelial cells. EPH-A2, or ECK (a receptor for ephrin-A1), is ectopically expressed in most melanoma cell lines; the pathology where this expression is first manifested and the possible role of the receptor in tumor progression are unknown. To determine these, we studied the expression of this ligand and receptor in biopsies of benign and malignant melanocytic lesions. EPH-A2 was not detected in normal melanocytes, benign compound nevi or advanced melanomas, though it was found in 2 of 9 biopsies of malignant melanoma in situ. Ephrin-A1 was present in occasional early lesions and in advanced primary melanomas (43%) and metastatic melanomas (67%). Expression of ephrin-A1 was induced in melanoma cells by pro-inflammatory cytokines. Our findings are consistent with 2 possible roles for ephrin-A1 in melanoma development: it may promote melanocytic cell growth or survival and induce vascularization in advanced melanomas. Both effects may be potentiated by inflammatory responses. Our data are consistent with earlier observations that an inflammatory infiltrate is associated with poor prognosis in thin primary melanomas.     

Ultraviolet radiation and the risks of cutaneous malignant melanoma and non-melanoma skin cancer: perceptions and behaviours of Danish and American adolescents.

The highest prevalence rates of skin malignancy in the northern hemisphere occur in Scandinavia and the United States (USA). Most Danes and Americans receive 50% of their lifetime ultraviolet (UV) radiation before the age of 21, making it important to address sun exposure risks with adolescents. The project was undertaken to determine differences between Danish and American adolescents in knowledge of sun exposure and skin malignancy, activities accounting for sun exposure, and means used for sun protection. Questionnaires regarding skin cancer and sun exposure were distributed to 674 secondary school age students in Hilleroed, Denmark, and to 483 similarly aged students in Winston-Salem, North Carolina, USA. Differences in responses between and within groups were compared. American adolescents had more knowledge of the characteristics and malignant potential of melanoma than did Danish adolescents. Danish youth and females from both countries were significantly more likely to engage in sunbathing and tanning bed use. Black Danish students reported significantly more sunburn and were more likely to sunbathe or use a tanning bed than were black American students. Danish students were more likely than Americans to use sunscreen, however, Americans were more likely to apply sun protective factor (SPF) 15 or greater. In conclusion, given that sunbathing and tanning bed use are associated with the development of precancerous lesions and skin malignancy, Danish teens are at increased risk. The rates of skin malignancy are relatively high in Scandinavia and efforts to improve understanding of exposure and cancer risks should be undertaken in adolescents.     

Vascular endothelial growth factor-A(165) induces progression of melanoma brain metastases without induction of sprouting angiogenesis.

We investigated the mechanisms of vascularization in a brain metastases model of malignant melanoma. Parenchymal metastases expressing little vascular endothelial growth factor-A (VEGF-A) co-opted the preexistent brain vasculature, leading to an infiltrative phenotype. Metastases of the human melanoma cell line Mel57, engineered to express recombinant VEGF-A(165), showed accelerated growth in a combined expansive and infiltrative pattern with marked central necrosis. This difference in growth profile was accompanied by dilation of co-opted intra- and peritumoral vessels with concomitant induction of vascular permeability. Our data show that modulation of preexistent vasculature can contribute to malignant progression without induction of sprouting angiogenesis.     

Carnosol, radiation and melanoma: a translational possibility

Purpose

To compare the genoprotective and radioprotective effect of carnosol (COL) against damage induced by ionizing radiation with similar effects produced by different antioxidant compounds.

Methods

The genoprotective effect was studied by means of the micronucleus test for antimutagenic activity in which the reduction in the frequency of micronuclei was evaluated in cytokinesis-blocked cells of human lymphocytes. The radioprotective effects were studied by cell viability test (MTT) in PNT2 (normal prostate) and B16F10 (melanoma) cell lines when they were administered before exposure to different X-ray doses (4, 6, 8, 10 and 0 Gy).

Results

Carnosol shows a significant genoprotective capacity (p < 0.001) against radiation with a protection factor of 50 %, and a dose-reduction factor of 4.3. Cell survival obtained with COL administered before exposure to 10 Gy of X-rays showed a protection factor of 55.1 %, eliminating 39 % of radiation-induced cell death in normal epithelial cells of prostate (PNT2) (p < 0.001). However, in the melanoma cell lines (B16F10) assayed, COL acted not as a radioprotector, but as a sensitizing agent increasing the cellular death by 34 % (p < 0.01) and producing an enhancement ratio of 2.12.

Conclusions

Carnosol may be developed as a radioprotective agent in the non-tumoral cells. However, in the B10F16 melanoma cells, melanogenesis is activated by COL leading to redistribution of the enzymatic balances of glutathione and cysteine-lyase production, which could compromise the intracellular redox defence system. This effect appears as an increase in the capacity of ionizing radiation-induced damage, and thus exhibits a paradoxical protective effect of COL on melanoma cells.