08) due to the low number of samples and the weak expression of TRP-2 in the metastases ( Figure 1B). In addition, we found also a significant decrease of TRP-2 positive cells in cell culture compared to their matched primary tumor tissue (p = 0.01; Figure 1C).

These findings indicate the survival benefit of TRP-2 negative cells in cell culture. Using our newly developed co-staining of Mib-1 and TRP2, we analyzed the proliferating (MIB-1 positive) melanoma cells depending on their TRP-2 expression in primary melanoma, and metastases (Figure 2A-D). In melanoma metastases, proliferating TRP-2 negative cells were significantly more frequent compared to the primaries (p = 0.01; Figure 1D), whereas non-proliferating TRP-2 positive cells were significantly less frequent in melanoma metastases compared to the primaries (p = 0.01). For the subgroups, which were Bafilomycin A1 in vitro either negative or positive for both markers, we found no significant difference find more between primary melanomas and metastases. Interestingly the percentage of TRP-2−/Mib-1+ cells significantly correlated with Breslow tumor thickness in the patient group with Breslow tumor thickness over 1 mm (p = 0.048; Spearman’s correlation coefficient 0,3). Furthermore, these cells were significantly correlated with Hif-1α expression (p = 0.03; Spearman’s correlation coefficient 0,3) and therefore with hypoxic condition in primary melanoma. In addition patients

who had less than 15% of TRP-2−/Mib-1+ in their primary melanoma had statistically an approaching significance for a better tumor specific survival (p = 0.05; Figure 1E). Melanoma patients’ cell cultures expressed significantly less Melan A than primary melanomas (p = 0.001) or metastases (p = 0.001; Figure 1 F). In addition TRP-2 was significantly less expressed in cell cultures if compared to primaries (p = 0.001) or to metastases (p = 0.02; Figure 1A). Hif-1α expression was significantly

higher in melanoma metastases (p = 0.04) and cell cultures (p = 0.0001) when compared to Ribose-5-phosphate isomerase primary melanomas (Figure 1G). Analysing all melanoma samples primary melanomas, metastases and melanoma cell cultures we found a significant correlation between Hif-1α expression and the the presence of TRP-2−/Mib-1+ cells (p = 0.002; Spearman’s correlation coefficient 0,2) as well as with proliferation (Mib-1) alone (p = 0.01 Spearman’s correlation coefficient 0,2). However, analysing separately the different groups, only a significant correlation between Hif-1α expression and the presence of TRP-2−/Mib-1+ cells in melanoma patient’s cell cultures persisted (p = 0.01; Spearman’s correlation coefficient 0,3). We found no significant correlation between Hif-1α, and TRP-2 expression neither in primary melanoma, melanoma metastases nor melanoma cell cultures as expected by cell line experiments. We treated primary human melanoma cell cultures with hypoxia for 72 hours and subsequently performed qRT-PCR for TRP-2 (Figure 3C).