e22056 Background: Within circulating tumor cells there may be a subset of cell with stem cell (tumor initiating) characteristics able to develop distant metastasis. Several markers including nestin and CD133 have been found to be possible candidates to identify such a kind of subpopulation in other experimental models. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients with cutaneous or uveal melanoma after depletion of the leukocytes fraction. Methods: Between 50 and 100 ml of peripheral blood were collected from 12 melanoma patients with various tumor burden as well as three healthy volunteers. Blood samples were enriched for tumour cells by CD45 depletion of the leukocyte fraction using magnetic beads separation (EasySep, Stem Cell Technologies. Inc.). The remaining material was stained with antibodies for the markers Melan-A/Mart-1 (Dako) and HMB45 (Dako), CD133 (Miltenyi Biotec) and nestin (R&D System) and analysed by flow cytometry (BD FACSCalibur). Ten ml of blood were further processed and CD133, nestin, Melan-A/Mart-1 transcripts were quantified by Real Time RT-PCR (LightCycler, Roche Diagnostic). Results: CD45-depleted fractions in healthy controls were negative for melanoma markers. Melan-A/Mart-1 and/or HMB45 positive cells were detectable in 11 out of 12 melanoma patients. The absolute number of melanoma cells identified ranged from 6 to 176 per 10 ml of blood. Nestin expressing cells were more represented compared to CD133 expressing cells (median 27.4%, range 0.3% to 65.1% vs. median 9.3%, range 0.1% to 16.8%) within the melanoma fraction of cells positive for Melan-A/Mart-1 and/or HMB45. In one patient two different melanoma cell populations were detectable. The population of cells with lower expression of the melanoma markers showed at the same time higher expression of nestin and CD133 (5.9% vs. 1.3% and 10.2% vs. 6.7% respectively). Nestin results were in good accordance to the FACS data (nestin: r=0.55; CD133: r=0.23; Pearson test). Conclusions: The novel negative separation technique allows reliable isolation of melanoma cells from peripheral blood of patients with metastatic disease. A significant fraction of melanoma cells in peripheral blood bears a stem cell phenotype. No significant financial relationships to disclose.