Correlation of stem cell marker nestin expression on circulating melanoma cells with extension of disease and survival.

Abstract

8551 Background: Within circulating melanoma cells (CMCs) there may be a subset of cells with stem cell characteristics able to develop distant metastasis. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients (pts) with metastatic melanoma. Stem cell marker assessment included nestin (NES) and CD133. Methods: Twenty-five ml blood were collected after informed consent from 32 consecutive pts affected by metastatic uveal (n = 14) or cutaneous (n = 18) melanoma and from (n = 6) healthy volunteers according to ethically approved protocols. Blood was enriched for tumour cells by CD45 depletion of the non-melanoma cell fraction using a magnetic bead separation technique. Potential melanoma cells were identified using flow cytometry with the markers MELAN-A and HMB45. Multiparameter cytometry was carried out to co-stain with the combinations of CD133 and NES. Five tissue samples from metastatic lesions of five different pts were stained with the same antibodies by immunohistochemistry. Results: No cells positive for the melanocyte markers were detected in blood from volunteers. In pts MELANA and/or HMB45 positive cells were detected in 28 samples (87.5%). The absolute number of melanoma cells ranged from 0 to 1233 (median 53) per 10 mL of blood. NES expressing cells were more represented compared to CD133 expressing cells (median 18.0% [0.3%-85.1%] vs. median 0.1% [0%-23.4%]). Percentage of NES-positive cells correlated significantly with tumor burden (p = 0.01) and number of metastatic sites (p = 0.03), whereas percentage of CD133-positive cells and absolute total number of cells did not. Cox regression analysis revealed levels of LDH (HR: 12.8 [1.35-121.5]; p = 0.02), number of metastatic sites (HR 3.87 [1.66-9.03]; p = 0.02), tumor burden (HR 5.72 [1.57- 20.9]; p = 0.01) and high level of NES expression (HR 3.5 [0.92-13.6]; p = 0.04) to be factors related to shorter overall survival. CD133 and NES expression profiles on CMCs and in matched metastatic tissue were similar. Conclusions: Melanoma cells in peripheral blood expressed stem cell associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. No significant financial relationships to disclose.