Background and objectives: There is growing evidence that a minor subset of melanoma cells drive melanoma progression and is responsible for the metastatic potentials. Thus, identification and elimination of these cancer initiating stem cells and understanding their mechanisms is paramount. With a novel approach, we attempted to identify tumor biomarkers specific for cancer initiating cells. A comprehensive immunological study of liver and lympnode metastatic tissues compared to the primary tumors was performed. Patients and Methods: Samples were excised from surgically removed cancerous tissues, lymphnode metastases and peripheral blood of patients with malignant melanomas (Ethical Permission No: ETT TUKEB 16462- 02/2010) (n= 118) and processed in cellular immunological and molecular genetic assays. Immunohistochemistry was performed on core biopsies of liver metastases (n=31) and tissue microarrays of primary and metastatic tumors (n: 32) with antibodies against various novel tumorassociated antigens and B cell markers. Fresh cell cultures of lymphnodes and cancerous tissues were immunofluorescence FACS sorted and analysed for characteristics relevant to cancer initiating cells. Results: Immunohistochemistry of paraffin-embedded and fresh frozen tissue sections identified a characteristic colocalisation of unique GD3 sialilated glycosphingolipids and CD20 positivity in primary melanomas and metastatic tissues. The double positive minor cell population (0.1% - 0,8 %) could be selected by immunofluorescence FACS in most of the cases with lymphnode metastases. They showed characteristic growth pattern and longterm tough cancerous outgowth potential, and were positive for CD133, Nestin, ABCB5, CD20 aswell as for the newly identified strong GD3 ganglioside (90% positivity). Further characterisation at gene expression and protein levels revealed unique characteristics of these minor cell populations. Conclusion: Our results provide novel markers to select stem cell like cancer initiating cells in malignant melanomas. This novel approach opens a door for specific detection and targeted elimination of cancer stem cells in patients with metastatic melanomas and provides basic material for antibody engineered specific, fully human immunoconjugates for diagnostics and therapy. Acknowledgements: The financial support of Harry J Lloyd Charitable Trust and the devoted work of the Oncodermatological, Surgical and Pathological Departments in the National Institute of Oncology are acknowledged.

Citation Format: Beatrix Kotlan, Gyorgy Naszados, Maria Godeny, Vanda Plotar, Laszlo Toth, Laszlo Gobor, Szabolcs Horvath, Miklos Kasler, Gabriella Liszkay, Francesco M. Marincola. A novel approach to select cancer initiating cells by targeting unique sialilated glycosphingolipids and CD20 in patients with metastatic melanomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2013-242