We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.

There is far less information available about the tumor infiltrating B (TIL-B) cells, than about the tumor infiltrating T cells. We focused on discovering the features and potential role of B lymphocytes in solid tumors. Our project aimed to develop innovative strategies to define cancer membrane structures. We chose two solid tumor types, with variable to considerable B cell infiltration. The strategy we set up with invasive breast carcinoma, showing medullary features, has been introduced and standardized in metastatic melanoma. After detecting B lymphocytes by immunohistochemistry, VH-JH, Vκ-Jκ immunoglobulin rearranged V region genes were amplified by RT-PCR, from TIL-B cDNA. Immunoglobulin variable-region genes of interest were cloned, sequenced, and subjected to a comparative DNA analysis. Single-chain variable (scFv) antibody construction was performed in selected cases to generate a scFv library and to test tumor binding capacity. DNA sequence analysis revealed an overrepresented VH3-1 cluster, represented both in the breast cancer and the melanoma TIL-B immunoglobulin repertoire. We observed that our previously defined anti GD3 ganglioside-binder antibody-variable region genes were present in melanoma as well. Our antibody fragments showed binding potential to disialylated glycosphingolipids (GD3 ganglioside) and their O acetylated forms on melanoma cancer cells. We conclude that our results have a considerable tumor immunological impact, as they reveal the power of TIL-B cells to recognize strong tumor-associated glycosphingolipid structures on melanomas and other solid tumors. As tumor-derived gangliosides affect immune cell functions and reduce the B lymphocytes' antibody production, we suspect an important B lymphocyte and cancer cell crosstalk mechanism. We not only described the isolation and specificity testing of the tumor infiltrating B cells, but also showed the TIL-B cells' highly tumor-associated GD3 ganglioside-revealing potential in melanomas. The present data help to identify new cancer-associated biomarkers that may serve for novel cancer diagnostics. The two-direction regulation mechanism between immune B cells and the tumor could eventually be developed into an innovative cancer treatment strategy.

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Background and objectivesMalignant and trophoblastic cells share common features in terms of migration and invasion, while they represent striking differences also (1). Our results on immune mechanisms in pregnancy failure (2) and use of immunotherapeutics (3) fostered the present new approach: Pregnancy derived compounds for potential growth controlling effect in metastatic melanomas were studied. MethodsPreimplantation factor (PIF), a novel peptide secreted by viable embryos was selected (4), as its immune regulatory effects were advantageous (5). Based on our tumorimmunological project (Ethical permission ETT TUKEB 1642-02/2010), minor tissue samples from surgically removed lymphnodes of patients with metastatic melanomas were processed. Primary cultures were set up in special conditions with or without immunomodulatory PIF. In a parallel system, we transfused human HT199 melanoma cells into immunodeficient NOD.CgPrkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and followed tumor burden in PIF-treated groups. ResultsWe found that PIF treatment delayed tumor outgrowth in time. A tendency of lower tumor volumes was seen both after administration of immunomodulator peptide and use of additional physical (static magnetic field) treatment. A synergistic effect could be achieved by this combination treatment strategy. Retained or primed expression of glycolipid based tumorassociated antigens on PIF treated melanoma cells could be defined by immunofluorescence FACS and confocal laser microscopy. ConclusionsOur results suggest an indirect mechanism of PIF on tumor growth. Retained or enhanced tumor antigen expression is advantageous, as cancerous cells become predisposed to be recognized, while induced activation of antigen presenting cells is beneficial for elimination. Our complementary strategy resulted as effective and provides a new potential modality for cancer control.

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