Beatrix Kotlan

Revealing cancer initiating cells in metastatic melanomas by harnessing the host′s anti tumor humoral immune mechanisms

Results Cell cultures grew out of the great majority of the starter metastatic tissue specimen and cancer initiating cells could be sorted (BD FACSAvia Sorter) by colocalized unique sialilated glycosphingolipids and anti CD20 binding capacity. Characteristic growth pattern, spheroid forming, CSC markers like CD133, Nestin, ABCB5, CD20 and unique GD3 gangliosides were found. Patients′ sera and selected patients′ Epstein Barr Virus transformed cell supernatants in bulk or after limiting dilution cloning were tested for cancer binding by ELISA and immunofluorescence. Our novel tumor infiltrating B cell antibody phage display technique and DNA sequence cluster analysis (Vector NTI Advance 11, Bioedit 7.0, ClustalX2.0.11) resulted in some antibody fragments, belonging to representative tumor binding antibody variable region subgroups, with defined silalilated glycosphingolipid specificity.

Conference overview: Cancer Immunotherapy and Immunomonitoring (CITIM): Moving forward

The immune system is a critical element involved in the control of tumor development and progression. While professionals have learned how to manipulate the immune system to generate tumor-specific immune responses, cancer immunotherapy has not yet delivered substantial clinical benefits. It has become increasingly clear that tumor-induced abnormalities in the immune system not only hamper tumor immunosurveillance, but also limit the efficacy of cancer immunotherapy. Meanwhile, the results of recent studies allow the belief that one is on the edge of a real breakthrough in this promising direction in cancer therapy. The 2nd International Conference ‘Cancer Immunotherapy and Immunomonitoring (CITIM)’ was the second meeting in Eastern Europe to specifically focus on the issue of immune regulation in the tumor environment, cancer immunotherapy, and immunomonitoring of immunotherapeutic clinical trials. This CITIM Conference held in Budapest, Hungary, was comprised from 12 plenary sessions, Best Abstract Award session, Poster session, and four Keynote lectures. Outstanding presentations and numerous productive discussions summarized the current place of the field and opened new directions for improving monitoring and therapy for patients with cancer.

Conference Scene: Immunotherapy reaches new milestones in cancer eradication

Biotherapy is widely considered as the fourth treatment modality for patients with cancer, and uses the constantly increasing knowledge in molecular biology, cell biology and immunology. Biotherapy uses naturally occurring biological molecules (e.g., cytokines and antibodies) or works by the manipulation of normal biological mechanisms (controlling or inhibiting tumor growth). Important achievements in anticancer drug development are immunotherapeutic strategies recently approved by the US FDA as well as clinical data of the cancer patients treated in clinical trials. There is a need to expand these novel cancer immunotherapeutic modalities for cancer patients all over the world. To meet that goal, it is essential to spread the information, to summarize the new clinical data and to draw the conclusions from the clinical and preclinical investigations. These frontline tasks can be well advanced by organizing international conferences in this domain in less scientifically developed countries, with a significant tumor burden statistics. Therefore, special efforts were done to organize the 2nd International Cancer Immunotherapy and Immunomonitoring Conference (CITIM-2011) in Hungary.

“Immune B Cells know it better”: tumorimmunological panel assay to define tumor-associated antigen binding antibodies in patients with metastatic melanomas.

Background Revealing novel cancer targeting biomarkers is a great challenge, and especially urging in cancer types with a more pronounced metastatic feature. We focus on potential anti-tumor immune reactions of the host. In order to harness the natural humoral immune response a novel immunological and molecular genetic panel assay has been developed for the investigation of patients with melanomas.

Natural immunomodulator preimplantation factor PIF affected cancer growth in malignant melanomas

Background and objectives Malignant 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. Methods

Minor tumor infiltrating B cells opened a door to reveal and eliminate cancer initiating cells in metastatic melanomas

Meeting abstracts The theory and investigations on cancer stem cells (CSCs) have received growing attention, as these cells are responsible for the failure of cancer therapeutic strategies and the return of cancer. A complex tumorimmunological study on primary and metastatic cancerous tissue

Anti-tumor antibody profile analysis to harness the potentials of B cells in melanomas and the natural humoral immune response

Meeting abstracts Natural humoral immune response and autoimmune mechanisms have great importance in keeping the balance of tumorimmunity, although it has not yet been fully understood. We aimed to reveal potential anti-tumor immune response by immunoglobulin (Ig) profile analysis of patients with

New strategy to reveal the black seeds of melanomas (cancer stem cells) with their vulnerable characteristics at cellular and molecular levels

Meeting abstracts A suitable approach to select cancer stem cells (CSC), the black seed of melanomas would enable their characterization and elimination. Cancerous tissues from primary and metastatic lesions of patients with malignant melanomas (n=150) were investigated by cell cultures and

PreImplantation factor (PIF) potentiates static magnetic field (SMF) effect to decrease tumor burden (melanoma murine model)

Background and objectives PreImplantation factor (PIF) secreted by viable embryos exerts essential regulatory role on global systemic immune response. Synthetic PIF (PIF) translates endogenous effects to immune disorder models. Metastatic melanoma displays tumor-immunological behaviour. Static magnetic field (SMF) affects inflammatory reactions. There is increased interest toward SMF’s potential antitumor effects. Herein examined a novel anti-melanoma strategy using combined physical and immune-based therapy. Methods Daily whole-body SMF exposure, combined with subcutaneous PIF administration was examined in engrafted HT199 melanoma cells’ progression, transplanted into NSG mice. PIF effect on unique tumor-associated antigen expression relevant for tumor proliferation/ invasion was examined in vitro using specific antibodies. Direct PIF anti-proliferative effects on several cancer cell lines were tested using MTT. Results PIF potentiates SMF beneficial effect by reducing tumor volume vs. control (Mmax=96%) on day 34. Metastatic spleen mass is reduced by SMF alone (M=59%) or combined with PIF (M=62%). Daily SMF exposure alone inhibits tumor outgrowth (Mmax=60%, F5.32 (P<0.002) =21.16) while in combination with PIF, effect is considerably potentiated (M=80%), F5.32(P<0.0004)=34.84). PIF did not impair tumor antigen expression nor reduced significantly cultured tumor cell lines’ proliferation. Conclusions Collectively, results indicate that PIF’s potentiating antitumoral effect is mainly immune-regulatory, synergizing with SMF’s pro-tumor necrosis properties. The preserved tumor-associated antigen expression is important for the maintained antitumor immune activity. Overall, combined physico / immune regulatory treatment represents a useful, promising novel avenue for anti-cancer strategy.

Abstract 242: A novel approach to select cancer initiating cells by targeting unique sialilated glycosphingolipids and CD20 in patients with metastatic melanomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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