Gunter Schuch

Treatment for advanced pancreatic cancer is still very unsatisfactory. Treosulfan is an alkylating agent used for conventional, as well as high-dose chemotherapy regimens, whereby plasma concentrations over 500 μg/ml can be achieved. We investigated the effects of treosulfan on pancreatic cancer cell lines. Using Panc-1, MIA PaCa-2 and Capan-2 cell lines, we investigated the in vitro cytotoxicity of treosulfan-alone and in combination with gemcitabine, 5-fluorouracil or irradiation. Treosulfan was potently cytotoxic against all pancreatic cancer cell lines at all concentrations (1-100 μg/ml). Combination of treosulfan and gemcitabine revealed strong synergistic effects independent of the sequence of drug administration. Similarly, synergism was observed with irradiation. Combination of treosulfan and 5-fluorouracil revealed antagonism. Treosulfan effectively kills pancreatic carcinoma cells in vitro and has synergistic activity in combination with gemcitabine and irradiation. These results warrant further investigation of treosulfan in the treatment of pancreatic cancer.

Evolving role of cetuximab in the treatment of colorectal cancer Gunter Schuch, Sebastian Kobold, Carsten BokemeyerDepartment of Oncology, Hematology, and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany In recent years, the monoclonal epidermal growth factor receptor (EGFR)-targeting antibody cetuximab was introduced into systemic therapy of colorectal cancer and gained an established role in the treatment of this disease. Cetuximab was shown to be active as a single agent in chemorefractory metastatic disease as well as in combination with varying chemotherapies. Recently, randomized trials demonstrated the activity of cetuximab combinations in the first-line setting of metastatic colorectal cancer. Interestingly, the activity of cetuximab was restricted to patients with KRAS wildtype tumors, as was seen with panitumumab, another EGFR antibody. While 60%–70% of tumors harbor KRAS wildtype genes, 30%–40% of tumors express oncogenic KRAS with mutations in codons 12 and 13 causing constitutive activation of signaling cascades downstream of EGFR and resistance to EGFR blockade. Since proof of KRAS wildtype status became a prerequisite for cetuximab treatment, KRAS testing is being established throughout the world. Future trials will address the question which part of the KRAS wildtype cohort will benefit from EGFR inhibition and how to identify those patients. Additionally, new strategies for treatment of KRAS mutated tumors are strongly needed. Recent developments and future strategies will be summarized.Keywords: cetuximab, colorectal cancer, KRAS

CEACAM-1 is involved in intercellular adhesion and is expressed in a variety of human tissues. In cases of malignant transformation, a down-regulation or loss of CEACAM-1 has been shown. In contrast, CEACAM-1 is not expressed in normal lung tissue or melanocytes. It has been demonstrated that an expression in these tissues is associated with the development of metastatic disease. The aim of the present investigation was to analyze a possible association between the expression of CEACAM-1 in pulmonary adenocarcinomas and their lymph node and hematogenous metastatic cells. CEACAM-1 expression was immunhistochemically evaluated in primary tumors, lymph nodes and distant metastases of 96 patients with metastatic pulmonary adenocarcinoma who had undergone surgery between 1999 and 2002. Expression of CEACAM-1 was shown in 78 out of 96 primary tumors (81.3%). A significant positive correlation was found between CEACAM-1 expression on cells of the primary tumor, lymph node metastases (p < 0.005) and hematogenous metastases (p = 0.03). CEACAM-1 expression did not correlate with stage, gender, grading or patients' age. Compared to patients with tumors not expressing CEACAM-1, patients with a CEACAM-1-expressing tumor had a shorter median overall survival (21 vs. 28 months) and progression-free survival (11.7 vs. 16.3 months). CEACAM-1 is expressed in most primary pulmonary adenocarcinomas. This investigation demonstrates that its expression is preserved in lymph node and hematogenous metastases, indicating that its expression is of functional significance for both metastatic sites. These results support the prognostic relevance of the expression of CEACAM-1 in pulmonary adenocarcinoma.

Background: The antiangiogenic agent cilengitide disrupts integrin binding to the extracellular matrix leading to apoptosis of activated endothelial cells. Integrins are also widely expressed in malignant glioma and integrin inhibitors may directly target tumor cells in this disease. Aim of the current study was to investigate effects of cilengitide on endothelial and glioma cells on molecular and cellular levels. Results: Cilengitide caused dose-dependent detachment of endothelial cells from cell culture dishes. Proliferation of endothelial cells was significantly inhibited while the proportion of apoptotic cells was increased. Incubation of integrin-expressing glioma cells with cilengitide caused rounding and detachment after 24 hours as observed with endothelial cells. Cilengitide inhibited proliferation and induced apoptosis in glioma cells with methylated MGMT promotor when given alone or in combination with temozolomide. In endothelial as well as glioma cells cilengitide inhibited phosphorylation of FAK, Src and Akt. Assembly of cytoskeleton and tight junctions was heavily disturbed in both cell types. Conclusion: Cilengitide inhibits integrin-dependent signaling, causes disassembly of cytoskeleton, cellular detachment and induction of apoptosis in endothelial and glioma cells thereby explaining the profound activity of integrin inhibitors in gliomas. The combination of cilengitide with temozolomide exerted additive effects in glioma cells as observed clinically.

Similar to cytotoxic drugs, a combination of antiangiogenic factors may lead to an improved treatment response and minimize resistance by targeting different pathways. Therefore, we investigated the effects of a combination of endogenous inhibitors using endostatin, soluble neuropilin-1 and thrombospondin-2 in a renal cell carcinoma model. Microencapsulated porcine aortic endothelial cells producing endostatin, soluble neuropilin-1 or thrombospondin-2 were tested in vitro and in a murine renal cell carcinoma alone or as a combination of the all 3 factors. Renca cells were applied subcutaneously for local therapy or injected intravenously in a metastatic model. Factors released from microbeads inhibited endothelial cell function but did not affect tumor cell proliferation in vitro. In vivo tumor growth was inhibited similarly by each angiogenic inhibitor alone (0.17, 0.18 and 0.18 gm in endostatin, soluble neuropilin-1 and thrombospondin-2 treated mice vs 1.3 gm in controls). The combination of all 3 inhibitors further decreased tumor weight (0.03 gm). In the metastatic model treatment with angiogenic inhibitors induced a significant reduction in the size and number of lung metastases with additive effects when factors were used in combination. The combination of angiogenic inhibitors was superior to single factors, suggesting additive activity. These data support the strategy of combining angiogenic inhibitors to accomplish a complete angiogenic blockade.

MediGene is developing EndoTAG-1, a formulation of paclitaxel encapsulated in positively charged lipid-based complexes, for the potential treatment of cancer. Phase II clinical trials in patients with advanced pancreatic cancer are ongoing.

Increased vessel density in the bone marrow of patients with acute myeloid leukemia as well as elevated expression of proangiogenic factors by leukemic cells implies a central role of angiogenesis in hematological malignancies. Endostatin (ES), a fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis that has shown therapeutic activity in solid tumors in various preclinical models. Using microencapsulation technology, we studied the therapeutic effect of ES in AML. While ES had no effect on proliferation of M1 murine leukemic cells in vitro, ES producing microbeads significantly inhibited growth of subcutaneous chloromas in SCID mice as compared to controls. In a leukemia model using M1 cells the concomitant treatment of mice with ES microbeads prolonged median survival significantly. Histological analysis revealed a decreased microvessel density and a reduced number of CD31-positive single cells, putatively endothelial progenitor cells, in the bone marrow of treated animals. Taken together, ES has inhibitory effects on neo-angiogenesis in the bone marrow and on progression of leukemia in vivo. These experiments suggest a possible therapeutic role of antiangiogenic gene therapy with ES in AML.

Circulating endothelial cells (CECs) are present in peripheral blood and have been shown to contribute to normal and pathological neovascularization. Antiangiogenic molecules can inhibit neovascularization in tumors and other sites, but their effect on CECs has not yet been determined. We hypothesize that angiogenic factors will increase the number of CECs, and conversely, antiangiogenic treatment will reduce these numbers. Mice treated with high levels of vascular endothelial growth factor (VEGF) showed increased numbers of Flk-1-positive cells in peripheral blood and endothelial cell colonies compared with vehicle-treated controls. These changes were accompanied by increased bone marrow neovascularization. In contrast, mice that received VEGF and endostatin had significantly lower numbers of CECs and reduced bone marrow vascularization. Endostatin-induced apoptosis was probably responsible for the decreased number of CECs. Systemic delivery of a VEGF antagonist, soluble Flt-1, also inhibited the VEGF-induced increase in CECs. These results were further confirmed in a Tie2/LacZ mouse model, in which endostatin reduced the number of beta-galactosidase-expressing peripheral blood mononuclear cells. We propose that endothelial progenitor cells are a novel target for endostatin and suggest that the relative numbers of CECs can serve as a surrogate marker for the biological activity of antiangiogenic treatment.