Search

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo. Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice. Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

BACKGROUND: The glycoprotein YKL-40 is synthesized both by cancer cells and by tumor-associated macrophages and plays a functional role in tumor progression. Consequently, high serum YKL-40 levels have been associated with a poor prognosis in patients with several cancer types. However, the role of YKL-40 has not been established in nonsmall cell lung cancer (NSCLC). METHODS: Pretreatment serum levels of YKL-40 were determined in 189 patients with NSCLC (143 men and 46 women; median age, 62 years;, age range, 41-76 years). Twelve percent of patients had stage IIIB disease, and 88% had stage IV disease. Ninety-eight patients received combined gemcitabine and vinorelbine, and 91 received combined gemcitabine, vinorelbine, and cisplatin as first-line chemotherapy. The median overall survival was 37 weeks. RESULTS: Patients had a median serum YKL-40 level of 209 ng/mL (range, 19-2153 ng/mL). No correlation was observed between overall survival and the type of chemotherapy regimen used, tumor stage, sex, or histologic types. Patients with high serum YKL-40 levels (greater than the median level for all patients [209 ng/mL]) had a significantly shorter survival than patients with serum YKL-40 levels below the median (median survival, 32 weeks vs 41 weeks; P = .007). In multivariate analysis, the serum YKL-40 level, the presence of bone lesions, and the serum lactate dehydrogenase level were independent, statistically significant prognostic factors. CONCLUSIONS: The pretreatment serum YKL-40 level was identified as a new, independent prognostic biomarker in patients with metastatic NSCLC and may help to determine the individual prognosis of these patients. Cancer 2010. © 2010 American Cancer Society.

Purpose To evaluate the association of germ-line polymorphisms of genes that may impact treatment outcome of platinum and fluorouracil combination chemotherapy in advanced gastric cancer (AGC). Patients and Methods Blood samples of 156 patients enrolled onto a phase III study comparing fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin were collected. Polymorphisms within genes of TS, MTHFR, MTR, OPRT, XPD, ERCC1, XRCC1, XPA, GSTP1, GSTT1, and GSTM1 were genotyped using polymerase chain reaction–based techniques. Results Median overall survival (OS) was 11.8 months (95% CI, 9.75 to 13.79 months) and median progression-free survival (PFS) was 5.8 months (95% CI, 4.99 to 6.61 months). The TS-3R/+6 haplotype (P = .004), the GSTT1 deletion polymorphism (P = .015), and genotypes of OPRT-Gly213Ala (P = .003) and XRCC1-Arg399Gln (P = .023) could be identified as independent predictors of OS. For PFS analyses, the TS-3R/+6 haplotye (P = .003) and MTR-A2756G (P = .01) were identified as independent positive predictors. The association between the GSTT1 deletion polymorphism and PFS showed only borderline significance (P = .053). Treatment related hematotoxicity in terms of grade 3/4 leukopenia was lowest among TS-3R/+6 haplotype carriers (P = .037). Grade 3/4 neutropenia was directly associated with the MTR-2756G/G genotype (P = .011), GSTP1-105Ile/Ile genotype (P = .02), and with the ERCC1-118T/8092C-haplotype (P = .042). In addition, significant associations between GSTP1-105Ile/Ile genotype and neurotoxicity and between the XPD-Asn312/751Gln haplotype and nephrotoxicity could be identified (P = .028 and P = .005, respectively). Conclusion These findings underline the hypothesis that germ-line polymorphisms may play an important role in individualizing chemotherapy in AGC and deserve further prospective evaluation in AGC patients.

Purpose This randomized study assessed whether the best overall response rate (ORR) of cetuximab combined with oxaliplatin, leucovorin, and fluorouracil (FOLFOX-4) was superior to that of FOLFOX-4 alone as first-line treatment for metastatic colorectal cancer. The influence of KRAS mutation status was investigated. Patients and Methods Patients received cetuximab (400 mg/m2 initial dose followed by 250 mg/m2/wk thereafter) plus FOLFOX-4 (oxaliplatin 85 mg/m2 on day 1, plus leucovorin 200 mg/m2 and fluorouracil as a 400 mg/m2 bolus followed by a 600 mg/m2 infusion during 22 hours on days 1 and 2; n = 169) or FOLFOX-4 alone (n = 168). Treatment was continued until disease progression or unacceptable toxicity. KRAS mutation status was assessed in the subset of patients with assessable tumor samples (n = 233). Results The confirmed ORR for cetuximab plus FOLFOX-4 was higher than with FOLFOX-4 alone (46% v 36%). A statistically significant increase in the odds for a response with the addition of cetuximab to FOLFOX-4 could not be established (odds ratio = 1.52; P = .064). In patients with KRAS wild-type tumors, the addition of cetuximab to FOLFOX-4 was associated with a clinically significant increased chance of response (ORR = 61% v 37%; odds ratio = 2.54; P = .011) and a lower risk of disease progression (hazard ratio = 0.57; P = .0163) compared with FOLFOX-4 alone. Cetuximab plus FOLFOX-4 was generally well tolerated. Conclusion KRAS mutational status was shown to be a highly predictive selection criterion in relation to the treatment decision regarding the addition of cetuximab to FOLFOX-4 for previously untreated patients with metastatic colorectal cancer.

Cancer of unknown primary site (CUP syndrome) is a particular challenge in oncology which occurs in about 5-10% of cancer patients. Here, we investigated clinicopathological and prognostic factors in patients with CUP syndrome in a retrospective analysis.136 patients with CUP syndrome who were treated in our Department of Oncology and Hematology were analyzed over a period of 10 years. Clinical and histopathological characteristics, response to chemotherapy, survival and prognostic factors were investigated in a retrospective analysis.83 of the patients (61%) received first-line chemotherapy, which induced an overall response rate of 19%. Altogether 37 different chemotherapy regimens were used. Median overall survival of all patients was 7.9 months. In multivariate Cox regression analysis, gender, Karnofsky performance status, treatment modality and extent of disease were identified as independent prognostic factors.Our analysis showed a poor prognosis for patients with CUP syndrome. The response rate to chemotherapy was low with no significant benefit for any of the investigated cytotoxic agents. Newer diagnostic and therapeutical approaches might contribute to an improvement of prognosis, and their value is currently investigated in prospective studies.

Purpose This study was designed to compare fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin in patients with advanced gastric cancer. Patients and Methods Patients with previously untreated advanced adenocarcinoma of the stomach or esophagogastric junction were randomly assigned to receive either fluorouracil 2,600 mg/m2 via 24-hour infusion, leucovorin 200 mg/m2, and oxaliplatin 85 mg/m2 (FLO) every 2 weeks or fluorouracil 2,000 mg/m2 via 24-hour infusion, leucovorin 200 mg/m2 weekly, and cisplatin 50 mg/m2 every 2 weeks (FLP). The primary end point was progression-free survival (PFS). Results Two hundred twenty patients (median age, 64 years; metastatic, 94%) were randomly assigned. FLO was associated with significantly less (any grade) anemia (54% v 72%), nausea (53% v 70%), vomiting (31% v 52%), alopecia (22% v 39%), fatigue (19% v 34%), renal toxicity (11% v 34%), thromboembolic events (0.9% v 7.8%), and serious adverse events related to the treatment (9% v 19%). FLP was associated with significantly less peripheral neuropathy (22% v 63%). There was a trend toward improved median PFS with FLO versus FLP (5.8 v 3.9 months, respectively; P = .077) and no significant difference in median overall survival (10.7 v 8.8 months, respectively). However, in patients older than 65 years (n = 94), treatment with FLO resulted in significantly superior response rates (41.3% v 16.7%; P = .012), time to treatment failure (5.4 v 2.3 months; P < .001), and PFS (6.0 v 3.1 month; P = .029) and an improved OS (13.9 v 7.2 months) as compared with FLP, respectively. Conclusion FLO reduced toxicity as compared with FLP. In older adult patients, FLO also seemed to be associated with improved efficacy.

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.

This mini review highlights the role of vascular stabilization which is apparently mediated by factors belonging to both the pro- and anti-angiogenic group. Knowingly angiopoietin-1 has pro-angiogenic properties while endostatin acts anti-angiogenic. But both factors suppress tumor growth in experimental tumor models. The crossing points of mechanisms of their action are the parameters vascular stabilization and vascular permeability, which are of high importance not only for tumor vascularization, but also for tissue vascularization in general. Both angiopoietin-1 and endostatin reduce the vascular permeability, but promote vascular stabilization. Vascular stabilization is significantly increased under experimental treatment of tumors with angiogenesis inhibitors such as endostatin. It inhibits tumor growth and probably tumor metastasis. The understanding of temporal and spatial sequences of vascular stabilization is an important challenge for scientists working on tumor vascularization and anti-angiogenic tumor therapy.

In acute myeloid leukemia (AML), autocrine or paracrine activation of receptor tyrosine kinases such as c-kit and FLT3 contributes to proliferation and apoptosis resistance of leukemic blasts. This provided the rationale for a multicenter clinical trial in patients with refractory AML with SU5416, a small molecule kinase inhibitor which blocks phosphorylation of c-kit, FLT3, VEGFR-1, VEGFR-2 (KDR) and VEGFR-3. The levels of VEGF mRNA expression were investigated in peripheral blood leukemic blasts taken from AML patients before and during treatment with SU5416. Rapid down regulation of VEGF was observed in AML blasts from 72% (13 of 18) of patients analysed. Patients initially expressing high VEGF-levels had a stronger downregulation and a higher clinical response rate (mean 865-fold, n = 10, P = 0,01) than patients initially expressing low VEGF-levels (mean four-fold, n = 8). These results suggest that abnormal high VEGF expression is downregulated by SU5416 treatment, and furthermore that decreases in VEGF mRNA levels may provide an early marker of therapeutic response with anti-angiogenic therapy. Additionally, protein expression of STAT5 and AKT was assessed by western blotting in these patient samples, as well as in the leukemia cell line, M-07e, treated in vitro with SU5416 as a model system. In the AML patient samples, parallel downregulation of both STAT5 and AKT was observed in several cases (STAT5 in four of 15; AKT in three of six examined patients). These effects were confirmed with the cell line M-07e after incubation with SU5416 in vitro using concentrations that are achievable in patients. In summary, our data show suppression of the expression of VEGF and key signal transduction intermediates in AML blasts during treatment with SU5416.

Summary Purpose: In the present study, we investigated the prognostic value of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 serum levels in patients with metastatic non-small cell lung cancer (NSCLC). Patients and methods: From September 1999 to June 2001, pretreatment serum levels of VEGF and MMP-9 were analysed in 194 patients of a randomized phase III trial with enzyme-linked immunoassays. Results: Patients with a VEGF serum level higher than the median serum level (10,995 pg/ml) had a significantly shorter overall survival than those with a lower serum level ( P = 0.04). The MMP-9 serum level did not correlate with survival. In a multivariate Cox regression analysis, only the pretreatment serum level of VEGF, the Karnofsky performance status, and the presence of bone metastases were identified as independent prognostic factors. Conclusions: The pretreatment VEGF serum level was identified as independent prognostic factor in this study and may help to assess individual risk and treatment profiles in patients with metastatic NSCLC.

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.

Background: We investigated the combination of docetaxel and cisplatin as first-line chemotherapy in patients with metastatic esophageal cancer. Patients and Methods: 16 chemotherapy-naive patients with distant metastases were included in the study (15 male, 1 female; median age: 58.5 years (range 37-69); median ECOG performance status: 1). 11 patients (69%) had esophageal cancer, and 5 patients (31%) had cancer of the gastroesophageal junction. Patients received docetaxel 75 mg/m2 and cisplatin 80 mg/m2 on day 1 every 3 weeks. A total of 55 chemotherapy cycles was administered. The median number of cycles was 3 (range 1-6). Results: The overall response rate was 31.3%. 4 out of 10 patients (40%) with squamous cell carcinoma and 1 out of 5 patients (20%) with adenocarcinoma responded to chemotherapy. The median overall survival was 29.6 weeks, and the median progression-free survival was 18.6 weeks. Hematological and non-hematological toxicities were moderate (neutropenia WHO grade III/IV: 42.9%, alopecia grade II/III: 64.3%, nausea/vomiting grade II/III: 57.2%, neurotoxicity grade II: 14.3%). Conclusion: The combination of docetaxel and cisplatin is an active regimen with moderate toxicity in the treatment of patients with metastatic esophageal cancer. This pilot study demonstrates the feasibility of a combination treatment containing a taxane and cisplatin in metastatic esophageal cancer.

Recent findings implied that the progression of hematologic malignancies, like that of solid tumors, is dependent on neovascularization. Recent studies on patients with acute myeloid leukemia (AML) showed increased levels of leukocyte-associated vascular endothelial growth factor (VEGF) and neovascularization of the bone marrow. Murine (32D, M1) and human (HEL, U937, and UKE-1) leukemic cell lines and freshly isolated leukemic cells were analyzed for the expression of VEGF and VEGF receptor mRNA. The expression of VEGF and VEGF receptors KDR and neuropilin-1 (NRP-1) was detected in these cells. In a murine chloroma model, delivery of VEGF165using microencapsulation technology resulted in enhanced tumor growth and vascularization, whereas treatment with a VEGF antagonist soluble NRP-1 (sNRP-1) inhibited tumor angiogenesis and growth. In a systemic leukemia model, survival of mice injected with adenovirus (Ad) encoding for Fc-sNRP-1 (sNRP-1 dimer) was significantly prolonged as compared with mice injected with Ad-LacZ. Further analyses showed a reduction in circulating leukemic cells and infiltration of liver and spleen as well as bone marrow neovascularization and cellularity. Taken together, these results demonstrate that angiogenic factors such as VEGF promote AML progression in vivo. The use of VEGF antagonists as an antiangiogenesis approach offers a potential treatment for AML. Finally, our novel in vivo drug delivery model may be useful for testing the activities of other peptide antiangiogenic factors.

We investigated an intensified conditioning regimen including fractionated total body irradiation (12 Gy), etoposide (30–45 mg/kg) and cyclophosphamide (120 mg/kg), followed by autologous (n = 5), allo-related (n = 13) or allo-unrelated (n = 6) bone marrow (n = 22) or peripheral stem cell (n = 2) transplantation in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. One patient received busulfan (16 mg/kg) instead of TBI. Nineteen patients were transplanted in 1CR, two in 2CR, one in 1PR and two in relapse. Major toxicity was mucositis grade II according to the Bearman scale in all patients. The treatment-related mortality was 25%, mainly due to infection or GVHD after allogeneic transplantation. After a median follow-up of 45 months (range 2–93), nine patients (37.5%) remain alive in CR. Nine patients (37.5%) relapsed and eight (33.3%) of these subsequently died. After autologous transplantation, four of five patients (80%) relapsed and died. Late relapse was seen after allogeneic, as well as autologous transplantation, at 33 and 59 months, respectively. The Kaplan–Meier estimate of leukemia-free survival for all patients is 38% at 3 years (95% CI: 18–58%) and 35% at 5 years (95% CI: 15–55%). For allogeneic transplants in first CR (n = 15) the estimate of disease-free survival was 46% at 3 years (95% CI: 19–73%) and 34% at 5 years (95% CI: 17–51%). Patients aged below 30 years had a better estimated overall survival at 3 years (61% vs 11%, P

In patients with metastatic non-small cell lung cancer (NSCLC), second-line chemotherapy induces response rates of less than 20% and median survival times between 5 and 8 months.In the case described here, a patient with metastatic NSCLC responded with complete remission of the primary tumor and the involved lymph nodes as well as partial remission of bone metastases to a second-line chemotherapy with docetaxel and carboplatin. Since April 2003 (33 months), no tumor progression has been observed. Until present, the patient received monthly infusions of pamidronic acid.Our case report indicates that in certain patients with metastatic NSCLC who did not respond to first-line regimens, second-line chemotherapy can induce outstanding tumor response and significantly improve survival. It also indicates that the role of bisphosphonates in the treatment of NSCLC should be further investigated in large clinical trials.

Scale-up of Distillation Columns. In spite of remarkable advances in the development of computer programs for calculations on distillation columns, it is frequently still impossible to design them without experimental testing. Economic constraints demand use of miniaturised test plant, thus enhancing the importance of laboratory measurements. A system is presented for scale-up of separation which required information from laboratory experiments, includes data from reference columns, and takes account of models of fluid dynamics and mass transfer. The special feature of this approach lies in the free choise of internals, both in the laboratory and in the designed column.

This chapter briefly reviews the types and source of stem cells in peripheral blood, their specific cell-surface markers, and factors that change their abundance in peripheral blood and focuses on the isolation and in vitro expansion of peripheral blood-derived mesenchymal cells (MCSs) and endothelial progenitor cells (EPCs). It also describes their therapeutic applications for regenerative medicine. One of the types of stem cells is hematopoietic stem cells (HSCs), which are characteristically quiescent, multipotent cells with the capacity for both self-renewal and differentiation, and another type, mesenchymal stem cells (MSCs) reside in bone marrow. Bone marrow-derived EPCs contribute to adult tissue neovascularization in several models including wound healing and cornea and tumor angiogenesis. Bone marrow-derived EPCs could be detected in normal organs including spleen, lung, liver, intestine, skin, hindlimb muscle, ovary, and uterus, indicating their participation in maintenance of physiological neovascularization. Hormonally induced ovulation cycles are also associated with localization of bone marrow-derived EPCs in the corpus lutea and in the uterus endometrium and stroma. In the case of MSCs, the lineage-committed cells can generate a variety of specialized mesenchymal tissues including bone, cartilage, muscle, marrow stroma, tendon, ligament, fat, and a variety of other connective tissues. MSCs may have a dramatic impact on the overall health status of individuals by controlling the body's capacity to naturally remodel, repair, and upon demand rejuvenate various tissues. In human clinical research, initial efforts are focused on applications of MSC-based tissue repair using cell delivery approaches.

Experience Gained with Multipurpose Plants. Multipurpose plants are used in many fields of production. Some products often have a short life cycle or are not in continuous demand; hence these plants undergo constant changes. In order to control these changes from an economical point of view, certain boundary conditions must be met. The following article describes which questions have to be asked and answered and which structures prove to be advantageous for such plants.

The aim of this pilot study was to evaluate the efficacy and safety of a chemotherapy containing docetaxel and oral trofosfamide as a 'metronomic' secondline treatment of patients with metastatic non-small cell lung cancer (NSCLC).21 patients with stage IV disease NSCLC who had progressed under first-line chemotherapy were enrolled. Previous chemotherapy was platinum-based in 15 patients (71.4%), whereas 6 patients (28.6%) had received platinum-free combination chemotherapy. Patients received docetaxel 25 mg/m(2) on days 1, 8, and 15 every 4 weeks plus trofosfamide 50 mg per day.A total of 62 chemotherapy cycles were administered. The median number of cycles per patient was 3. The overall response rate to chemotherapy was 19%, median overall survival was 6.9 months, the median progression-free survival 2.9 months, the 1-year survival rate 28.6%, and the 2-year survival rate 7.1%. No grade IV toxicity was observed.Our results suggest that the combination of docetaxel and trofosfamide in a metronomic schedule is active and well tolerable as second-line therapy in patients with metastatic NSCLC. The concept of metronomic chemotherapy promises to be a valuable addition to the existing treatment options in NSCLC and warrants further investigation in phase III studies.

Purpose Bone marrow neoangiogenesis plays an important pathogenetic and possible prognostic role in acute myeloid leukemia (AML). Members of the vascular endothelial growth factor (VEGF) and angiopoietin family represent the most specific inducers of angiogenesis secreted by AML blasts. We therefore correlated expression of angiogenic factors with clinical variables. Patients and Methods We investigated the expression of VEGF-A, VEGF-C, angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the receptor Tie2 by quantitative polymerase chain reaction in a cohort of 90 patients younger than 61 years with de novo AML entered into the German AML Süddeutsche Hämoblastose Gruppe Hannover 95 trial. Uni- and multivariate analyses were performed using clinical and gene expression variables. Results Univariate analysis of overall survival indicated the following variables as prognostic factors: good response on a day-15 bone marrow examination after initiation of induction chemotherapy, karyotype, and high Ang2 expression. In multivariate analysis, only bad response and log Ang2 expression remained of statistical significance, with a hazard ratio of 3.51 (95% CI, 1.91 to 6.47) and 0.75 (95% CI, 0.61 to 0.91), respectively. Subgroup analysis suggested that the prognostic impact of Ang2 expression was especially evident in cohorts with low VEGF-C and Ang1 mRNA levels. Conclusion These results show that expression of Ang2 represents an independent prognostic factor in AML. Additional research into interactions of angiogenic cytokines in the pathogenesis of bone marrow angiogenesis in AML is warranted.

Summary Background: Magic Roundabout (MR; ROBO 4) is a recently discovered gene which encodes a protein that derived its name form the structural homology with the roundabout family of genes. Genes of the roundabout family comprise neuronal-specific cell surface receptors that are involved in axon guidance. MR in contrast showed endothelial specificity in vitro and in vivo using immunohistochemistry and in situ hybridisation. The putative role of MR as an endothelial analogue of Roundabout in angiogenesis makes it an attractive target for anti-angiogenic cancer therapy. Patients and methods: Using specific antibodies against MR peptide, we screened pretreatment sera from 193 patients with advanced non-small cell lung cancer (NSCLC) for MR-protein levels. Results: Patients with serum levels of MR-protein lower than median ( E 450nm =0.652) had a median survival of 41.0 weeks whereas those with a higher serum level had a considerably shorter median survival of 32.4 weeks ( P =0.05). The pretreatment serum level of MR-protein achieved no significance in a univariate Cox regression analysis. Conclusions: This is the first study to present clinical data that link MR expression with outcome in patients with NSCLC, whether the correlation of pretreatment serum levels of MR and survival can be attributed to MR dependent angiogenesis remains to be investigated.

19 patients who failed the target collection of at least 2.5 × 106 CD34+ cells/kg underwent further mobilization procedures either with granulocyte-colony-stimulating factor (G-CSF) alone after failure to chemotherapy plus G-CSF (group 1), or with chemotherapy plus G-CSF (group 2), or with high-dose G-CSF (24 μg/kg) alone (group 3) after failure to respond to standard dose of G-CSF (10 μg/kg) alone. In all groups, an increase in median CD34+ cell yield could be observed following alternative procedures (1.1- to 1.9 × 106 kg; p = 0.02). The highest increase in CD34+ cell harvest was achieved in group 1 (0.85 to 2.2 × 106 kg), followed by group 2 (1.2 to 1.7) and group 3 (1.0 to 1.4), but without statistically significant difference between the mobilization technologies. All patients with more than 1.0 × 106 CD34+ cells/kg in the first apheresis procedure reached the overall target of 2.5 × 106 CD34+ cells/kg after a second or subsequent mobilization procedure. In contrast, only 3 of 8 patients (37%) with less than 1.0 × 106 CD34+ cells in the first harvest could reach the target of 2.5 × 106 CD34+ cells after further mobilization attempts.

e21527 Background: HEH is a rare tumor of the liver with an unpredictable malignant potential. Surgical resection or liver transplantation is recommended in locally advanced disease and has been successfully performed in selected cases with extrahepatic manifestations. Systemic therapy, however, is not standardized. Small cohorts have been treated with interferon, chemotherapy and angiogenesis inhibitors such as thalidomide with various results. Case report: A 33-year old caucasian previously healthy male was admitted to hospital with newly diagnosed suspicious lesions in liver and spleen. Further analyses showed additional pulmonal nodules. Biopsy revealed a tumor with predominant epithelioid cells, positive for CD31 and CD34 and negative for CD117, HHV8, AFP and CEA, classified as a HEH. Disease progressed during first line treatment with adriamycin and ifosfamide (2 cycles given). Based on a case report with encouraging results with thalidomide we treated our patient with lenalidomide, which is known to have less side effects while being more effective in the myeloma setting. After 6 courses of lenalidomide 25mg daily (21/28), regression of the splenic lesions was detected. Retrospective analysis after 9 cycles demonstrated stable disease in comparison to the recent investigation, but an overall progression of 22% according to RECIST criteria in the liver was observed. Due to the excellent tolerance we increased the daily dose to 30 mg (21/28) and 6 months later a slight regression in the lung and overall stable disease in the liver was observed. The splenic lesion disappeared completely. The patient was listed for liver transplantation and after another 4 months on lenalidomide 30 mg, he was successfully transplanted recently. Because of the remaining extrahepatic lesions it is planned to continue lenalidomide therapy in combination with the immunosuppressant rapamycine. Conclusions: We report the first case of successful therapy with lenalidomide in HEH. Higher doses than 25mg seem to be more effective. We grateful thank Celgene for offering us lenalidomide for this off label use. No significant financial relationships to disclose.

18105 Background: An emerging notion for the treatment of advanced solid malignancies in adults is to manage it as a chronic disease and aim to keep tumor burden and associated symptoms at the lowest possible level. One of the most promising strategies to achieve this is to target the tumor vasculature using modified, “metronomic”, dosing schedules. We conducted a pilot study of weekly docetaxel in combination with daily trofosfamide as a metronomic second-line regimen in patients (pts) with metastatic non-small cell lung cancer (NSCLC). Methods: From March 2003 to November 2005, 21 pts with stage IV disease who had one prior chemotherapy were enrolled. Pts received docetaxel 25 mg/m2 on days 1, 8 and 15, every 4 weeks plus trofosfamide 50mg per day. Results: Median age: 60 years; gender: 18 male (85.7%), 3 female (14.3%); median Karnofsky PS: 80%; histologic subtypes: 8 squamous cell carcinomas (38.1%), 7 adenocarcinomas (33.3%), 6 large cell carcinomas (28.6%); previous chemotherapy: platinum-based in 15 pts (71.4%), platinum-free in 6 pts (28.6%). A total of 62 chemotherapy cycles were administered. The median number of cycles per pt was 3 (range 1 - 8). The intent-to-treat overall response rate was 19% (95% CI 5.5 - 41.9) 1 CR (4.8%), 2 PR (14.3%), 9 SD (42.9%), 8 PD (38.1%). The median overall survival from onset of second-line therapy was 6.9 months (95% CI 5.0 - 13.9 months), the median progression-free survival 2.9 months (95% CI 1.58 - 6.28 months), the 1-year survival rate 28.6% (95% CI 14.5 - 56.2%), and the 2-year survival rate 7.1% (95% CI 1.26 - 40.6%). Hematological and non-hematological toxicities were mild. No grade 4 toxicity was observed. Conclusions: To the best of our knowledge, this is the first trial that combines docetaxel and trofosfamide for the treatment of NSCLC. Our results suggest that the concept of metronomic chemotherapy is a valuable addition to the treatment of NSCLC. The combination of docetaxel and trofosfamide is active and well tolerated as second-line therapy in pts with metastatic NSCLC. No significant financial relationships to disclose.

15172 Background: Cholangiocarcinoma or gallbladder cancer are often diagnosed at an advanced stage with limited treatment options. Methods: Between 1994 and 2004, 94 patients (pts) (47 male, 47 female) with advanced biliary tract cancer were treated at the Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf. Clinical and histopathological characteristics, response to chemotherapy, and survival were investigated in a retrospective analysis. Median age was 59 years (range 30–80) and median Karnofsky performance status was 90%. Predominant histologic type was adenocarcinoma (94.7%). Primary tumor sites were extrahepatic bile duct (29.9%), gallbladder (28.7%), intrahepatic bile duct (10.6%), ampulla of Vater (2.1%), not specified (28.7%). Predominant localizations of metastases were liver (73 pts (77.7%)), lymph nodes (49 pts (52.1%)) and the peritoneum (14 pts (14.9%)). 33 pts (35.1%) underwent surgery of the primary tumor at time of diagnosis. Results: 72 of 94 pts (76.6%) received a first-line chemotherapy, all together 10 different chemotherapy regimens were used. The median number of cycles was 2.5 (range 1 - 12). A single agent chemotherapy with gemcitabine was the most often adminstered regimen (23 pts (31.9%)), followed by carboplatin and etoposide plus whole body hyperthermia (12 pts (16.7%)) and 5- fluorouracil and folic acid (10 pts (13.9%)). The overall response rate was 8.3% (95% CI 3.1 - 17.3) (34.7% SD, 47.2% PD, 9.7% not evaluable). Second-line chemotherapy was given in 27 patients, which induced no tumor response, but a stable disease rate of 22.2%. Median time to follow- up was 44.8 months. Survival was calculated for all 94 pts since time of diagnosis. Median overall survival was 12.2 months and median progression-free survival 9.2 months. The median overall survival time for the 72 pts who were treated with chemotherapy was 14.0 months, and for the 22 pts who did not receive chemotherapy 10.7 months (p=0.2). Conclusions: Our analysis showed a poor prognosis for patients with advanced biliary tract cancer. Response rate to chemotherapy was low. Therefore, well tolerated cytotoxic agents should be used and new treatment strategies (including molecular targeted therapy) should be further investigated. No significant financial relationships to disclose.

15648 Background: Combinations of cytotoxic drugs lead to increased activity and minimize resistance compared to single agents in tumor therapy. Similarly, antiangiogenic treatment could be improved by combinations targeting different pathways. We investigated a combination of endogenous inhibitors using endostatin (ES), soluble Neuropilin-1 (sNP-1), and thrombospondin-2 (TSP-2) in a model of renal cell carcinoma. Methods: Porcine aortic endothelial cells have been engineered for stable production of angiogenic inhibitors by lipofection and were encapsulated in sodium alginate microbeads. Proliferation of human umbilical vein endothelial cells or Renca renal carcinoma cells was examined after incubation with different microbeads. Similarly, effects of inhibitors on endothelial cell function were tested in tube formation and in vitro wound assays. Microbeads were implanted into SCID mice with subcutaneously growing tumors derived from Renca cells or in mice developing lung metastases after intravenous injection of tumor cells. Results: Factors released from microbeads inhibited endothelial cell function but had no effect on tumor cell proliferation in vitro. In vivo, subcutaneous tumor growth was inhibited similarly by each angiogenic inhibitor alone. After 30 days mean tumor weight was 1.3 g in controls and 0.17, 0.18, 0.18g in ES, sNP-1, and TSP-2 treated mice, respectively. Tumor weight in mice treated with all three inhibitors was further reduced to 0.03g. Histological analyses confirmed antiangiogenic activity by inhibition of microvessel density in treated tumors. In a metastastic model treatment with angiogenic inhibitors induced a significant reduction in size and number of lung metastases with additive effects when factors were used in combination. Conclusions: We conclude that combination therapy targeting multiple angiogenic pathways has synergistic activity and could help to avoid resistance to single inhibitors in tumor treatment. No significant financial relationships to disclose.

20010 Background: Bone marrow derived endothelial progenitor cells (ECPs) contribute to neoangiogenesis in cancer patients. Elevated numbers of circulating endothelial cells and EPCs can be detected in the peripheral blood of tumor patients. Recently, proliferating lymphatic vessels have been described in human cancers such as melanoma and head and neck tumors. Therefore, we investigated whether circulating lymphatic endothelial cells can be detected in cancer patients. Methods: We developed a sensitive immunocytochemical approach using a monoclonal antibody against the lymphendothelial specific hyaluronic receptor LYVE. After enrichment by Ficoll density gradient centrifugation, 7 × 10(5) peripheral blood mononuclear cells (PBMNCs) from 23 patients with metastatic cancer (6 gastrointestinal, 4 lung, 3 thymus, 3 thyroid, 1 mamma, 1 pancreas, 2 renal, 1 adrenal, 2 urothelial cancer, 1 PNET, 1 NET) and healthy individuals (n = 7) were spun onto glass slides. Two million PBMNs from each patient were stained for LYVE using the APAP technique. Isotype antibodies were used as controls. Cytospins were analyzed with the automated cellular imaging system (ACIS; ChromaVision Medical Systems). The method was validated with spiked blood samples. Results: Circulating LYVE+ lymphatic endothelial cells could be detected in 4 of 7 healthy subjects (57%) and in 16 of 23 patients (69%) with metastatic cancer. The mean number of lymphendothelial cells was significantly higher in cancer patients (15 cells/1 × 10(6) PBMNCs [range 0–276] vs. 1.0 cells/1 × 10(6) PBMNCs [range 0–2]). As a control, circulating tumor cells were enumerated after staining with a cytokeratin antibody (A45-B/B3). Circulating tumor cells could not be detected in healthy controls but in 13 of 23 of cancer patients (mean 1.4 cells/1 × 10(6) PBMNCs range [0–9]). Conclusion: Circulating lymphendothelial cells can be detected in patients with metastatic cancer and healthy subjects. Tumor patients have higher levels of circulating lymphendothelial cells than normal controls. These cells may participate in the generation of lymphatic vessels within tumor manifestations. No significant financial relationships to disclose.

7199 Background: Magic Roundabout (MR; ROBO 4) is a recently discovered gene which encodes for a protein that possess structural homology with the roundabout family of proteins. Genes of the rounda...