Torsten Kessler

Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia

Vascular endothelial growth factor (VEGF) and its cellular receptor VEGFR-2 have been implicated as the main endothelial pathway required for tumor neovascularization. However, the importance of the VEGF/VEGFR-2 system for angiogenesis in hematologic malignancies such as AML remains to be elucidated. In 32 patients with newly diagnosed untreated AML, we observed by immunohistochemical analysis of bone marrow biopsies significantly higher levels of VEGF and VEGFR-2 expression than in 10 control patients (P <0.001). In contrast, VEGFR-1 staining levels in AML patients were in the same range as in the controls. Expression of VEGF and VEGFR-2 was significantly higher in patients with a high degree of microvessel density compared to those with a low degree (VEGF: P =0.024; VEGFR-2: P =0.040) and correlated well with bone marrow microvessel density (rs=0.566 and 0.609, respectively; P <0.001). Furthermore, in patients who achieved a complete remission following induction chemotherapy VEGFR-2 staining levels decreased into the normal range. In conclusion, our results provide evidence for increased expression of VEGF/VEGFR-2 of leukemic blasts and correlation with angiogenesis in the bone marrow of AML patients. Thus, VEGF/VEGFR-2 might constitute promising targets for antiangiogenic and antileukemic treatment strategies in AML.

Infarction of tumor vessels by NGR-peptide-directed targeting of tissue factor: experimental results and first-in-man experience.

We induced thrombosis of blood vessels in solid tumors in mice by a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA, targeting aminopeptidase N (CD13) and the integrin alpha(v)beta(3) (CD51/CD61) on tumor vascular endothelium. The designed fusion protein tTF-NGR retained its thrombogenic activity as demonstrated by coagulation assays. In vivo studies in mice bearing established human adenocarcinoma (A549), melanoma (M21), and fibrosarcoma (HT1080) revealed that systemic administration of tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels as shown by histologic analysis. tTF-NGR, but not untargeted tTF, induced significant tumor growth retardation or regression in all 3 types of solid tumors. Thrombosis induction in tumor vessels by tTF-NGR was also shown by contrast enhanced magnetic resonance imaging (MRI). In the human fibrosarcoma xenograft model, MRI revealed a significant reduction of tumor perfusion by administration of tTF-NGR. Clinical first-in-man application of low dosages of this targeted coagulation factor revealed good tolerability and decreased tumor perfusion as measured by MRI. Targeted thrombosis in the tumor vasculature induced by tTF-NGR may be a promising strategy for the treatment of cancer.

Inhibition of tumor growth by RGD peptide-directed delivery of truncated tissue factor to the tumor vasculature.

Selective activation of blood coagulation in tumor vessels with subsequent tumor infarction is a promising anticancer strategy. To this end, a fusion protein consisting of the extracellular domain of tissue factor [truncated tissue factor (tTF)] was fused to the peptide GRGDSP selectively targeting αv-integrins on tumor endothelial cells. tTF-RGD retained its thrombogenic and integrin-binding activity in vitro. In vivo studies in mice bearing human adenocarcinomas (CCL185), melanoma (M21), and fibrosarcoma (HT1080) revealed that i.v. administration of tTF-RGD induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in all three types of solid tumors. No apparent side effects, such as thrombosis, in other organs or other treatment-related toxicities were observed. Reduced tumor blood flow in tTF-RGD–treated animals as determined by contrast-enhanced magnetic resonance imaging underlines the proposed mechanism. In conclusion, we consider RGD peptide–directed delivery of tTF as alternative to previously used antibody fusion proteins. Small peptide-directed delivery of coaguligands does not cause immunologic side effects and those caused by accumulation in the reticuloendothelial system. This is the first report to describe the induction of selective thrombosis in tumor vessels by RGD peptide–directed delivery of tTF, which may be a promising strategy for the treatment of cancer.

Circulating angiopoietin-2 is a strong prognostic factor in acute myeloid leukemia

Angiogenesis plays an important role in solid tumors and hematologic malignancies. The angiopoietins act as essential regulators in this process. We investigated the impact of circulating angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and soluble Tie2 (sTie2) on overall survival in patients with acute myeloid leukemia (AML). Ang-1, Ang-2 and sTie2 were measured in plasma samples from 68 AML patients and 11 controls using enzyme-linked immunosorbent assay. Circulating levels of Ang-2 and sTie2 (median (range): 1098.0 (361.4–4147.6) pg/ml and 3.40 (1.21–10.00) ng/ml, respectively) were significantly elevated in AML patients as compared to controls (307.9 (199.7–1225.0) pg/ml and 2.88 (1.71–3.29) ng/ml; P<0.001 and P=0.014). In a univariate Cox proportional hazards model, higher levels of Ang-2 and sTie2 were predictive of poor survival. In multivariate analyses, Ang-2 and cytogenetics proved to be independent prognostic factors, with a relative risk of 4.07 (95% confidence interval (CI) 1.88–8.81) and 2.70 (95% CI 1.25–5.81), respectively. The 3-year survival rate for AML patients with Ang-2 levels⩾1495.6 pg/ml was only 14.7% compared to 64.7% for those with Ang-2 levels<1495.6 pg/ml. These data provide evidence that circulating Ang-2 represents an independent prognostic factor in AML and may be used as a prognostic tool in the risk-adapted management of AML.

Angiogenesis Inhibition in Cancer Therapy

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. VEGF gene transcription is induced in particular in hypoxic cells. In developmental angiogenesis, the role of VEGF is demonstrated by the finding that the loss of a single VEGF allele results in defective vascularization and early embryonic lethality. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Platelet-derived growth factor (PDGF) is mainly believed to be an important mitogen for connective tissue, and also has important roles during embryonal development. Its overexpression has been linked to different types of malignancies. Thus, it is important to understand the physiology of VEGF and PDGF and their receptors as well as their roles in malignancies in order to develop antiangiogenic strategies for the treatment of malignant disease.

Vascular endothelial growth factor and its receptor as drug targets in hematological malignancies.

Angiogenesis is defined as formation of new blood vessels from the preexisting vasculature, a process which is essential for malignant tumor growth. While this has been accepted for solid forms of cancer there is now emerging evidence that progression of hematological malignancies also requires the induction of new blood vessels. Vascular endothelial growth factor (VEGF) is known to be an essential regulator of physiological and pathological angiogenesis. Numerous preclinical and clinical studies have validated VEGF as target for antiangiogenesis and anticancer therapy. With regard to hematological malignancies a stimulating effect of VEGF for proliferation, survival and migration of leukemia cells could be demonstrated. Bone marrow of leukemia patients shows an increased microvessel density as well as VEGF expression. Complete remissions in acute myeloid leukemia (AML) have been reported by targeting the receptor tyrosine kinase system of VEGF. While the pathophysiology behind the contribution of VEGF to leukemia progression is not yet completely understood, VEGF and its receptors may provide promising targets not only in solid tumors but also hematological malignancies such as AML.

Nivolumab in a patient with refractory Hodgkin's lymphoma after allogeneic stem cell transplantation.

Nivolumab in a patient with refractory Hodgkin’s lymphoma after allogeneic stem cell transplantation

Osteopontin is a prognostic factor for survival of acute myeloid leukemia patients

Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.

Prediction of antiangiogenic treatment efficacy by iron oxide enhanced parametric magnetic resonance imaging

Rationale and Objectives:Tools for monitoring modern target-specific antiangiogenic and antivascular therapies are highly desirable because treatment strategies are time consuming, expensive, and yet sometimes ineffective. Therefore, the aim of this experimental study was to evaluate the predictive value of steady-state ultrasmall particles of iron oxide (USPIO; SH U 555 C)-enhanced magnetic resonance imaging (MRI) for early assessment of antivascular tumor-treatment effectiveness. Methods:Mice were inoculated with an HT-1080 fibrosarcoma xenograft and subjected to target-specific antivascular therapy using a selective thrombogenic vascular-targeting agent (truncated tissue factor fused to RGD peptide) or saline as control. Four to 8 hours after treatment, the USPIO-induced change in the transverse relaxation rate &Dgr;R2* was measured by MRI, and the vascular volume fraction (VVF) was calculated by calibrating &Dgr;R2* of the tumor by &Dgr;R2* of muscle tissue. Treatment response was defined by histologic grading of vascular thrombosis and tumor necrosis. Results:After thrombogenic treatment, half of the HT-1080 xenograft-bearing animals showed only minor (=nonresponder) whereas the other half showed extensive tumor thrombosis (=responders). For responders, a significant decrease of &Dgr;R2* and VVF was observed compared with the control group (&Dgr;R2*: controls: 16 ± 1 s−1 vs. responder: 4 ± 2 s−1; P < 0.001) whereas &Dgr;R2* and VVF remained nearly unchanged for nonresponders (&Dgr;R2*: nonresponder 14 ± 2 s−1). VVF and &Dgr;R2* values correlated inversely with the histologic grading of vascular thrombosis and tumor necrosis (VVF: r = −0.8; &Dgr;R2*: r = −0.71; P < 0.01). Conclusion:USPIO-enhanced MRI allows a noninvasive, early assessment of treatment efficacy of thrombogenic vascular-targeting agents.

Generation of Fusion Proteins for Selective Occlusion of Tumor Vessels

Selective activation of blood coagulation in tumor vessels with subsequent thrombosis and tumor infarction is a promising strategy in cancer therapy. To this end, different fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) were fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or cyclic derivates of these peptides, which selectively target alpha(v)-integrins or aminopeptidase N (CD13), respectively. Rationale for this strategy is the fact that these surface receptors are preferentially expressed on tumor endothelial cells. The tTF constructs were expressed in Escherichia coli BL21 (DE3). The integrity of the fusion proteins was evaluated by SDS-PAGE, immunoblotting and mass spectrometry. The screening process for the activity contained coagulation assays as well as purified receptor binding assays. The fusion proteins which retained their thrombogenic and binding activity were evaluated further. In vivo studies in nude mice bearing established different malignant human tumors revealed that i.v. administration of tTF-RGD or tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels, which was demonstrated by histological analysis. Furthermore, treatment studies showed that the targeted tTF fusion proteins but not untargeted tTF proteins induced significant tumor growth retardation in human adenocarcinoma of the breast in a nude mice model without apparent side effects such as thrombosis in liver, kidney, heart or lung at therapeutic dose levels. Finally, we illustrate the upscaling process of fusion protein fabrication in order to produce the amounts needed for clinical studies. Thus, generation and screening of active fusion proteins, which induce selective thrombosis in the tumor vasculature, may be a promising strategy for the development of new drugs as cancer therapeutics.