Rolf M. Mesters

Impact of Thrombophilia on Risk of Arterial Ischemic Stroke or Cerebral Sinovenous Thrombosis in Neonates and Children A Systematic Review and Meta-Analysis of Observational Studies

Background— The aim of this study was to estimate the impact of thrombophilia on risk of first childhood stroke through a meta-analysis of published observational studies. Methods and Results— A systematic search of electronic databases (Medline via PubMed, EMBASE, OVID, Web of Science, The Cochrane Library) for studies published from 1970 to 2009 was conducted. Data on year of publication, study design, country of origin, number of patients/control subjects, ethnicity, stroke type (arterial ischemic stroke [AIS], cerebral venous sinus thrombosis [CSVT]) were abstracted. Publication bias indicator and heterogeneity across studies were evaluated, and summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated with fixed-effects or random-effects models. Twenty-two of 185 references met inclusion criteria. Thus, 1764 patients (arterial ischemic stroke [AIS], 1526; cerebral sinus venous thrombosis [CSVT], 238) and 2799 control subjects (neonate to 18 years of age) were enrolled. No significant heterogeneity was discerned across studies, and no publication bias was detected. A statistically significant association with first stroke was demonstrated for each thrombophilia trait evaluated, with no difference found between AIS and CSVT. Summary ORs (fixed-effects model) were as follows: antithrombin deficiency, 7.06 (95% CI, 2.44 to 22.42); protein C deficiency, 8.76 (95% CI, 4.53 to 16.96); protein S deficiency, 3.20 (95% CI, 1.22 to 8.40), factor V G1691A, 3.26 (95% CI, 2.59 to 4.10); factor II G20210A, 2.43 (95% CI, 1.67 to 3.51); MTHFR C677T (AIS), 1.58 (95% CI, 1.20 to 2.08); antiphospholipid antibodies (AIS), 6.95 (95% CI, 3.67 to 13.14); elevated lipoprotein(a), 6.27 (95% CI, 4.52 to 8.69), and combined thrombophilias, 11.86 (95% CI, 5.93 to 23.73). In the 6 exclusively perinatal AIS studies, summary ORs were as follows: factor V, 3.56 (95% CI, 2.29 to 5.53); and factor II, 2.02 (95% CI, 1.02 to 3.99). Conclusions— The present meta-analysis indicates that thrombophilias serve as risk factors for incident stroke. However, the impact of thrombophilias on outcome and recurrence risk needs to be further investigated.

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.

Prognostic Relevance of Increased Angiogenesis in Osteosarcoma

Purpose: The purpose of this work was to evaluate the prognostic relevance of microvessel density (MVD) for response to chemotherapy and long-term outcome in osteosarcoma. Experimental Design: Pretherapeutic tumor biopsies of 60 patients with high-grade central osteosarcoma, who were treated according to multimodal neoadjuvant protocols of the German-Austrian-Swiss Cooperative Osteosarcoma Study Group, were evaluated for intratumoral MVD. MVD was correlated with demographic and tumor-related variables, response, and survival. Results: The median intratumoral MVD was 52 microvessels per 0.26-mm2 field area (interquartile range, 31–77 microvessels per 0.26-mm2 field area). At a median follow-up period of 3.5 years, patients with a high (>median) MVD had significantly higher 5- and 10-year overall survival rates (84%) than patients with low (≤median) MVD (49%; P = 0.0029). Furthermore, increased relapse-free survival for patients with high MVD (P = 0.0064) was observed. In a subgroup analysis of 44 patients with primary high-grade central osteosarcoma of the extremities without primary metastases and good surgical remission, high MVD was associated with 5- and 10-year overall survival rates of 91% compared with 58% for low MVD (P = 0.034). Cox regression analysis revealed that MVD was an independent prognostic factor for survival. A good response to chemotherapy (histologic grading scale of Salzer-Kuntschik) correlated significantly with a high MVD (P = 0.006). Conclusions: Increased angiogenesis is a prognostic indicator for higher survival and response rates to chemotherapy in patients with osteosarcoma. Thus, measurement of MVD might be useful in decisions selecting patients for future neoadjuvant treatment.

S100A2 Induces Metastasis in Non–Small Cell Lung Cancer

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non–small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Bevacizumab reduces VEGF expression in patients with relapsed and refractory acute myeloid leukemia without clinical antileukemic activity

Bevacizumab reduces VEGF expression in patients with relapsed and refractory acute myeloid leukemia without clinical antileukemic activity

A phase 2 pilot study of pegfilgrastim and filgrastim for mobilizing peripheral blood progenitor cells in patients with non-Hodgkin’s lymphoma receiving chemotherapy

This phase 2 randomized, double-blind trial examines the efficacy of pegfilgrastim and filgrastim for mobilizing peripheral blood progenitors cells following chemotherapy in patients with non-Hodgkin’s lymphoma. The results of the study suggest that pegfilgrastim may provide a suitable alternative to filgrastim in this setting. Background Growth factors are frequently used to aid peripheral blood progenitor cell mobilization from bone marrow. This phase 2 study examined the efficacy and safety of pegfilgrastim for mobilizing peripheral blood progenitors cells for autologous transplantation. Design and Methods Patients with non-Hodgkin’s lymphoma received one cycle of mobilizing chemotherapy (ifosfamide, carboplatin and etoposide, ICE). Twenty-four hours later they were randomized, double-blind, to receive a single dose of pegfilgrastim 6 mg or 12 mg, or filgrastim 5 μg/kg/day (until the end of leukapheresis). Following leukapheresis (collection phase), patients rested or received one or two ‘salvage’ cycles of ICE. High-dose BEAM chemotherapy was then given before peripheral blood progenitor cell transplantation. The primary end-point was the patients’ mean yield of CD34+ cells/kg during the collection phase. Results Ninety patients were randomized and received a study drug; 63% completed the collection phase. The patients’ mean (95% CI) CD34+ cell harvest per leukapheresis was 0.8 (0.5–1.4), 0.8 (0.5–1.6) and 1.2 (0.7–2.0)×106 cells/kg for the pegfilgrastim 6 mg, pegfilgrastim 12 mg and filgrastim groups, respectively. Twenty (69%), 17 (59%) and 23 (72%) patients in these three groups achieved the targeted minimum harvest (≥2×106 cells/kg). The mean total harvests were 1.7, 1.4 and 2.2×106 cells/kg, respectively. Post-transplantation, the median days to absolute neutrophil count recovery (≥0.5×109/L) were 12, 11, and 11, respectively. Pegfilgrastim and filgrastim were generally well tolerated. Conclusions Pegfilgrastim (6 or 12 mg) was effective for mobilizing peripheral blood progenitors cells in patients with non-Hodgkin’s lymphoma. These data may aid the design of studies to clarify optimal dosing and leukapheresis with pegfilgrastim.

The syndrome of hemophagocytic lymphohistiocytosis in primary immunodeficiencies: implications for differential diagnosis and pathogenesis

Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome defined by clinical and laboratory criteria. Current criteria were created to identify patients with familial hemophagocytic lmyphohistiocytosis in immediate need of immunosuppressive therapy. However, these criteria also identify patients with infection-associated hemophagocytic inflammatory states lacking genetic defects typically predisposing to hemophagocytic lymphohistiocytosis. These patients include those with primary immunodeficiencies, in whom the pathogenesis of the inflammatory syndrome may be distinctive and aggressive immunosuppression is contraindicated. To better characterize hemophagocytic inflammation associated with immunodeficiencies, we combined an international survey with a literature search and identified 63 patients with primary immunodeficiencies other than cytotoxicity defects or X-linked lymphoproliferative disorders, presenting with conditions fulfilling current criteria for hemophagocytic lymphohistiocytosis. Twelve patients had severe combined immunodeficiency with <100/μL T cells, 18 had partial T-cell deficiencies; episodes of hemophagocytic lymphohistiocytosis were mostly associated with viral infections. Twenty-two patients had chronic granulomatous disease with hemophagocytic episodes mainly associated with bacterial infections. Compared to patients with cytotoxicity defects, patients with T-cell deficiencies had lower levels of soluble CD25 and higher ferritin concentrations. Other criteria for hemophagocytoc lymphohistiocytosis were not discriminative. Thus: (i) a hemophagocytic inflammatory syndrome fulfilling criteria for hemophagocytic lymphohistiocytosis can be the initial manifestation of primary immunodeficiencies; (ii) this syndrome can develop despite severe deficiency of T and NK cells, implying that the pathophysiology is distinct and not appropriately described as “lympho”-histiocytosis in these patients; and (iii) current criteria for hemophagocytoc lymphohistiocytosis are insufficient to differentiate hemophagocytic inflammatory syndromes with different pathogeneses. This is important because of implications for therapy, in particular for protocols targeting T cells.

CYTOKINE RESPONSE TO INFECTION IN PATIENTS WITH ACUTE MYELOGENOUS LEUKAEMIA FOLLOWING INTENSIVE CHEMOTHERAPY

Summary. Septic shock is the major cause of treatmentrelated death in patients with acute myelogenous leukaemia (AML) undergoing intensive chemotheray. Interleukins (IL)‐1β, −6, −8, and tumour nerosis factor α (TNF‐α) have been implicated as mediators of septic shock, with circulating leucocytes being considered a major source for their release. However, Plasma cytokine levels of leucocytopnic patients with evolving sepsis have not been studied. We have prospectively measured plasma cytokines during chemotherapy‐induced leucocytopenia (< 1 × 109/1) in 50 patiens with AML. Cytokine levels in patients with severe sepsis (n=5) or septic shock (n=8) were cmpared to those measured in 13 matched patients with uncmplicated febrile infections. In evolving septic shock, IL‐6, IL‐8 and TNF‐α peaked within 48 h of fever onset at levels reported for nonleucocytopenic patients and distinctively higher than during uncomplicated febrile episodes (P < 0.05). Peak concentrations measured within 48 h after onset of fever were related to fatal outcome. IL‐1β was detected in less than 5% of all samples. Cytokine concentrations were unrelated to leucocyte counts and markers of neopterphil or monocyte activation (elastase and neeopterin levels, respectively). We conclude that cytokine release associated with evolving septic shock in patients with AML does not depend on circulating leucocytes.

In vivo optical imaging of CD13/APN-expression in tumor xenografts.

The metalloexopeptidase CD13/aminopeptidase N (APN) has been shown to be involved in cancer angiogenesis, invasion, and metastasis. Therefore, a CD13/APN-targeted NGR-peptide was labeled with the cyanine dye Cy 5.5 and applied to image tumor xenografts with different APN-expression levels using both planar and tomographic optical imaging methods. In vitro, the peptide-dye conjugate showed a clear binding affinity to APN-positive HT-1080 cells, while negative MCF-7 cells and predosing with the free NGR-peptide revealed little to no fluorescence. In vivo, tumor xenografts (n>or=5) were clearly visualized by two-dimensional (2-D) planar fluorescence reflectance imaging (FRI) and three-dimensional (3-D) fluorescence mediated tomography (FMT) up to 24 h after injection. FMT also allowed us to quantify fluorochrome distribution in deeper tissue sections, showing an average fluorochrome concentration of 306.7+/-54.3 nM Cy 5.5 (HT-1080) and 116.0+/-18.3 nM Cy 5.5 (MCF-7) in the target tissue after 5 h. Competition with the free NGR-peptide resulted in a reduction of fluorochrome concentration in HT-1080 tumor tissue (195.3+/-21.9 nM; 5 h). We thus conclude that NGR-Cy 5.5 combined with novel tomographic optical imaging methods allows us to image and quantify tumor-associated CD13/APN expression noninvasively. This may be a promising strategy for a sensitive evaluation of tumor angiogenesis in vivo.