Johannes Fischer

Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dose-dense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial

BACKGROUNDnBreast cancer with extensive axillary-lymph-node involvement has a poor prognosis after conventional treatment. In trials with historical controls, high-dose chemotherapy produced improved outcomes. We compared an intensive double-cycle high-dose chemotherapy regimen with an accelerated conventionally dosed regimen in high-risk breast cancer in a multicentre trial.nnnMETHODSnPatients with at least nine positive nodes were randomly assigned either two courses of accelerated (2-week intervals, with filgrastim support), conventionally dosed epirubicin and cyclophosphamide followed by two courses of high-dose chemotherapy (epirubicin, cyclophosphamide, and thiotepa supported by peripheral-blood progenitors) or four identical cycles of epirubicin and cyclophosphamide followed by three cycles of accelerated cyclophosphamide, methotrexate, and fluorouracil. The primary endpoint was event-free survival. Analyses were done both by intention to treat and per protocol.nnnFINDINGSn403 patients were enrolled; 201 were assigned high-dose chemotherapy and 202 conventional treatment. The mean number of positive nodes was 17.6, and median follow-up was 48.6 months. 4-year event-free survival (intention-to-treat analysis) was 60% (95% CI 53-67) in the high-dose chemotherapy group and 44% (37-52) in the control group (p=0.00069). The corresponding overall survival was 75% (69-82) versus 70% (64-77; p=0.02). There were no treatment-related deaths.nnnINTERPRETATIONnOur finding of significant improvements in both event-free and overall survival for high-dose chemotherapy compared with a dose-dense conventional regimen contrasts with the results of other studies. The discrepancy might be due partly to design differences (tandem, brief induction) between our regimen and those studied in other trials. This approach merits further study.

Clinical-scale Generation of Dendritic Cells in a Closed System

Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 ± 0.2 × 1010 (mean ± SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 ± 2.3%, monocytes were enriched to 94.3 ± 2.2%. CD14+ cell recovery was 67.0 ± 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1&bgr;, IL-6, TNF&agr;, and PGE2, cells (82.0 ± 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 ± 4.7% and viability was 91.9 ± 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFN&ggr; but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.

A single dose of 6 or 12 mg of pegfilgrastim for peripheral blood progenitor cell mobilization results in similar yields of CD34+ progenitors in patients with multiple myeloma

BACKGROUND:u2002 Current regimens for peripheral blood progenitor cell (PBPC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of granulocyte–colony‐stimulating factor (G‐CSF) starting shortly after cytotoxic therapy. Recently a polyethylene glycol–conjugated G‐CSF (pegfilgrastim) was introduced that has a substantially longer t1/2 than the original formula.

Hepatocyte Growth Factor/c-MET Axis-mediated Tropism of Cord Blood-derived Unrestricted Somatic Stem Cells for Neuronal Injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.

Phenotypic and functional comparison of monocytes from cord blood and granulocyte colony-stimulating factor–mobilized apheresis products

OBJECTIVEnIt is well established that T cells are effector cells in graft-vs-host disease (GVHD), yet the contribution of graft monocytes is less well characterized. Therefore, monocytes in cord blood (CB) and granulocyte colony-stimulating factor-mobilized apheresis products (G-AP), two stem cell grafts associated with reduction of acute and chronic GVHD and relative reduction of acute GVHD, respectively, were compared phenotypically and functionally.nnnMATERIALS AND METHODSnThe frequencies, phenotypes, and pinocytosis activities of monocytes from CB and G-AP were determined by flow cytometry and their allostimulatory potential in a primary mixed leukocyte reaction.nnnRESULTSnG-AP contained significantly more monocytes than CB (24.9% +/- 7.1% vs 8.8% +/- 1.5% CD14+ and 62.4 +/- 27.5 x 10(6) vs 0.9 +/- 0.2 x 10(6) CD14+ cells/mL). Monocytes from both sources revealed similar phenotypes. They expressed CD4, CD11a, CD11b, CD11c, CD18, CD32, CD33, CD45R0, CD48, CD50, CD54, CD58, CD64, CD86, CD102, CD116, CD123, and HLA-DR; showed no expression of CD1a and CD83; and weak expression of CD16, CD45RA, and CD80. The levels of CD80 and CD86 expression were comparable; however, in contrast to G-AP monocytes, CB monocytes lacked CD40. There was no difference in pinocytosis activity and allostimulatory capacity of CB and G-AP monocytes.nnnCONCLUSIONSnMonocytes in CB and G-AP are phenotypically and functionally comparable. The only difference observed is the lack of CD40 on CB monocytes.

Comparison of soluble CD40L concentrations and release capacities in apheresis and prestorage pooled platelet concentrates.

INTRODUCTIONnSoluble CD40L (sCD40L) is expressed by platelets and is involved in the stabilization of arterial thrombi. Additionally, it was shown that sCD40L accumulation occurred in stored blood products triggering adverse transfusion reactions like TRALI. To study the influence of the preparation technique on sCD40L accumulation and platelet function we examined CD40L concentrations in prestorage pooled platelet concentrates compared to apheresis products. In addition, sCD40L release capacity was determined as a marker for platelet viability.nnnMATERIAL AND METHODSnsCD40L concentrations were determined in prestorage pooled platelet concentrates (n = 8) and in platelet apheresis concentrates (n = 8) before and after platelet stimulation (recalcification and clot formation) at day 1, 3 and 5 under routine storage conditions. sCD40L concentrations were determined by a commercially available ELISA kit.nnnRESULTSnsCD40L concentrations in storage medium increased over time in prestorage pooled platelet concentrates (from 1,185 pg/mL ± 87 pg/mL at day 1 to 4,464 pg/mL ± 212 pg/mL at day 5) as well as in apheresis products (from 581 pg/mL ± 124 pg/mL at day 1 to 2,718 pg/mL ± 154 pg/mL at day 5) in a hyperbolic manner. Recalcification and clot formation caused an increase in sCD40L concentrations (for example 3,842 pg/mL ± 769 pg/mL before platelet activation to 31,219 pg/mL ± 2,063 pg/mL after platelet activation at day 3), and we observed comparable release capacities for both preparation techniques, however, decreasing over storage time up to 50% (day 5) of the respective control value (day 1).nnnCONCLUSIONSnAmounts of sCD40L accumulation and release capacity during storage of platelet concentrates were dependent on storage duration, but showed no relevant differences regarding the preparation technique. After 5 days of storage, CD40L basal levels were increased, in contrast sCD40L release capacity was decreased. By recalcification and clot formation sCD40L release capacity could be easily induced and is assumed to be used as a marker for platelet viability.

Transient appearance of postoperative EDTA-dependent pseudothrombocytopenia in a patient after gastrectomy

Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is a well known phenomenon. Antiplatelet antibodies cause platelet clumping in EDTA anticoagulated blood samples, and blood count analysers calculate a spurious low platelet count. We describe a case of a transient appearance of EDTA-PTCP in a patient after gastrectomy. A 58-year-old man underwent partial gastrectomy in for gastric cancer. Preoperatively, his platelet count was in a normal range, and the surgical procedure was performed without bleeding complications. At day 10 after surgery the patient showed a low platelet count, which could be identified as EDTA-PTCP. The phenomenon disappeared in a following postoperative time interval of 2 months. In cases of recently occurring thrombocytopenias EDTA-PTCP should always be considered as a possible cause of low platelet count, in particular in cases of inconspicuous clinical findings. Appropriate laboratory analysis should be applied.

Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering

One of the major challenges in tissue engineering is to supply larger three‐dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)‐derived endothelial colony‐forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with Good Manufacturing Practice (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue‐culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo‐expanded ECFCs are a novel, very potent cell source for scaffold‐based tissue engineering. Copyright

Reducing costs in flow-cytometric counting of residual white blood cells in blood products: utilization of a single-platform bead-free flow-rate calibration method.

BACKGROUND: Commercial flow‐cytometric methods for counting residual white blood cells (rWBCs) in leukoreduced blood products use calibration beads for estimation of the measured sample volume. A bead‐free flow‐rate calibration method is developed and validated.

Methods for Separate Isolation of Cell-Free DNA and Cellular DNA from Urine-Application of Methylation-Specific PCR on both DNA Fractions

The analysis of genomic DNA is widely-used for research, forensic and diagnostic purposes. Here we describe reliable methods for isolation of cell-free DNA and cellular DNA from urine. Both DNA fractions are suitable for PCR and Methylation-Specific PCR (MSP) amplification, leading to consistent and reproducible results. A kinetics analysis de- scribes the decline of efficiency of MSP performed with urinary DNA which had been stored at room temperature with and without proteinase K for various time periods.