Christian Junghanß

Addition of sorafenib versus placebo to standard therapy in patients aged 60 years or younger with newly diagnosed acute myeloid leukaemia (SORAML): a multicentre, phase 2, randomised controlled trial

BACKGROUND Preclinical data and results from non-randomised trials suggest that the multikinase inhibitor sorafenib might be an effective drug for the treatment of acute myeloid leukaemia. We investigated the efficacy and tolerability of sorafenib versus placebo in addition to standard chemotherapy in patients with acute myeloid leukaemia aged 60 years or younger. METHODS This randomised, double-blind, placebo-controlled, phase 2 trial was done at 25 sites in Germany. We enrolled patients aged 18-60 years with newly diagnosed, previously untreated acute myeloid leukaemia who had a WHO clinical performance score 0-2, adequate renal and liver function, no cardiac comorbidities, and no recent trauma or operation. Patients were randomly assigned (1:1) to receive two cycles of induction therapy with daunorubicin (60 mg/m(2) on days 3-5) plus cytarabine (100 mg/m(2) on days 1-7), followed by three cycles of high-dose cytarabine consolidation therapy (3 g/m(2) twice daily on days 1, 3, and 5) plus either sorafenib (400 mg twice daily) or placebo on days 10-19 of induction cycles 1 and 2, from day 8 of each consolidation, and as maintenance for 12 months. Allogeneic stem-cell transplantation was scheduled for all intermediate-risk patients with a sibling donor and for all high-risk patients with a matched donor in first remission. Computer-generated randomisation was done in blocks. The primary endpoint was event-free survival, with an event defined as either primary treatment failure or relapse or death, assessed in all randomised patients who received at least one dose of study treatment. We report the final analysis. This trial is registered with ClinicalTrials.gov, number NCT00893373, and the EU Clinical Trials Register (2008-004968-40). FINDINGS Between March 27, 2009, and Nov 28, 2011, 276 patients were enrolled and randomised, of whom nine did not receive study medication. 267 patients were included in the primary analysis (placebo, n=133; sorafenib, n=134). With a median follow-up of 36 months (IQR 35·5-38·1), median event-free survival was 9 months (95% CI 4-15) in the placebo group versus 21 months (9-32) in the sorafenib group, corresponding to a 3-year event-free survival of 22% (95% CI 13-32) in the placebo group versus 40% (29-51) in the sorafenib group (hazard ratio [HR] 0·64, 95% CI; 0·45-0·91; p=0·013). The most common grade 3-4 adverse events in both groups were fever (71 [53%] in the placebo group vs 73 [54%] in the sorafenib group), infections (55 [41%] vs 46 [34%]), pneumonia (21 [16%] vs 20 [14%]), and pain (13 [10%] vs 15 [11%]). Grade 3 or worse adverse events that were significantly more common in the sorafenib group than the placebo group were fever (relative risk [RR] 1·54, 95% CI 1·04-2·28), diarrhoea (RR 7·89, 2·94-25·2), bleeding (RR 3·75, 1·5-10·0), cardiac events (RR 3·46, 1·15-11·8), hand-foot-skin reaction (only in sorafenib group), and rash (RR 4·06, 1·25-15·7). INTERPRETATION In patients with acute myeloid leukaemia aged 60 years or younger, the addition of sorafenib to standard chemotherapy has antileukaemic efficacy but also increased toxicity. Our findings suggest that kinase inhibitors could be a useful addition to curative treatment for acute myeloid leukaemia. Overall survival after long-term follow-up and strategies to reduce toxicity are needed to determine the future role of sorafenib in treatment of this disease. FUNDING Bayer HealthCare.

Comparative in Vivo Confocal Microscopical Study of the Cornea Anatomy of Different Laboratory Animals

Purpose: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals. Matherials and Methods: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified. Results: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman’s membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet’s membrane. Conclusions: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.

Survey and analysis of the efficacy and prescription pattern of sorafenib in patients with acute myeloid leukemia

Sorafenib is a multi-kinase inhibitor with activity against several intracellular kinases which may play a role in the pathogenesis of acute myeloid leukemia (AML). In vitro data and results from early clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. However, clinical data are still sparse, and there are only a few reported cases of monotherapy. The aim of the present research was to collect clinical data on efficacy and safety in a systematic way by conducting a survey on clinical experience with sorafenib. Thirty institutions were asked to document all patients treated with sorafenib diagnosed with AML. Of all 29 evaluable patients, six (21%) responded to sorafenib containing treatment by achieving a complete remission (CR, n = 2) or complete remission with incomplete platelet recovery (CRi, n = 4). In 23 patients receiving sorafenib as monotherapy, the CRi rate amounted to 13% and no CRs were documented. Of the 18 FLT-ITD positive patients with sorafenib monotherapy, two patients achieved a CRi (11%). In five FLT3-ITD negative cases, one CRi was documented (20%). Our results suggest the potential ability of the drug to induce remissions in refractory or relapsed AML even when given as monotherapy.

Prognostic Role of Circulating Tumor Cells during Induction Chemotherapy Followed by Curative Surgery Combined with Postoperative Radiotherapy in Patients with Locally Advanced Oral and Oropharyngeal Squamous Cell Cancer

Background The prognostic role of circulating tumor cells (CTCs) after induction chemotherapy using docetaxel, cisplatin and fluorouracil (TPF) prior to surgery and adjuvant (chemo)radiation in locally advanced oral squamous cell cancer (OSCC) was evaluated. Methods In this prospective study, peripheral blood samples from 40 patients of the phase II study TISOC-1 (NCT01108042) with OSCC before, during, and after treatment were taken. CTCs were quantified using laser scanning cytometry of anti– epithelial cell adhesion molecule–stained epithelial cells. Their detection was correlated with clinical risk factors, recurrence-free (RFS) and overall survival (OS). Results Before starting the treatment CTCs were detected in 32 of 40 patients (80%). The median number at baseline was 3295 CTCs/ml. The median maximal number of CTCs during treatment was 5005 CTCs/ml. There was a significant increase of CTCs before postoperative radiotherapy compared to baseline before 1st cycle of IC (p = 0.011), 2nd cycle of IC (p = 0.001), 3rd cycle of IC (p = 0.004), and before surgery (p = 0.002), but not compared to end of therapy (p = 0.118). CTCs at baseline >median was also associated to risk of recurrence (p = 0.014). Maximal CTCs during therapy >median was more frequently observed in tumors of the oral cavity (p=0.022) and related to higher risk of death during follow-up (p = 0.028). Patients with CTCs at baseline >median value had significant lower RFS than patients with CTCs at baseline <median value (p = 0.025). Patients with maximal CTCs values >median during the complete course of therapy had a significantly lower OS than patients with values <median (p = 0.049). Finally, the multivariate analysis revealed that OS was significantly lower in patients with maximal CTCs during treatment higher than the median value (HR=6.151; CI: 1.244-30.420). Conclusions Baseline CTCs and maximal CTCs during therapy both seem to be good prognostic markers for OSCC when treated by TPF induction chemotherapy, surgery, and postoperative (chemo)radiation.

Biophysical effects in off-resonant gold nanoparticle mediated (GNOME) laser transfection of cell lines, primary- and stem cells using fs laser pulses

Gold nanoparticle mediated (GNOME) laser transfection is a powerful technique to deliver small biologically relevant molecules into cells. However, the transfection of larger and especially negatively charged DNA remains challenging. The efficiency for pDNA was 0.57% using parameter that does not influence the endo- and exogenous DNA. In order to gain a deeper understanding of the actual molecule uptake process, the uptake efficiency was determined using molecules of different sizes. It was evaluated that uncharged dextran molecules (2000 kDa) were delivered with an efficiency of 68%. The intracellular distribution of injected molecules was visualized and larger molecules were primary found in the cytoplasm. Patch clamp measurements suggested a permeabilization time up to 15 minutes. The uptake efficiency depended on the size and charge of the molecule to deliver as well as the cell size. A minor role for transfection plays the cell type since primary stem cells were successfully transfected. The perforation efficiency of semi-adherent and suspension cells is influenced by the cell and molecule size.

Systems Biology Approaches in Aging Research

Aging is a systemic process which progressively manifests itself at multiple levels of structural and functional organization from molecular reactions and cell-cell interactions in tissues to the physiology of an entire organ. There is ever increasing data on biomedical relevant network interactions for the aging process at different scales of time and space. To connect the aging process at different structural, temporal and spatial scales, extensive systems biological approaches need to be deployed. Systems biological approaches can not only systematically handle the large-scale datasets (like high-throughput data) and the complexity of interactions (feedback loops, cross talk), but also can delve into nonlinear behaviors exhibited by several biological processes which are beyond intuitive reasoning. Several public-funded agencies have identified the synergistic role of systems biology in aging research. Using one of the notable public-funded programs (GERONTOSYS), we discuss how systems biological approaches are helping the scientists to find new frontiers in aging research. We elaborate on some systems biological approaches deployed in one of the projects of the consortium (ROSage). The systems biology field in aging research is at its infancy. It is open to adapt existing systems biological methodologies from other research fields and devise new aging-specific systems biological methodologies.

Ultrashort laser pulse cell manipulation using nano- and micro- materials

The delivery of extra cellular molecules into cells is essential for cell manipulation. For this purpose genetic materials (DNA/RNA) or proteins have to overcome the impermeable cell membrane. To increase the delivery efficiency and cell viability of common methods different nano- and micro material based approaches were applied. To manipulate the cells, the membrane is in contact with the biocompatible material. Due to a field enhancement of the laser light at the material and the resulting effect the cell membrane gets perforated and extracellular molecules can diffuse into the cytoplasm. Membrane impermeable dyes, fluorescent labelled siRNA, as well as plasmid vectors encoded for GFP expression were used as an indicator for successful perforation or transfection, respectively. Dependent on the used material, perforation efficiencies over 90 % with a cell viability of about 80 % can be achieved. Additionally, we observed similar efficiencies for siRNA transfection. Due to the larger molecule size and the essential transport of the DNA into the nucleus cells are more difficult to transfect with GFP plasmid vectors. Proof of principle experiments show promising and adequate efficiencies by applying micro materials for plasmid vector transfection. For all methods a weakly focused fs laser beam is used to enable a high manipulation throughput for adherent and suspension cells. Furthermore, with these alternative optical manipulation methods it is possible to perforate the membrane of sensitive cell types such as primary and stem cells with a high viability.

FocusHeuristics – expression-data-driven network optimization and disease gene prediction

To identify genes contributing to disease phenotypes remains a challenge for bioinformatics. Static knowledge on biological networks is often combined with the dynamics observed in gene expression levels over disease development, to find markers for diagnostics and therapy, and also putative disease-modulatory drug targets and drugs. The basis of current methods ranges from a focus on expression-levels (Limma) to concentrating on network characteristics (PageRank, HITS/Authority Score), and both (DeMAND, Local Radiality). We present an integrative approach (the FocusHeuristics) that is thoroughly evaluated based on public expression data and molecular disease characteristics provided by DisGeNet. The FocusHeuristics combines three scores, i.e. the log fold change and another two, based on the sum and difference of log fold changes of genes/proteins linked in a network. A gene is kept when one of the scores to which it contributes is above a threshold. Our FocusHeuristics is both, a predictor for gene-disease-association and a bioinformatics method to reduce biological networks to their disease-relevant parts, by highlighting the dynamics observed in expression data. The FocusHeuristics is slightly, but significantly better than other methods by its more successful identification of disease-associated genes measured by AUC, and it delivers mechanistic explanations for its choice of genes.

Pathway maps enable straightforward yet customized and semi-automated yet insightful analyses of omics data

To explore the molecular processes underlying some biological theme of interest based on public data, gene lists are used herein as input for the construction of annotated pathway maps, employing Cytoscape apps, and then high-throughput (“omics”) gene expression data are overlaid onto these maps. Seeded with a published set of marker genes of the senescence-associated secretory phenotype and the genes of the cellular senescence KEGG pathway, a gene/protein interaction network and annotated clusters (a “pathway map”) of cellular senescence are derived. The map can be amended, by adding some application-specific genes, and overlaid with gene expression data describing cellular senescence of fibroblasts and with disease-related gene expression data associated with prostate and pancreatic cancer, and with ischemic stroke, allowing insights into the role of cellular senescence in disease. Some gene expression data are derived from the “Biomarker Benchmark repository”. The pathway map approach can be followed in principle for any biological theme of interest, fostering much-needed independence from the investigator-biased expert networks usually used for overlaying gene expression data.

Gold nanoparticle mediated cell manipulation using fs and ps laser pulses for cell perforation and transfection

Manipulation of cells requires the delivery of membrane-impermeable substances like genetic materials or proteins into the cytoplasm. Thus delivery of molecules over the cell membrane barrier is one of the key technologies in molecular biology. Many techniques concerning especially the delivery foreign DNA have been developed. Notwithstanding there still is a range of applications where these standard techniques fail to raise the desired results due to low efficiencies, high toxicity or other safety issues. Especially the transfection of sensitive cell types like primary and stem cells can be problematic. Here we present an alternative, laser based technique to perforate the cell membrane and thus allowing efficient delivery of extra cellular molecules: Gold nanoparticles (GNP) are brought into close contact with the cell, were the laser-GNP interaction leads to membrane perforation. This allows the utilisation of a weakly focused laser beam leading to fast scanning of the sample and thus to a high throughput. To investigate the GNP-laser interaction in more detail we have compared membrane perforation obtained by different laser pulse lengths. From our results we assume strong light absorption for ps laser pulses and relatively small particles as the initiating perforation mechanism, whereas an enhanced near field scattering occurs at 200 nm GNP when using fs laser pulses. SEM and ESEM imaging were applied to give a deeper insight in the GNP-cell interaction and the effects of laser radiation on the GNP. Additionally dextran- FITC derivatives of varying sizes were used to investigate the impact of molecule size on delivery efficiency.