Design of a multi-signature ensemble classifier predicting neuroblastoma patients' outcome

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is the most common pediatric solid tumor of the sympathetic nervous system. Development of improved predictive tools for patients stratification is a crucial requirement for neuroblastoma therapy. Several studies utilized gene expression-based signatures to stratify neuroblastoma patients and demonstrated a clear advantage of adding genomic analysis to risk assessment. There is little overlapping among signatures and merging their prognostic potential would be advantageous. Here, we describe a new strategy to merge published neuroblastoma related gene signatures into a single, highly accurate, Multi-Signature Ensemble (MuSE)-classifier of neuroblastoma (NB) patients outcome.</p> <p>Methods</p> <p>Gene expression profiles of 182 neuroblastoma tumors, subdivided into three independent datasets, were used in the various phases of development and validation of neuroblastoma NB-MuSE-classifier. Thirty three signatures were evaluated for patients' outcome prediction using 22 classification algorithms each and generating 726 classifiers and prediction results. The best-performing algorithm for each signature was selected, validated on an independent dataset and the 20 signatures performing with an accuracy > = 80% were retained.</p> <p>Results</p> <p>We combined the 20 predictions associated to the corresponding signatures through the selection of the best performing algorithm into a single outcome predictor. The best performance was obtained by the Decision Table algorithm that produced the NB-MuSE-classifier characterized by an external validation accuracy of 94%. Kaplan-Meier curves and log-rank test demonstrated that patients with good and poor outcome prediction by the NB-MuSE-classifier have a significantly different survival (p < 0.0001). Survival curves constructed on subgroups of patients divided on the bases of known prognostic marker suggested an excellent stratification of localized and stage 4s tumors but more data are needed to prove this point.</p> <p>Conclusions</p> <p>The NB-MuSE-classifier is based on an ensemble approach that merges twenty heterogeneous, neuroblastoma-related gene signatures to blend their discriminating power, rather than numeric values, into a single, highly accurate patients' outcome predictor. The novelty of our approach derives from the way to integrate the gene expression signatures, by optimally associating them with a single paradigm ultimately integrated into a single classifier. This model can be exported to other types of cancer and to diseases for which dedicated databases exist.</p

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

BACKGROUND: The most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase. RESULTS: We developed a photoaffinity label ((3)H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of (3)H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α(1)-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the (3)H-meta-PAL labeled sGC was fragmented by CNBr digest. The (3)H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α(1)-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the (3)H-meta-PAL. CONCLUSIONS: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the α(1)-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators

Anrechnung Teil 2: Ein Einblick in die Praxis

Diese Handreichung ergänzt den separat veröffentlichten ersten Teil zum Thema Anrechnung um Praxiserfahrungen. Vier Projekte geben einen Einblick in ihr schrittweises Vorgehen bei der Entwicklung und Umsetzung von Anrechnungsmodellen in die Praxis. Es handelt sich hierbei um Verbund- und Einzelprojekte, die Verfahren der individuellen oder pauschalen Anrechnung für bestimmte Studiengänge oder eine hochschulweite Anrechnungsrahmenordnung entwickelt und erprobt haben. Die Handreichung soll Projekten wertvolle Hinweise zur Auseinandersetzung mit dem Thema Anrechnung liefern und in Bezug auf eventuell auftretende Hürden frühzeitig sensibilisieren

Inflammatory Phenotype of Intrahepatic Sulfatide-Reactive Type II NKT Cells in Humans With Autoimmune Hepatitis

Background: Natural Killer T (NKT) cells are CD1d-restricted innate-like T cells that can rapidly release stored cytokines upon recognition of lipid antigens. In mice, type I NKT cells seem to promote liver inflammation, whereas type II NKT cells seem to restrict hepatitis. Here, we aimed at characterizing the role of human type I and type II NKT in patients with autoimmune hepatitis (AIH).Methods: NKT cells were analyzed by flow cytometry in peripheral blood and liver of AIH patients and control groups. α-galactosylceramide-loaded or sulfatide-loaded tetramers were used to detect type I or II NKT cells, respectively. Hepatic CD1d was stained by in situ-hybridization of liver biopsies.Results and Conclusions: Type II NKT cells were more prevalent in human peripheral blood and liver than type I NKT cells. In AIH patients, the frequency of sulfatide-reactive type II NKT cells was significantly increased in peripheral blood (0.11% of peripheral blood leukocytes) and liver (3.78% of intrahepatic leukocytes) compared to healthy individuals (0.05% and 1.82%) and patients with drug-induced liver injury (0.06% and 2.03%; p &lt; 0.05). Intrahepatic type II NKT cells of AIH patients had a different cytokine profile than healthy subjects with an increased frequency of TNFα (77.8% vs. 59.1%, p &lt; 0.05), decreased IFNγ (32.7% vs. 63.0%, p &lt; 0.05) and a complete lack of IL-4 expressing cells (0% vs. 2.1%, p &lt; 0.05). T cells in portal tracts expressed significantly more CD1d-RNA in AIH livers compared to controls. This study supports that in contrast to their assumed protective role in mice, human intrahepatic, sulfatide-reactive type II NKT cells displayed a proinflammatory cytokine profile in patients with AIH. Infiltrating T cells in portal areas of AIH patients overexpressed CD1d and could thereby activate type II NKT cells

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20∆CNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL as adhesion molecule expressed by A20-deficient CNS-ECs. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20∆CNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in wildtype mice. In addition, blocking of ICOSL on primary mouse brain microvascular endothelial cells (pMBMECs) impaired the adhesion of T cells in vitro. Taken together, we here propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation

Establishing stereotactic body radiotherapy with flattening filter free techniques in the treatment of pulmonary lesions - initial experiences from a single institution

Background: Stereotactic body radiotherapy (SBRT) using flattening filter free (FFF)-techniques has been increasingly applied during the last years. However, clinical studies investigating this emerging technique are still rare. Hence, we analyzed toxicity and clinical outcome of pulmonary SBRT with FFF-techniques and performed dosimetric comparison to conventional techniques using flattening filters (FF). Materials and methods: Between 05/2014 and 06/2015, 56 consecutive patients with 61 pulmonary lesions were treated with SBRT in FFF-mode. Central lesions received 8 × 7.5 Gy delivered to the conformally enclosing 80%-isodose, while peripheral lesions were treated with 3 × 15 Gy, prescribed to the 65%-isodose. Early and late toxicity (after 6 months) as well as initial clinical outcomes were evaluated. Furthermore, [deleted] plan quality and efficiency were evaluated by analyzing conformity, beam- on and total treatment delivery times in comparison to plans with FF-dose application. Results: Median follow-up time was 9.3 months (range 1.5–18.0 months). Early toxicity was low with only 5 patients (8.9 %) reporting CTCAE 2° or higher side-effects. Only one patient (1.8 %) was diagnosed with radiation-induced pneumonitis CTCAE 3°, while 2 (3.6 %) patients suffered from pneumonitis CTCAE 2°. After 6 months, no toxicity greater than CTCAE 2° was reported. 1-year local progression-free survival, distant progression-free survival and overall survival were 92.8 %, 78.0 %, and 94.4 %, respectively. While plan quality was similar for FFF- and FF-plans in respect to conformity (p = 0.275), median beam-on time as well as total treatment time were significantly reduced for SBRT in FFF-mode compared to FF-mode (p ≤ 0.001, p ≤ 0.001). Conclusions: Patient treatment with SBRT using FFF-techniques is safe and provides promising clinical results with only modest toxicity at significantly increased dose delivery speed

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells

Objective Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. Design Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. Results NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. Conclusion Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis

Extensive alterations of the whole-blood transcriptome are associated with body mass index: results of an mRNA profiling study involving two large population-based cohorts

Background: Obesity, defined as pathologically increased body mass index (BMI),is strongly related to an increased risk for numerous common cardiovascular and metabolic diseases. It is particularly associated with insulin resistance, hyperglycemia, and systemic oxidative stress and represents the most important risk factor for type 2 diabetes (T2D). However, the pathophysiological mechanisms underlying these associations are still not completely understood. Therefore, in order to identify potentially disease-relevant BMI-associated gene expression signatures, a transcriptome-wide association study (TWAS) on BMI was performed. Methods: Whole-blood mRNA levels determined by array-based transcriptional profiling were correlated with BMI in two large independent population-based cohort studies (KORA F4 and SHIP-TREND) comprising a total of 1977 individuals. Results: Extensive alterations of the whole-blood transcriptome were associated with BMI: More than 3500 transcripts exhibited significant positive or negative BMI-correlation. Three major whole-blood gene expression signatures associated with increased BMI were identified. The three signatures suggested: i) a ratio shift from mature erythrocytes towards reticulocytes, ii) decreased expression of several genes essentially involved in the transmission and amplification of the insulin signal, and iii) reduced expression of several key genes involved in the defence against reactive oxygen species (ROS). Conclusions: Whereas the first signature confirms published results, the other two provide possible mechanistic explanations for well-known epidemiological findings under conditions of increased BMI, namely attenuated insulin signaling and increased oxidative stress. The putatively causative BMI-dependent down-regulation of the expression of numerous genes on the mRNA level represents a novel finding. BMI-associated negative transcriptional regulation of insulin signaling and oxidative stress management provide new insights into the pathogenesis of metabolic syndrome and T2D

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.Norwegian PSC Research Center German Ministry of Education and Research (BMBF) through the National Genome Research Network (NGFN) Integrated Research and Treatment Center - Transplantation 01EO0802 PopGen biobank NIH DK 8496