Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study.

BACKGROUND Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. METHODS We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. FINDINGS Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. INTERPRETATION This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. FUNDING Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD.

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Alginate-coated chitosan nanogels differentially modulate class-A and class-B CpG-ODN targeting of dendritic cells and intracellular delivery.

UNLABELLED CpG-oligodeoxynucleotides (CpG-ODNs) interact with dendritic cells (DCs), but evidence is less clear for CpG-ODN admixed with or incorporated into vaccine delivery vehicles. We loaded alginate-coated chitosan-nanogels (Ng) with class-A or class-B CpG-ODN, and compared with the same CpG-ODNs free or admixed with empty Ng. Experiments were performed on both porcine and human blood DC subpopulations. Encapsulation of class-A CpG-ODN (loading into Ng) strongly reduced the CpG-ODN uptake and intracellular trafficking in the cytosol; this was associated with a marked deficiency in IFN-α induction. In contrast, encapsulation of class-B CpG-ODN increased its uptake and did not influence consistently intracellular trafficking into the nucleus. The choice of CpG-ODN class as adjuvant is thus critical in terms of how it will behave with nanoparticulate vaccine delivery vehicles. The latter can have distinctive modulatory influences on the CpG-ODN, which would require definition for different CpG-ODN and delivery vehicles prior to vaccine formulation. FROM THE CLINICAL EDITOR This basic science study investigates the role of class-A and class-B CpG-oligodeoxynucleotides loaded into alginate-coated chitosan nanogels, demonstrating differential effects between the two classes as related to the use of these nanoformulations as vaccine delivery vehicles.

Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo.

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

BACKGROUND Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays. METHODS Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays. RESULTS Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes. CONCLUSION Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.

Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

Comparison of peak cardiopulmonary performance parameters on a robotics-assisted tilt table, a cycle and a treadmill

Robotics-assisted tilt table (RATT) technology provides body support, cyclical stepping movement and physiological loading. This technology can potentially be used to facilitate the estimation of peak cardiopulmonary performance parameters in patients who have neurological or other problems that may preclude testing on a treadmill or cycle ergometer. The aim of the study was to compare the magnitude of peak cardiopulmonary performance parameters including peak oxygen uptake (VO2peak) and peak heart rate (HRpeak) obtained from a robotics-assisted tilt table (RATT), a cycle ergometer and a treadmill. The strength of correlations between the three devices, test-retest reliability and repeatability were also assessed. Eighteen healthy subjects performed six maximal exercise tests, with two tests on each of the three exercise modalities. Data from the second tests were used for the comparative and correlation analyses. For nine subjects, test-retest reliability and repeatability of VO2peak and HRpeak were assessed. Absolute VO2peak from the RATT, the cycle ergometer and the treadmill was (mean (SD)) 2.2 (0.56), 2.8 (0.80) and 3.2 (0.87) L/min, respectively (p < 0.001). HRpeak from the RATT, the cycle ergometer and the treadmill was 168 (9.5), 179 (7.9) and 184 (6.9) beats/min, respectively (p < 0.001). VO2peak and HRpeak from the RATT vs the cycle ergometer and the RATT vs the treadmill showed strong correlations. Test-retest reliability and repeatability were high for VO2peak and HRpeak for all devices. The results demonstrate that the RATT is a valid and reliable device for exercise testing. There is potential for the RATT to be used in severely impaired subjects who cannot use the standard modalities.

The International Surface Pressure Databank version 2

The International Surface Pressure Databank (ISPD) is the world's largest collection of global surface and sea-level pressure observations. It was developed by extracting observations from established international archives, through international cooperation with data recovery facilitated by the Atmospheric Circulation Reconstructions over the Earth (ACRE) initiative, and directly by contributing universities, organizations, and countries. The dataset period is currently 1768–2012 and consists of three data components: observations from land stations, marine observing systems, and tropical cyclone best track pressure reports. Version 2 of the ISPD (ISPDv2) was created to be observational input for the Twentieth Century Reanalysis Project (20CR) and contains the quality control and assimilation feedback metadata from the 20CR. Since then, it has been used for various general climate and weather studies, and an updated version 3 (ISPDv3) has been used in the ERA-20C reanalysis in connection with the European Reanalysis of Global Climate Observations project (ERA-CLIM). The focus of this paper is on the ISPDv2 and the inclusion of the 20CR feedback metadata. The Research Data Archive at the National Center for Atmospheric Research provides data collection and access for the ISPDv2, and will provide access to future versions.

Feasibility of cardiopulmonary exercise testing and training using a robotics-assisted tilt table in dependent-ambulatory stroke patients

BACKGROUND: We evaluated the feasibility of an augmented robotics-assisted tilt table (RATT) for incremental cardiopulmonary exercise testing (CPET) and exercise training in dependent-ambulatory stroke patients. METHODS: Stroke patients (Functional Ambulation Category ≤ 3) underwent familiarization, an incremental exercise test (IET) and a constant load test (CLT) on separate days. A RATT equipped with force sensors in the thigh cuffs, a work rate estimation algorithm and real-time visual feedback to guide the exercise work rate was used. Feasibility assessment considered technical feasibility, patient tolerability, and cardiopulmonary responsiveness. RESULTS: Eight patients (4 female) aged 58.3 ± 9.2 years (mean ± SD) were recruited and all completed the study. For IETs, peak oxygen uptake (V'O2peak), peak heart rate (HRpeak) and peak work rate (WRpeak) were 11.9 ± 4.0 ml/kg/min (45 % of predicted V'O2max), 117 ± 32 beats/min (72 % of predicted HRmax) and 22.5 ± 13.0 W, respectively. Peak ratings of perceived exertion (RPE) were on the range "hard" to "very hard". All 8 patients reached their limit of functional capacity in terms of either their cardiopulmonary or neuromuscular performance. A ventilatory threshold (VT) was identified in 7 patients and a respiratory compensation point (RCP) in 6 patients: mean V'O2 at VT and RCP was 8.9 and 10.7 ml/kg/min, respectively, which represent 75 % (VT) and 85 % (RCP) of mean V'O2peak. Incremental CPET provided sufficient information to satisfy the responsiveness criteria and identification of key outcomes in all 8 patients. For CLTs, mean steady-state V'O2 was 6.9 ml/kg/min (49 % of V'O2 reserve), mean HR was 90 beats/min (56 % of HRmax), RPEs were > 2, and all patients maintained the active work rate for 10 min: these values meet recommended intensity levels for bouts of training. CONCLUSIONS: The augmented RATT is deemed feasible for incremental cardiopulmonary exercise testing and exercise training in dependent-ambulatory stroke patients: the approach was found to be technically implementable, acceptable to the patients, and it showed substantial cardiopulmonary responsiveness. This work has clinical implications for patients with severe disability who otherwise are not able to be tested.