Final Report Summary - BESTAGEING (Biomarker Research Alliance for Diagnosing Heart Disease in the Ageing European Population)
Executive Summary:
The ageing of the European population represents a growing health care problem, threatening future economic growth and quality of life of our society. In particular, cardiovascular diseases show a marked increase with age and have remained the leading cause of morbidity and mortality in the elderly. Unfortunately, cardiovascular disorders and their associated risks are often difficult to diagnose in the elderly due to many age-related confounders and co-morbidities, leading to substantial uncertainties in diagnostic classification and therapeutic decision making with huge impact on patients’ outcomes. Hence, there is an unmet need for novel biomarkers to enable a more accurate diagnosis, sub-classification, risk assessment and treatment guidance for both acute and chronic cardiovascular diseases in the elderly.
The BestAgeing consortium aimed to improve this unsatisfactory situation by developing and validating novel omics-based biomarkers that may complement or substitute traditional biomarkers for more accurate or earlier diagnosis, better risk assessment and appropriate guidance of therapies, overcoming limitations inherent with traditional biomarkers – supporting healthy ageing in Europe. By combining leading experts in the field of “classical” biomarker development, high-throughput technologies, omics-biomarker research, research intensive SME and industry, and clinical institutions, we are confident that our efforts led to novel European medical technologies that will improve the efficacy and efficiency of our care for elderly patients, which will also impact on the competiveness of research-intensive European industries and socioeconomic wealth in Europe
Project Context and Objectives:
The ageing of Europe is challenging our health care systems and economic growth. It is estimated that by 2080, the number of people aged over 65 in Europe will have almost doubled from now 28,8 % to 52,3 % of the total population (EC eurostats). Therefore, strategies to improve population health and to increase healthy life years must be a keystone for a sustainable Europe. Cardiovascular diseases are by far the leading cause of morbidity and mortality in industrialized nations, and they soon will become the most prevalent cause of death worldwide. Due to remarkable progress in prevention and acute cardiac patient care, cardiovascular diseases nowadays manifest significantly later in life. As such, the incidence of coronary artery disease (CAD), myocardial infarction (MI) and heart failure (HF) – often occurs in rapid sequence within one patient – increases nearly exponentially with age.
The diagnosis of HF and CAD syndromes in the elderly are demanding due to a high rate of atypical symptoms, overlapping clinical manifestations of comorbid states, and silent disease stages. Confounding age-related alterations in the cardiovascular system such as cardiac hypertrophy or conduction abnormalities often impair interpretation of the electrocardiogram and clinical imaging. Further, results of clinical chemistry with e.g. natriuretic peptides or troponins are more difficult to interpret due to age-related changes in kidney function and muscle mass as well as co-existence of multi-organ diseases, often leading to uncertainties in clinical decision making, hindering effective treatment. Moreover reference values for biomarkers in an aged population are largely missing. Thus there is an unmet need for novel, specific and sensitive biomarkers for diagnosis, risk prediction, and treatment guidance for acute and chronic cardiovascular diseases in the elderly population.
The main objectives of BestAgeing are:
1.) The Validation of novel omics-based biomarkers in elderly patients in Europe.
The BestAgeing project brought together partners which have conducted extensive biomarker discovery studies using current state-of-the-art or even proprietary “beyond state-of-the-art” omics-technologies and so previous work has generated panels of omics-biomarkers that have been available for analytical and clinical validation and assay technologies that enable true translation into clinical application. From these undertakings, several highly promising biomarker candidates have been pursued within the BestAgeing project. First we validated the previously identified omics-biomarkers in available retrospective and prospective cohorts. Additionally, we defined comparative reference markers, statistical validation concepts and we developed a dedicated IT infrastructure which enables a sophisticated analysis of the complex omics and phenotype data. As in the omics-field standardization and quality control (QC) are essential prerequisites for successful validation of biomarkers by the different technologies, we established various Standard Operating Procedures (SOPs) to assure standardization processes for sampling, storage and exchange of biomaterials.
2. Development of novel assays for selected omics-biomarkers.
Although highly efficient screening technologies for thousands of miRNAs, metabolites, and even whole-genomes have been developed in the last decade, only few assays measuring single analytes or distinct features out of the omics-entities are ready for clinical application. For instance, there is currently no methodology available to measure a distinct miRNA pattern in a turnaround time of less than 1h. However clinical decision-making in ACS patients will need reporting of test results within 90 minutes as proposed by ESC guidelines. For chronic CVD conditions, turnaround times of hours to days are tolerated, however even these require technically robust, validated, and certified assay systems at centralized lab facilities. Hence, development and optimization of analytical technologies is clearly required. BestAgeing partners were aware of these challenges and aimed to develop assay technologies that allow fast and precise detection of omics-features. Both the SME and industry partners of BestAgeing have extensive research and development capabilities for biomedical assays. The development of assays and analyzers for dedicated omics-marker testing will also be of benefit for other disciplines. The specific assay technologies shall circumvent existing and unsolved limitations of current methodologies, such as missing analytical robustness, standardization, and turnaround times. Two main goals of the BestAgeing project were the development of new miRNA test assays and the development and optimization of a targeted, quantitative mass spectrometry-based method for the simultaneous quantification of 20-50 metabolite biomarkers. Our partner Siemens Healthcare AG aimed to provide a method which allows rapid information on the miRNA status of AMI (Acute Myocardial Infarction) – suspected patients. It was possible to develop a de-centralized and automated system within the BestAgeing project. Only a minimum of hands-on procedures would be acceptable for such a rapid test and the turnaround time has to be in the range of one hour or even shorter. Thus an automated sample preparation has been developed. The second goal was also achieved: during the BestAgeing project phase Numerious human plasma samples derived from various heart failure-associated cohorts has analyzed applying MxP® CLP +/- MxP® Broad Profiling and MxP® Quality Control metabolomics technology. The analyses of metabolomics data successfully confirm the added value of the novel metabolic biomarker panel called MxP® CLP suitable for the improved diagnosis of heart failure with reduced ejection fraction.
3. Development of multi-marker strategies based on diverse omics and established biomarkers as well as advanced imaging modalities for diagnosis, risk prediction and treatment guidance in elderly patients.
In clinical practice, multiple markers are often analyzed in aggregate to better understand progression of disease processes or to test for the relative contribution of different disease pathways. Thus the combined measurement of myocardial necrosis markers with different kinetics in blood such as myoglobin, CKMB and troponins provides information on the temporal progression of myocardial infarction and on quality of reperfusion. The analysis of markers reflecting different disease pathways such as troponins for necrosis, BNP for myocardial stress and GDF-15 for pro-inflammatory activation provides incremental prognostic information in ACS and CHF. Patients with ACS and both, elevated BNP and troponin, are at a particular high risk for cardiac death and myocardial infarction and benefit most from revascularization therapy. However multi-marker strategies in current state-of-the-art clinical care do not include omics markers or decision support systems. It is expected that the added information inherent to metabolite or miRNA profiles and the genetic and epigenetic variables will improve diagnosis and risk stratification beyond available protein markers. Multi-marker strategies accounting for disease specific changes on different levels may enhance the predictive power over single biomarkers, a hypothesis that will be tested in our elderly HF cohorts. Our aim was not only to test the added benefit of omics in a multi-marker approach, but also to develop statistical methods and computer algorithms / applications that predict the optimal combination and aid in the interpretation of multiple markers from the same or even different omics-technologies. Overall, classical machine learning and network analysis technologies should be explored and novel systems biology approaches developed. Additionally, the gathered information and potential of the evaluated markers should be used to model the potential socioeconomic impact of the novel biomarkers.
Project Results:
Our BestAgeing study designs addressed the most frequent and severe cardiovascular diseases of elderly patients by incorporating the appropriate phenotype and reference cohorts and biomaterials from European populations. The strength of the BestAgeing consortium is our extensive previous work on biomarker identification and validation, our long-standing expertise in technology development and omics-based research, as well as our experience in translational research and test development as exemplified by the invention, the successful translation, and continuous refinement of cardiac Troponin T to a world-wide standard of good clinical practice in diagnosing myocardial infarction and prognosis of cardiac risk.
Work Package 1 - Definition of most promising omics-biomarker candidates and database development
The WP1 comprises four main objectives focused on the most promising biomarker candidates and the development of a dedicated database for clinical and selected omics data of all BestAgeing studies. The first objective is the definition / selection of the most promising omics based biomarker candidates which had been initially identified by the consortium. This selection was based on proven value of the markers (statistical measures in profiling studies, biological replication), previously demonstrated analytical quality (technical reproducibility), diagnostic and prognostic power, IP protection, addressed disease cohorts of elderly patients including the clinical need in previous screening studies of the consortium partners and all related biomaterial and pre-analytical requirements. Based on first results of the validation studies the initial list has revised e.g. markers that were published for proven diagnostic or prognostic value have been added; and markers that turned out to be non-informative (no statistically significant (added) diagnostic or prognostic information) have been eliminated. The major changes are related to the selection of miRNA markers where several new potential markers were added and few deleted in the phenotypes acute coronary syndrome, coronary artery disease and heart failure. Besides the changes in miRNA marker candidates, RBM20 was added as an important and promising candidate in the revised list of protein markers.
Beside the revision of the biomarker candidates list, the selection and definition of reference marker for comparative analyses (objective two) was made. For comparison with the novel biomarkers, BestAgeing defined a panel of already established or currently investigated high sensitive cardiac biomarkers, clinical risk scores, and imaging modalities together with their associated phenotypes that are needed as best standard reference markers and phenotypes for comparative analyses. We have identified, based on current literature and clinical use in routine, three established biomarkers that will be primarily used as biomarker comparators: (1) high sensitivity cardiac Troponin (T and/or I); (2) B-type natriuretic peptides (BNP and/or NT-proBNP); and (3) high sensitivity C-reactive protein (CRP).
Complementary, the clinical characterisation for the cardiovascular phenotypes addressed in BestAgeing: Acute Coronary Syndrome (ACS), Coronary Artery Disease (CAD), Heart Failure with preserved ejection fraction (HFpEF), Dilated Cardiomyopathy (DCM) and Systolic Dysfunction as well as for “healthy” controls was re- evaluated and defined in the beginning of the project.
The third main objective aims to establish specific validation concepts for each omics-entity considering their specific needs in preanalytic and analytic processes of samples as well as statistics. Validation of the selected omics-panel in disease cohorts and controls will be performed at the responsible partner sites. Applied methods include qrtPCR, Luminex, next-generation sequencing, Fluidigm, Massarray, Infinium, mass-spectrometry, and immuno-assays. For all markers sensitivity, specificity, AUC and predictive power were assessed. Diagnostic and prognostic performance of novel markers has been benchmarked for diagnostic classification against current gold-standard (e.g. troponins for ACS, natriuretic peptides for HF, imaging phenotypes, etc.), for risk and outcome prediction against defined primary and secondary endpoints and for treatment monitoring against clinical scores and advanced clinical phenotyping/imaging. Another result generated in WP1 is the development of the basic requirements for validation concepts and for diagnostic and prognostic studies which are available for the entire consortium. WP1 will advise all project leaders in planning and implementing retrospective and prospective cohorts including basics of ethics requirements which need to be implemented at different BestAgeing partner sites.
Establishment of a consortium database for data integration and analyses (objective 4) is a pivotal objective for successful multi-center studies in BestAgeing. The database contains a minimum and extended dataset for the phenotypes and the application is deployed in a secure datacenter where all groups of the consortium have access to. In the first two periods the database was developed, tested and redefined and is from this time on fully operational. In the second reporting period the database could be finalized after the evaluation of the consortium. All groups of the consortium have access to the data base, which is accessible via the web: https://www.bestageing.org. This final version includes last revised definitions of the markers, diagnoses, studies, diagnostic complementary tests, events, etc. A regularly update is done over the entire project life-time and will be done beyond this. Up to date 2.393 patient with their respective clinical data are listed.
As this work package sets the basis for the collaborative work in WP2-8, all objectives and tasks were imbedded in the first two years of the project term. All objectives and tasks have been successfully fulfilled.
Work Package 2 - Standardization, quality control, exchange of biomaterials
In the field of omics-biomarker research, standardization and quality control (QC) are essential prerequisites for successful validation of biomarkers by the different technologies. The on-going quality control of the biomaterials and analytical procedures is central to obtain the best possible results. The BestAgeing validation concept consists of two main components: the retrospective (i.e. existing) cohorts and prospective (i.e. newly to be recruited) cohorts of the Beneficiaries. Open exchange between all partners of expertise, experimental results on pre-analytics and discussion led to the definition of a uniform procedure for sample collection, handling, processing and storage aimed to obtain the maximum number of –omics analyses with a minimum number of different matrices, aliquots and total volume for the prospective cohort. This guaranteed both clinical validation of a high number of new –omics biomarkers for cardiovascular diseases and the ethic requirement of a maximal amount of blood to be obtained from any single patient.
The first objective is the standardization of sampling and storage conditions as omics-biomarkers include fragile molecules such as RNAs or metabolites. In the first year Standard Operating Procedures (SOPs) for all processes including collection, preanalytics, shipping, storage and analysis of samples were defined. The second objective specifically aims to establish stable and robust clinical routines for RNA-based biomarker analysis from whole blood and serum. According to the working plan, the sample group investigates different sampling, extraction and storage protocols and established a SOP valid for the BestAgeing consortium. Objective 3 focuses on quality control of biomaterials and data integrity quality control of sampling, storage, and assessment of the condition of biomaterials. Therefore the samples were first checked centrally at the Heidelberg CardioBiobank upon arrival. To investigate center specific effects the prospectively collected samples have been analyzed with a metabolite based quality assay performed by partner MTXH. Beside that specific assessment of the quality of samples was also done at the different sites where the respective omics-technologies were applied. Results were integrated in the website (intranet) and regularly assessed for completeness and data integrity. Exchange of biomaterials represents objective four in WP2 to setup rules for data collection, pseudonymization of patient data and biosamples, and organization of sharing of biomaterials and data. In the course of the project the consortium decided that the Heidelberg CardioBiobank (HCB, part of the SMCU) takes over the responsibilities for the storage and shipping of all samples. Therefore the HCB developed SOPs for the on local storage of the samples and organized certified shipping processes to Heidelberg and later on to the analyzing partner sites. The consortium benefited from the long-term experience in handling and processing of biomaterial and of the basically existing SOPs and quality control mechanisms. Furthermore the HCB provides comprehensive and state-of-the-art infrastructure in Biobanking. This includes experienced study nurses, technicians, documentalists and scientific staff as well as cutting edge technical equipment such as custom specific robotics cryotank for >1 Mio samples, robotics for sample aliquoting, nucleic acid extraction etc.. As WP 2 is fundamental for standardization processes and therefore for the reproducibility of results, all objectives has been successfully fulfilled. All in all, the extensive work on the final definition of SOPs for standardized pre-analytics, a pre-requisite to ensure the highest quality for -omics based biomarkers discovery using prospective cohorts of patients leads to detailed insights of the consequences of preanalytical processes. This elaborately work have been published in 2017 in a peer review scientific journal thus reaching the objective to share our knowledge with the scientific community (Basso D, Padoan A, Laufer T, Aneloni V, Moz S, Schroers H, Pelloso M, Saiz A, Krapp M, Fogar P, Cornoldi P, Zambon CF, Rossi E, La Malfa M, Marotti A, Brefort T, Weis TM, Katus HA, Plebani M. Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling. Clin Biochem. 2017 Mar;50(4-5):186-193).
Work Package 3 - Assay-technology development
Usually for omics analyses highly expensive and complicated analytical methods and equipment is required. BestAgeing not only aims to validate novel omics based biomarkers in large multicenter studies but also to develop analytical tools and assays to ultimately asses these markers in a clinical setting. In WP3 SME and industrial partners have been working on assays for miRNAs, (epi-) genomics, and metabolomics assays.
In the first and second funding period the assessment of various technologies for miRNA validation and quantification have been made. As result of the testing, Multiplicom selected the FireFly Bioworks method as the most flexible and efficient method for quantitative analysis of miRNAs in a clinical setting. In collaboration with partner 11 (ACSB), a first custom Firefly panel has been composed and tested for miRNA biomarker validation. In addition, to the versatile and easy-to-use Firefly Bioworks the BestAgeing partner HBx (CBC) successfully implemented, validated and optimized the high-throughput qRT-PCR platform Fluidigm Biomark HD. Ideally suited for the high throughput validation studies this platform provides excellent complementation to the Firefly Bioworks technology and additionally facilitates the immediate translation of the microRNA biomarkers from the validation studies to installed qRT-PCR platforms in most (clinical) labs.
In the third funding period the above mentioned systems were used to analyse the first sets of retrospective and prospective patients samples. Thus HBx successfully implemented and validated Fluidigm’s high throughput qRT-PCR (Biomark HD) technology for microRNA biomarker measurements of liquid biopsy samples. Also optimized protocols (SOPs) for the high throughput qRT-PCR microRNA assessment of body fluid samples have been established to facilitate immediate engagement into the analyses of validation cohorts. Robust and reliable protocols for the processing of different sample materials (i.a. Serum, Plasma and PAXgene samples) and RNA input amounts have been developed and validated on the Fluidigm HT-qRT-PCR Platform.
In addition to the Firefly Platform and on-going in house developed technologies, Multiplicom has investigated the miRNA multiplex quantification capabilities of the MyCartis technology platform, a novel technology acquired in house during the first trimester of 2016.
Siemens developed a first high level extraction system concept for the isolation of miRNA: In order to provide rapid information on the miRNA status of AMI (Acute Myocardial Infarction) – suspected patients, a de-centralized and automated system has to be provided. Only a minimum of hands-on procedures would be acceptable for such a rapid test and the turnaround time should be in the range of one hour or even shorter. Siemens developed an automated sample preparation module with a disposable cartridge using Siemens Versant chemistry and an automated miRNA detection module based on a double strand ligation assay on a CMOS-chip. The feasibility test on a miRNA POC-test format was performed with both modules, respectively. The results showed the miRNA extraction took about 30 minutes and yield of miRNA was sufficient for the CMOS-chip based detection; the miRNA detection had a time to result of 30 minutes and detection limit was up to 1 pM with a dynamic range between 1 pM to 1 nM. The developed methods have been led to several patent applications.
Work Package 4 - Validation of protein and reference biomarkers in elderly
This WP focuses on the role of protein biomarkers in the diagnosis and prognosis of cardiovascular diseases. For many years proteins have been the major source for informative circulation biomarkers. They are generally easy to measure and are excellent tools for discriminating between healthy and diseased subjects and for risk prediction. In recent years many other molecules such as non-coding RNAs and metabolites have been identified as promising novel classes of biomarkers. Proteins however, are still the gold standard biomarkers for many diseases including cardiovascular diseases. Therefore the SME partner ACSB has validated the diagnostic and prognostic value of several novel proprietary single protein markers aimed at improving further stratification of heart failure patients into meaningful clinical entities. Before multimarker panels for diagnosis and prognosis of heart failure can be established, individual (novel) biomarkers have to be validated separately in discovery cohorts. We have validated formin-binding protein 1; amyloid precursor protein; collagen type IV alpha 1; and RNA binding motif protein 20 in two respective discovery cohorts, which allows the combination of these markers with established markers Galectin-3 (Gal-3) and NT-proBNP for the diagnosis and prognosis of heart failure. Individual biomarkers have been validated in larger patient cohorts but before a third discovery cohort was chosen from the available cohorts from the BestAgeing consortium to confirm previous protein biomarker measurements. To allow valid comparison between patient and controls, samples were chosen from partner institutions SERMAS (Madrid) and AMC (Amsterdam) that had both collected biosamples from both patients as well as control persons. In addition to the biomarkers Gal-3; FNBP1; APP; COL4A1 and RBM20, NT-proBNP have been measured if these data are unavailable from cohorts. In addition, a subset of controls from IMCB (Tartu) has been validated to be used as controls for larger patient cohorts.
A further task focused then on the generation of a combined protein and miRNA multi-marker biomarker panel. Predicting the prognosis of individual heart failure patients is vital to individualize treatment. At present, many classification models for heart failure have been proposed. In these models, demographics (age and sex), severity of the disease, renal insufficiency, blood pressure, diabetes, sodium are among the most frequently used parameters for HF risk stratification. However, these parameters, alone or combined, often fall short of accurate prediction or are too elaborate to be widely clinically implemented. Therefore, additional simple and objective measures to stratify HF patients according to their risk are needed. Biomarkers, such as natriuretic peptides (NT-proBNP) myocardial stretch) or high-sensitivity cardiac troponins (markers of myocyte injury) have shown value to refine risk prediction in heart failure. MicroRNAs (miRNAs) are small non-coding RNAs that influence protein translation and represent a level of biologic regulation that was essentially unknown a decade ago. To this end both previously identified and validated miRNAs and protein biomarkers were analyzed in two large prognostic heart failure cohorts.
Partner ACSB also used first cohorts to assess the importance of novel ASCB proprietary biomarkers to differentiate between controls and HF, and more importantly, to differentiate between different stages of HF. The group in Heidelberg has already addressed the effect of age on diagnostic and prognostic performance of high-sensitivity cardiac troponin T (hs-cTnT). Recently, the Katus group showed that elevated hs-cTnT is more common in the elderly due to higher prevalence of non-ACS conditions and significantly impairs diagnostic performance in discriminating non-ST-segment elevation myocardial infarction. In a recent study in 2015 the prognostic value of elevated hs-cTnT levels in a low risk outpatient population with cardiovascular disease was investigated. Also in this cohort, both in univariate and in multivariate analyses, hs-cTnT was independently associated with age and gender.
During the project, partner UNIPD established the reference limits of the two new protein markers TWEAK and HMGB1 and verified that in healthy subjects they are not influenced by variations in anthropometric parameters, gender, age or biochemical tests of liver and renal function, and of heart failure. These findings support further analysis in patients’ cohorts.
Work Package 5 - miRNA biomarkers for CAD, ACS, and HF in elderly
Main Focuses of WP5 was the validation of already identified single markers or miRNA signatures in appropriate cohorts and biomaterials from the BestAgeing consortium and compare these with established biomarkers and clinical variables.
One of the tasks in this this WP was the development and validation of miRNA biomarkers expressed in the blood cells of whole blood samples collected in PAXgene Blood RNA tubes. Thus, the miRNA-signatures derived from this approach relate to the expression of miRNAs in all blood cells, including erythrocytes, leukocytes and thrombocytes. Utilizing a first discovery cohort of 15 CAD-patients and 15 CAD-controls patient samples were analyzed on whole-miRNome Agilent microarrays resulting in 23 miRNA-biomarker signature candidates for diagnosis of CAD. HBDx validated these candidates on both Fluidigm high-throughput qPCR platform and standard StepOne 96-well qPCR platform for comparison using TaqMan chemistry and HBDx’s established qPCR SOPs. Unfortunately, 11 out of 23 microRNAs could not be detected on Fluidigm and 9 out of 23 microRNA could not be detected on StepOne platform. These results may be due to huge chemical, physical and technical differences between the microarray and qPCR based method. Based on this results and consideration of costs and benefits it was decided to generate genome-wide miRNA profiles using Agilent’s SurePrint microarray technique in the case study. HBDx has gained great expertise for Agilent’s microarray technology over the last decade and is repeatedly certified by Agilent for microRNA profiling using Agilent microarray slides. Additionally, HBDx is ISO 17025 certified by the DAkkS (Deutsche Akkreditierungsstelle GmbH) for the complete miRNA profiling process (RNA extraction, quality control and Agilent miRNA profiling). For miRNnome microarray screening study HBDx received PAXgene samples of 536 CAD-patients and 175 CAD-controls from BestAgeing partners in total. HBDx extracted total RNA from these samples and performed quantification and quality control of corresponding RNA eluates. Finally, 511 CAD-patient and 168 CAD-control samples (679 in total) were measured on Agilent microRNA microarrays (comprising 2,549 in miRBase v21 published microRNAs) for microRNA profiling. Raw data files of all measured samples were transferred to partner USAAR for bioinformatics analysis (WP8).
Using EDTA plasma blood taken from the coronary venous sinus (CVS) as miRNA-source, Partner GUF analyzed ACS samples on Agilent’s SurePrintG3 Human miRNA (8x60K) microarrays leading to a selection of a panel of 6 miRNA biomarker candidates for further validation. For validation CVS blood of a cohort of 17 patients suffering an ACS vs. 17 controls via TaqMan qRT-PCR resulted in a panel of 4 miRNAs with robust diagnostic information to identify ACS. To test the prognostic ability of this panel, the 4 miRNA were determined in EDTA plasma of 198 patients with ACS including 49 patients who died within 200 days in a case control setting. Herein, the panel showed a significant association with prognosis, amendatory to the clinically used TIMI risk score with comparable magnitude to the established biomarker BNP. For validation of the diagnostic potential of these 4 miRNA biomarker candidates will be carried out from the prospectively collected ACS patient samples within the BestAgeing consortium in further studies.
Partner ACSB focused on the validation of miRNA biomarkers for heart failure (HF). Twelve circulating miRNAs were previously either identified in plasma from patients with HF or selected based on their presence in the myocardium. These biomarkers have now been validated in plasma samples from a large cohort consisting of 1380 patients with chronic heart failure. To understand the extent of normal variation of circulating miRNA variation in general population, 268 healthy control individuals has been selected from the Estonian Genome Center Biobank (partner UT) and determined the expression levels as well as expression variability of circulating miRNAs by QIAGEN Human & Plasma miScript miRNA PCR Array.
Additionally Partners UKL-HD and HBx successfully fractionated whole blood samples into the cellular components most relevant for ACS and HF and analyzed the miRNA-profiles of these blood cell subfractions by next generation sequencing. Herein, a method for analyzing miRNA-profiles in 6 leukocyte blood cell subfractions by combining antibody-based microbead technology and next generation sequencing was established and respective miRNA biomarkers have been identified.
UKL-HD and HBDx established the study protocol for validating miRNA-biomarkers in ACS-patients. Herein the input from the other clinical recruitment centers were integrated, to result in a study protocol useable by all partner for sample collection.
The successful collaborative work in WP5 is proven by the seven publications in peer review scientific journals.
Work Package 6 - Validation of metabolomic biomarkers for HF in elderly
The main focus of Work Package 6 was the validation of previously identified and IP-protected biomarkers from metabolite profiling analyses in Heart Failure patients. Within BestAgeing these metabolomics biomarkers have been validated in large multicenter European cohorts in the group of elderly patients. Specific study synopses have been designed to address the most relevant clinical use cases by WP6 leader Metanomics Health (MTXH), University Hospital Heidelberg (UKL-HD) and the entire consortium.
The novel lipid biomarker panel (Cardiac Lipid Panel; CLP plus NT-proBNP), developed by Metanomics Health, in combination with the established marker NT-proBNP, together with the newly developed simplified analytical method has the potential to facilitate the early diagnosis of systolic heart failure in both outpatient and clinical settings, enabling a timely treatment of the patients. In total, MTXH has analyzed so far 4069 human plasma samples derived from various heart failure-associated cohorts, and present metabolomics data successfully confirm the added value of the novel metabolic biomarker panel called MxP® CLP for the improved diagnosis of heart failure with reduced ejection fraction. The added value of Metanomics Health’s MxP® CLP for the diagnosis of heart failure with reduced ejection fraction was also confirmed by our key opinion leaders. MxP® CLP biomarker is able to reliably diagnose heart failure with reduced ejection fraction at an earlier disease stage. As a consequence, four-year survival rates are expected to become significantly shifted upwards to nearly 90% in the future. Through the earlier detection and improved risk stratification, admission rates and secondary diseases might be reduced, leading to a considerable decline of associated healthcare costs in the future. From a cost perspective, the biomarker-based decision for patients at-risk with a subsequent echocardiography - confirming the diagnosis of a reduced left ventricular ejection fraction (<50%) - is advantageous in comparison to the currently prevailing diagnostic methods. Thus far, all available data/studies with regard to the MxP® CLP biomarker performance indicate that this will hold true for the combination of CLP and NT-proBNP.
Work Package 7 - Genetic and epigenetic markers for HF diagnosis and risk assessment
Heart Failure (HF) shows remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of HF, either by genetic or epigenetic factors that may interact with environmental stimuli. Work Package 7 aimed to validate genetic but also epigenetic biomarkers identified in previous studies and dissect their value for diagnosis, risk stratification and therapy monitoring. We previously detected DNA methylation differences and genetic variants in several pathways related to HF, but also in genes with yet unknown function in this context. Since DNA methylation was previously accessed in myocardial tissue, we also aimed to identify correlating markers (cell-free DNA, blood DNA) in peripheral blood. This is not an issue for genetic markers (rare and common variants) that are routinely assessed through peripheral blood.
To study the impact of common genetic modifiers on heart failure risk prediction two GWAS (Genome-wide association studies) have been performed by BestAgeing partners including n = 5279 patients with HF (non-ischemic) and n = 8708 controls. This represents to our knowledge the largest study population on the genetic factors in HF so far. By using Affymetrix and Illumina genotyping arrays, the individual substudies obtained genome-wide SNP data. Using Taqman genotyping, candidate loci were validated in several replication cohorts. We found out that over 3000 individual protein coding transcripts are influenced by genetic variation, which is a remarkable finding. Furthermore a set of new loci in exonic regions those contribute to DCM and serve as additional risk alleles in a combined score have been identified.
The process of identification of methylation markers was performed in several stages. On the one previously identified markers have been tested in fcDNA and whole-blood. On the other hand, we have added additional markers identified in a larger screening cohort and by high-resolution methylation measurement (450,000 vs. 27,000 methylation positions). Specific isolation protocols for DNA (and RNA in parallel) from biopsies and from whole blood were developed and established in cooperation with Qiagen (Germany). ). This approach provided us information about genome-wide methylation of CpG methylation sites in promoter, transcription factor binding sites and enhancers / Isolater regions. It was also possible to validate successfully a panel of epigenetic biomarkers in peripheral blood that show superior performance compared to natriuretic peptides.
The successful collaborative work within this WP is proven by more than ten published scientific articles within the project duration.
Work Package 8 - Development of multi-marker strategies and economic impact modelling
The main focus of Work Package 8 is to develop strategies and provide tools for analyzing multi-omics data sets for the consortium to establish standardized and easy-to-apply tools for clinicians related to the different clinical use cases. In the first part of the project WP8 focused on the stability of markers and translation towards clinics. Therefore the cell type specificity, the clinical application platform and the stability with respect to transportation and batch effects have been considered. The gained respective information has been added to our knowledge management system (miEAA) for assessing the validity of biomarkers. Besides confounding factors such as age and gender now also other disease associations, stability of the biomarkers with respect to transportation as well as cell type specificity can be assessed. miEAA is freely available to all consortium members as web service.
As a statistical and bioinformatics tool the WP 8 leader developed a software for evaluating biomarkers within the BestAgeing consortium. The available solution integrates simple basics such as power calculations for single and combined markers over standard diagnostic performance measures such as AUC values up to complex multi-marker classification systems. As decision trees are powerful models for classification tasks, WP8 designed a decision tree based approach. Besides a high performance of these classifiers they can displayed graphically as flow chart representing a set of rules on which basis the prediction should be done. This allows physicians to evaluate diagnostic multi marker approaches in much more detail as compared to e.g. SVMs. Our solution allows for handling mixed types of input data, i.e. the input may be numeric (e.g. gene expression) or categorical (e.g. presence or absence of specific single nucleotide polymorphisms). Beyond this, even image-based features can be integrated. Furthermore, the model handles missing values in case that a certain parameter is not available for a patient.
Another major goal of WP8 is the development of a concept for multi-marker strategies. While the task of combining quantitative (or pseudo-quantitative) markers to patterns has been already successfully achieved in earlier project phases partner USAAR focused in the last year on combination of different omics entities. This effort starts with a new approach for combining the information content of genetic aberrations on small scales (e.g. SNVs, small in/dels) but also considering larger genomic aberrations. The respective approach relies on decision trees (manuscript in preparation). Beyond, they implemented complex statistical models for inferring marker cascades, not only including genes but also messenger RNAs, miRNAs and proteins. A manuscript describing the stoichiometry-based approach is in review.
The software that has been implemented in the project is used by up to 800 individuals per week, on average over 2,000 researchers worldwide are working with our software per month. Altogether, over 1,000 research organizations, most of them in Germany, the UK and the USA are analyzing data or performing studies with the respective tools. This makes the dissemination of BestAgeing results a huge success and we will work further to ensure long time sustainability and availability of the implemented solutions.
During the project we already published 25 manuscripts with data from the BestAgeing project. 5 manuscripts with data and resources from BestAgeing are currently under review or under revision. Moreover, 3 manuscripts are currently finalized.
Work Package 9 - Project Management and Coordination
The main responsibility of Work Package 9 is the coordination and management of the consortium. In the implementation phase WP9 was responsible for the draft, negotiation and final agreement of the Consortium Agreement. A second achievement of the first funding period was the fast and coordinated distribution of community funds to the beneficiaries including some participants where special clause 10 had to be considered.
As an all-day available support service for all project partners, the SMCU coordinates and evaluates the scientific activities, interactions and progresses within the consortium. Furthermore the SMCU takes care of the administrative and legal issues of the consortium as a whole and also of the regulations / requirements of certain partner sites. This includes assistance and guidance on staff hiring regulations, financial controlling, reporting processes (deliverables, annual reporting), organization of meetings and telephone conferences and public relations.
The second and third funding period was focused on planning and starting prospective studies. Unfortunately the start of the recruitments at all clinical partner sites were delayed because the ethics committees from several partner sites came up with specific requests which were unforeseen as the studies are planned as observational studies. In order to fast and efficiently solve the various issues with the different local ethics application the Coordinator took the role as trial leader and supported the partners in their local implementation of ethics votes. The coordinator and her group then invested much effort to provide “master ethics application” documents for ethics application, patient informed consent, patient information etc. and to preparing partner specific drafts of the before mentioned documents and to support the partners in the negotiation process of all ethics related issues.
WP9 was also responsible for establishing the project website and web-based tools to support the scientific (periodic) report as well as the financial reports (form C). The dedicated website (www.bestageing.eu) is fully operational since June 2013 and is regularly updated by the SMCU. The website provides comprehensive information on structure, aims and current achievements of the project (including publications) for scientists, journalists, patient groups and the general public. Besides the public part the website also contains a secure internal part restricted to participants. This password protected intranet section provides a comprehensive document repository containing all relevant documents such as the DoW, the Grant Agreement, the Consortium Agreement, meeting protocols, agendas as well as the large number of specialized SOPs developed by the consortium, deliverables and the periodic reports.
During the project time a further task has been added: the organization of the biomaterial transport and storage, as well as the quality control became also part of the SMCU. The consortium decided to store the biomaterial at the Heidelberg CardioBiobank (HCB) which now operates as a trustee for the BestAgeing samples. As well as the SMCU the HCB is also headed by Dr. Tanja Weis and therefore the BestAgeing consortium decided to benefit from the knowledge and experience of the SMCU and the HCB. The HCB provides comprehensive and state-of-the-art infrastructure in Biobanking. This includes experienced study nurses, technicians, documentalists and scientific staff as well as cutting edge technical equipment such as custom specific robotics cryotank for >1 Mio samples, robotics for sample aliquoting, nucleic acid extraction etc. Within this additional duty, the scientific staff of the SMCU developed BestAgeing specific SOPs for all recruiting partner sites containing the handling and sampling of the biomaterial, the shipping and the labelling of the samples to ensure equalized and optimized standards for all partner sites.
Potential Impact:
The aim of BestAgeing was to use advanced biotechnology and the joint expertise of a consortium of expert clinicians and scientists to improve the health of elderly patients in Europe. Up to date, most efforts to translate omics-biomarker candidates into clinical practice have had little impact on patient care. This is partly explained by the small size of most studies, but also reflects a failure to integrate clinical, basic research, technological, and bioinformatics expertise from academia and industry into coherent and comprehensive biomarker development strategies. With its multi-disciplinary integrated translational approach, BestAgeing can overcome these limitations by 1) capitalizing on innovative omics candidates identified by the members of the alliance, 2) bringing together academic and industry partners with the necessary methodological expertise, 3) applying and validating the novel markers to population and patient cohorts all over Europe, and 4) accelerating bench-to-bedside translation by highly synergistic work packages.
1.4.1 Medical implications
BestAgeing impacts on the most prevalent disorders in the elderly. In industrialized countries more than one of three adults have one or more types of CVD and by 2030 it is for example projected that 40.5% of the US population will have some form of CVD. In male and female subjects aged 60-79 and 80+ prevalence of CVD in 2008 was at an impressive rate of 72.6/71.9% and 80.1/86.7%, respectively. Analyses of Framingham Health Study data among participants free of CVD at age 50 years furthermore showed a lifetime risk for developing CVD was 51.7% for men and 39.2% for women. It is in addition projected that heart failure will become an epidemic disorder in Europe due to the success of acute cardiac care and increased life expectancy following acute cardiac events. Thus CVD is a major threat to public health in ageing Europe and there is an urgent need to prevent cardiovascular events or to initiate effective treatment as early as possible. BestAgeing aimed to detect CVD earlier and more precisely for a better care of patients and to identify subjects at increased risk with a high predictive power to initiate preventive and treatment measures against acute and chronic CVD.
Furthermore the proper diagnosis of a disease is the prerequisite for any therapeutic intervention and thus the quality of diagnostic tools determines the success of preventive and curative therapies. For example after we introduced the cardiac troponin T (cTnT) assay, 25% of ACS patients had to be reclassified from unstable angina to NSTEMI. If these newly identified NSTEMI patients were then treated according to the guidelines, it is estimated that 17,000 lives are being saved per year in the US alone. The further refinement of the sensitivity of the troponin assays by us and others (high sensitivity troponin) enabled the detection of an additional 10% of patients with NSTEMI amongst patients with suspected ACS. Guideline adjusted treatment for these patients reduced death and MI rate from 39% to 21% within 1 year (Mills et al. JAMA 2011). Similar data are also observed in heart failure cohorts. Thus by use of the high sensitivity cTnT assay an additional 25% of high risk dyspnea patients can be detected, which are missed by the conventional cTnT assay (Jacobs et al. Clin Chem 2011). These findings highlight the indispensable value of precise diagnostic modalities for optimal patient care and a better patient outcome.
In the recent years substantial technical progress was achieved in the dissection of complex biological systems by omics technologies. The comprehensive analyses of e.g. the metabolome or miRNome in blood have provided unexpected clues to the existence of disorders and their functional implication. These novel biomarkers will expand the diagnostic armamentarium beyond conventional protein markers and hold the potential for added diagnostic value. The BestAgeing consortium was assembled to bring novel omics-based diagnostic modalities into clinical practice to better address the following clinical scenarios: First and most importantly we expected that the selected omics-markers will enable a more precise and more sensitive diagnosis of sub-clinical CVD as compared to available diagnostic tools. While measures for primary prevention are often ignored, diagnosis of affection status- even in early disease stages - is an accepted starting point for treatment and life style changes. Second we anticipated that the novel markers will enable an earlier diagnosis of disease progression and by that indicate an increased risk and urgent treatment. Thus for example plaque vulnerability may be detected before vessel occlusion by miRNAs from blood cells or heart failure may be identified by metabolic signatures in subjects with yet BNP negative myocardial dysfunction. Third it was estimated that the omics marker results will aid in the selection and intensity of treatment strategies since earlier detection of reversible ischemia or heart failure by genomic markers, epigenetic alterations, circulating proteins, metabolites or miRNAs will mandate a more aggressive diagnostic work up and optimization of treatment. Fourth, by analyses of multiple markers such as DNA, miRNA, metabolites and proteins reflecting different molecular disease signatures and cellular adaption processes, substantial information will be obtained on the underlying myocardial disease and its systemic impact, aiding risk stratification and therapeutic decision making.
The next aim was to improve risk stratification for heart failure in the ageing populations. Heart failure prevalence increases markedly in the elderly and at present is the most frequent admission diagnosis to internal medicine hospitals in subjects >65 years of age. Cardiomyopathies, which are genetic disorders in a large proportion of patients, account for 30-40% of all heart failure cases. In previous work, we could identify genetic variants associated with a markedly increased risk for progressive myocardial dysfunction, which may be relevant to the progression of any form of heart failure. Therefore in it was planned to use next generation and high throughput DNA sequencing to seek for mutations in known disease genes of heart failure and known genetic risk variants. It is predicted that the proper identification of subjects at high risk for heart failure and disease progression will lead to better patient care and improved outcome.
Despite the major contribution of evidence-based medicine derived from large randomized clinical trials for an improved patient outcome, it is important to better account for the individuality of the affected subjects and their specific treatment response. Thus, for example in oncology only a third of the patients do respond to a specific therapy. Importantly, in these patients cause specific treatment and monitoring with companion diagnostics proved to be a major step towards a better patient care with improved treatment response and outcome. In BestAgeing we tried to better describe the individual “molecular background” of subjects at risk for CVD and provide clues as to the underlying causes of the disorder. It should also be possible to better assess the individual (mal)-adaptive response to CVD and predict treatment response by omics technologies. Thus the multi-marker approach proposed in BestAgeing tends to enable a better description of disease processes as well as individual response to injury and treatment. BestAgeing thus aims to promote novel strategies for a more individualized therapy in the care of CVD and elderly subjects.
The analyses of disease associated patterns of different omics-markers which has been proposed by BestAgeing should provide novel strategies in biomarker validation and clinical chemistry testing, but also generate novel hypotheses on disease mechanisms, dys-regulated pathways and potentially useful therapeutic targets in CAD, ACS, HF, and healthy ageing. Therefore the academic groups within BestAgeing share the data generated within the consortium with each other and the scientific community and attempt to dissect novel disease pathways and treatment options under alternative research programs in cooperation with biomedical industry. The novel omics-based biomarkers should provide clues to the biology of ageing as exemplified by the age associated variation of expression of distinct miRNAs. The analysis of these molecular markers in large cohorts enables to compare patients of different ages and to reveal ageing-related physiological and pathophysiological changes. The omics profiles may also identify similar dysregulated pathways in clinically distinct syndromes such as heart failure and chronic coronary artery diseases but also non-cardiac disorders like inflammatory diseases as indicators of commonalities in disease mechanisms. Thus it is predicted that BestAgeing will deliver new targets for treatment of CVD and generate novel metabolomic, genomic, epigenomic and miRNomic data in ageing.
Systems biology approaches have gained particular interest in the last decade since they aid in the discrimination of relevant from irrelevant data obtained in complex biological systems. In many cases, comprehensive systems biology techniques require for profiling datasets from diverse omics-technologies, connecting the different systems of cellular regulation and identifying connected components with key biological relevance. The biomarker candidates validated in BestAgeing were in part retrieved by using such techniques, which lead to the identification of promising markers reflecting key disease processes in CAD, ACS and HF. Although the data we will assess in the validation experiments only represent a smaller subset of the complexity of the cardiovascular systems, by combining those already proven as relevant datasets we could address possible interactions between genomic, epigenomic and transcriptomic regulatory pathways derived from systems biology studies.
Individual genetic background and life style, which strongly affects epigenetic mechanisms and metabolism, is not taken into account for individualized treatment and therapy control for CVD. During the last years, significant insight into the role of genetics and gene regulation could be achieved. In parallel the role of genomics, epigenomics, transcriptomics, metabolomics and miRNomics for the individual disease has been recognized. Additionally, genome wide association studies and functional genomics analyses gained exciting insights into causes and mechanisms of individual susceptibility to therapy response or the range from non-response to hypersensitivity, respectively. Our scientific approach comprises different omics technologies that built the base for future achievements in companion diagnostics by biomarkers indicative for therapy response or failure. We are convinced that some of our omics based biomarkers or their combination will significantly contribute to improved clinical phenotyping, earlier diagnosis, improved individual prognosis and more precise outcome prediction of ACS, CAD and HF in elderly and ageing patients. We are also confident that this work will promote omics-technologies in general and systems biology studies of CVD in particular and underline their usability and value for translational research.
1.4.2 Structural impact
Concerted action against CVD: The BestAgeing consortium brought together scientists, physicians and businessmen from different European regions each experienced in distinct fields such as omics research, test development, epidemiology, and clinical research. Although bilateral cooperation amongst many members of BestAgeing has existed for many years, the consortial structure was unique and enabled not only to fulfil the goals of this project but also stimulated basic and translational science of each individual member. Thus we have a close interaction of the experts in genome, miRNom, metabolome and protein analysis and with this expertize we harmonized the sampling, storage, and analytical strategies across the different omics-fields. The interaction of SME with academia is crucial for a successful translation of a test development into clinical practice, which requires consideration of not only technical feasibilities and business options on one side but also medical needs and clinical workflow on the other side. We also experienced the incremental value of an early synergistic developmental approach of SME and big diagnostic industry as implemented in this consortium and the significance of detailed knowledge in the different fields by each of the academic member. Thus the concerted action of this consortium covered the entire spectrum of technologies, biomaterials, basic sciences, clinical medicine and business is not only benefitting research of each participant but is required for the translation of omics-biomarkers into clinical practice.
One goal of the BestAgeing project was to provide a unique collection of patient cohorts and omics-grade biomaterials. High-sensitive omics-technologies have enabled completely novel ways for biomarker identification, test development and validation. However, each omics-technology has specific pre-analytical requirements on sample quality, preparation, storage and processing. The required quality of input material is a limitation to the broader application of these technologies to disease cohorts. BestAgeing has long-standing experience in omics-technologies and hence each partner could contribute a high number of biosamples from retrospective cohorts with “omics-grade quality” to the project. However, we recognized that the pre-analytical requirements of most omics-methodologies needed to be standardized and adjusted for routine clinical use, which solved within this project. Hence, we also saw the need to recruit elderly patients prospectively to investigate under optimal pre-analytical conditions the different omics-markers in the same well-characterized individual. Therefore we recruited with low additional costs European elderly-cohorts with high quality clinical phenotyping and “omics-grade” biomaterials, which could be shared with others. The established SOPs and methodologies for pre-analytical sample management and quality control procedures was available for all BestAgeing partners and the scientific community to leverage the potential of omics-biomarkers for all disease entities.
During the last years, a wealth of high-throughput technologies has emerged allowing the rapid parallel analysis of a multitude of molecules in complex mixtures. Substantial work has led to the validation of distinct genomic, epigenomic, proteomic and metabolomic markers or signatures in proof-of-principle studies. However, despite substantial progress in precision and turnaround time of high-throughput technologies the translation of omics into clinical practice was far from reality. For clinical use it is required to reduce omics results to a manageable number disease specific or risk predictive variables or to provide convenient algorithms for rapid analyses of complex data. Furthermore in clinical care robust and optimally standardized analytical test systems are needed, which provide the relevant results in a clinically useful turnaround time. In the WP2 and 3 our consortium focused on the optimization of available analytical techniques and on the development of novel assay formats for omics-derived biomarkers and their validation. This is an important part of our translational research, which benefits from the close interaction not only of the SMEs and the academic groups with long lasting experience in test development and validation but also from the ongoing interaction with big diagnostic industry, which is a prerequisite for a successful implementation of omics biomarkers in clinical chemistry laboratories. Thus our work in test development and optimization of analytical hardware is crucial for omics based diagnostics in other disease areas as well and will be an important step towards personalized medicine.
1.4.3 Socioeconomic impact
European lead in innovative diagnostics and creation of a network of excellence:
When planning the composition the BestAgeing consortium, we were aware that for a successful translation of novel biomarkers we need to include not only strong, technology driven SME partners, but also incorporate bigger players in the diagnostic industry. As such, we were able to enforce our consortium with Metanomics Health and Siemens Healthcare as active partners that added not only regulatory but also worldwide marketing expertise to the project. This should allow successful translation of diagnostic assays all the way through to implementation into the clinical routine, supported by the networks of a global player. The collaboration between our well-connected European partners also resultes in additional benefits for the consortium and the individual partners. Each of our partners has an outstanding track record in his field and is involved in several European and other international networks. We envisaged active networking inside and outside of BestAgeing to promote best scientific quality and excellence. Such a network of excellence enables a truly translational research and technology driven innovation in Europe. The joining of clinical expertise from different countries and partners dedicated to research for CVD, the identification of novel biomarkers, development and validation of diagnostic modalities, experience in omics technologies and ageing research resulted in a unique research environment.
Healthy ageing is a critical determinant for economic growth and prosperity in Europe. The economical productivity for instance will increasingly depend on the contribution of elderly workers. Already today however CVD accounts for 20.4% of disability adjusted life years (DALY) in developed countries and for 32.8% of all deaths (Murray et al. Lancet 1997). Not surprisingly due to the trend towards demographic ageing in Europe and the increasing prevalence of CVD with age, it is predicted that CVD will remain the leading cause of death in industrialized countries and continue to contribute markedly to morbidity and health care costs. It is therefore paramount to detect affected subjects early, to identify those at high risk for cardiac events properly, and to provide sensitive diagnostic tools for immediate therapeutic interventions.
Considering the rising expenses for health-care, results from health economic research will likely influence medical decision-making in the future. Hence, payers and health care providers in Europe need to be provided with robust information on optimal resource allocation to support healthy-ageing. This ambitious aim can be achieved in BestAgeing, since we have longstanding experience and insights into relevant national and international data sources and economic measures of diagnostic and therapeutic procedures. With our concomitant activities for public outreach, we plan to communicate our scientific work, the results of biomarker development, and the potential medical and economic consequences.
1.4.3. Main Dissemination activities and exploitation of results
The dissemination activities were one major part of WP9 (Project Management and Coordination) beside the task to offer a central project management facility for supporting the interdisciplinary European consortium in various administrative and scientific businesses. This comprehensive work is realized by the existing “Scientific Management and Coordination Unit (SMCU)” at the Department of Internal Medicine III in Heidelberg. At the beginning of the project the SMCU developed a dedicated webpage (www.bestageing.eu) which serves as professional platform for internal and external sharing of information. The website provides a frequently updated, large variety of up to date information on structure, aims and current achievements (including publications) to scientists, journalists, patient groups and the general public. Furthermore a professional information desk and information brochures (figure 1) were designed and realized. The information desk has been presented at least twice per year at the meetings of the German Society of Cardiology. At this meetings clinicians, scientists, nurses and technical assistants as well as patients were informed and had the chance to discuss directly the aims/results and challenges of the BestAgeing project.
Furthermore the SMCU organizes special press releases and printed information brochures for different target groups. To do so, the connection to all established information channels via the press offices of the partners is given. One other important information source for the public community and physicians and scientists is the BestAgeing webpage www.bestageung.eu (Fig.2) . Beside a secure intranet area for BestAgeing partners the website contains a public accessible area providing target specific information including the latest news and research releases. Beside the dissemination activities of the SMCU, several partners presented their results originated within the frame of the BestAgeing project with a poster or an oral presentation at high-ranking international meetings. The successful, constructive and productive execution of the BestAgeing project is also reflected by the numerous (> 37) scientific publications generated within the last years. And it is expected that several publications will follow as the generated data serves as the base for further research activities.
One main goal was the successful identification of omics-based biomarker and the translation of their application in the daily clinical routine. To proof the effective approach of the BestAgeing project, we can present eight patent applications up to date.
List of Websites:
www.bestageing.eu
The ageing of the European population represents a growing health care problem, threatening future economic growth and quality of life of our society. In particular, cardiovascular diseases show a marked increase with age and have remained the leading cause of morbidity and mortality in the elderly. Unfortunately, cardiovascular disorders and their associated risks are often difficult to diagnose in the elderly due to many age-related confounders and co-morbidities, leading to substantial uncertainties in diagnostic classification and therapeutic decision making with huge impact on patients’ outcomes. Hence, there is an unmet need for novel biomarkers to enable a more accurate diagnosis, sub-classification, risk assessment and treatment guidance for both acute and chronic cardiovascular diseases in the elderly.
The BestAgeing consortium aimed to improve this unsatisfactory situation by developing and validating novel omics-based biomarkers that may complement or substitute traditional biomarkers for more accurate or earlier diagnosis, better risk assessment and appropriate guidance of therapies, overcoming limitations inherent with traditional biomarkers – supporting healthy ageing in Europe. By combining leading experts in the field of “classical” biomarker development, high-throughput technologies, omics-biomarker research, research intensive SME and industry, and clinical institutions, we are confident that our efforts led to novel European medical technologies that will improve the efficacy and efficiency of our care for elderly patients, which will also impact on the competiveness of research-intensive European industries and socioeconomic wealth in Europe
Project Context and Objectives:
The ageing of Europe is challenging our health care systems and economic growth. It is estimated that by 2080, the number of people aged over 65 in Europe will have almost doubled from now 28,8 % to 52,3 % of the total population (EC eurostats). Therefore, strategies to improve population health and to increase healthy life years must be a keystone for a sustainable Europe. Cardiovascular diseases are by far the leading cause of morbidity and mortality in industrialized nations, and they soon will become the most prevalent cause of death worldwide. Due to remarkable progress in prevention and acute cardiac patient care, cardiovascular diseases nowadays manifest significantly later in life. As such, the incidence of coronary artery disease (CAD), myocardial infarction (MI) and heart failure (HF) – often occurs in rapid sequence within one patient – increases nearly exponentially with age.
The diagnosis of HF and CAD syndromes in the elderly are demanding due to a high rate of atypical symptoms, overlapping clinical manifestations of comorbid states, and silent disease stages. Confounding age-related alterations in the cardiovascular system such as cardiac hypertrophy or conduction abnormalities often impair interpretation of the electrocardiogram and clinical imaging. Further, results of clinical chemistry with e.g. natriuretic peptides or troponins are more difficult to interpret due to age-related changes in kidney function and muscle mass as well as co-existence of multi-organ diseases, often leading to uncertainties in clinical decision making, hindering effective treatment. Moreover reference values for biomarkers in an aged population are largely missing. Thus there is an unmet need for novel, specific and sensitive biomarkers for diagnosis, risk prediction, and treatment guidance for acute and chronic cardiovascular diseases in the elderly population.
The main objectives of BestAgeing are:
1.) The Validation of novel omics-based biomarkers in elderly patients in Europe.
The BestAgeing project brought together partners which have conducted extensive biomarker discovery studies using current state-of-the-art or even proprietary “beyond state-of-the-art” omics-technologies and so previous work has generated panels of omics-biomarkers that have been available for analytical and clinical validation and assay technologies that enable true translation into clinical application. From these undertakings, several highly promising biomarker candidates have been pursued within the BestAgeing project. First we validated the previously identified omics-biomarkers in available retrospective and prospective cohorts. Additionally, we defined comparative reference markers, statistical validation concepts and we developed a dedicated IT infrastructure which enables a sophisticated analysis of the complex omics and phenotype data. As in the omics-field standardization and quality control (QC) are essential prerequisites for successful validation of biomarkers by the different technologies, we established various Standard Operating Procedures (SOPs) to assure standardization processes for sampling, storage and exchange of biomaterials.
2. Development of novel assays for selected omics-biomarkers.
Although highly efficient screening technologies for thousands of miRNAs, metabolites, and even whole-genomes have been developed in the last decade, only few assays measuring single analytes or distinct features out of the omics-entities are ready for clinical application. For instance, there is currently no methodology available to measure a distinct miRNA pattern in a turnaround time of less than 1h. However clinical decision-making in ACS patients will need reporting of test results within 90 minutes as proposed by ESC guidelines. For chronic CVD conditions, turnaround times of hours to days are tolerated, however even these require technically robust, validated, and certified assay systems at centralized lab facilities. Hence, development and optimization of analytical technologies is clearly required. BestAgeing partners were aware of these challenges and aimed to develop assay technologies that allow fast and precise detection of omics-features. Both the SME and industry partners of BestAgeing have extensive research and development capabilities for biomedical assays. The development of assays and analyzers for dedicated omics-marker testing will also be of benefit for other disciplines. The specific assay technologies shall circumvent existing and unsolved limitations of current methodologies, such as missing analytical robustness, standardization, and turnaround times. Two main goals of the BestAgeing project were the development of new miRNA test assays and the development and optimization of a targeted, quantitative mass spectrometry-based method for the simultaneous quantification of 20-50 metabolite biomarkers. Our partner Siemens Healthcare AG aimed to provide a method which allows rapid information on the miRNA status of AMI (Acute Myocardial Infarction) – suspected patients. It was possible to develop a de-centralized and automated system within the BestAgeing project. Only a minimum of hands-on procedures would be acceptable for such a rapid test and the turnaround time has to be in the range of one hour or even shorter. Thus an automated sample preparation has been developed. The second goal was also achieved: during the BestAgeing project phase Numerious human plasma samples derived from various heart failure-associated cohorts has analyzed applying MxP® CLP +/- MxP® Broad Profiling and MxP® Quality Control metabolomics technology. The analyses of metabolomics data successfully confirm the added value of the novel metabolic biomarker panel called MxP® CLP suitable for the improved diagnosis of heart failure with reduced ejection fraction.
3. Development of multi-marker strategies based on diverse omics and established biomarkers as well as advanced imaging modalities for diagnosis, risk prediction and treatment guidance in elderly patients.
In clinical practice, multiple markers are often analyzed in aggregate to better understand progression of disease processes or to test for the relative contribution of different disease pathways. Thus the combined measurement of myocardial necrosis markers with different kinetics in blood such as myoglobin, CKMB and troponins provides information on the temporal progression of myocardial infarction and on quality of reperfusion. The analysis of markers reflecting different disease pathways such as troponins for necrosis, BNP for myocardial stress and GDF-15 for pro-inflammatory activation provides incremental prognostic information in ACS and CHF. Patients with ACS and both, elevated BNP and troponin, are at a particular high risk for cardiac death and myocardial infarction and benefit most from revascularization therapy. However multi-marker strategies in current state-of-the-art clinical care do not include omics markers or decision support systems. It is expected that the added information inherent to metabolite or miRNA profiles and the genetic and epigenetic variables will improve diagnosis and risk stratification beyond available protein markers. Multi-marker strategies accounting for disease specific changes on different levels may enhance the predictive power over single biomarkers, a hypothesis that will be tested in our elderly HF cohorts. Our aim was not only to test the added benefit of omics in a multi-marker approach, but also to develop statistical methods and computer algorithms / applications that predict the optimal combination and aid in the interpretation of multiple markers from the same or even different omics-technologies. Overall, classical machine learning and network analysis technologies should be explored and novel systems biology approaches developed. Additionally, the gathered information and potential of the evaluated markers should be used to model the potential socioeconomic impact of the novel biomarkers.
Project Results:
Our BestAgeing study designs addressed the most frequent and severe cardiovascular diseases of elderly patients by incorporating the appropriate phenotype and reference cohorts and biomaterials from European populations. The strength of the BestAgeing consortium is our extensive previous work on biomarker identification and validation, our long-standing expertise in technology development and omics-based research, as well as our experience in translational research and test development as exemplified by the invention, the successful translation, and continuous refinement of cardiac Troponin T to a world-wide standard of good clinical practice in diagnosing myocardial infarction and prognosis of cardiac risk.
Work Package 1 - Definition of most promising omics-biomarker candidates and database development
The WP1 comprises four main objectives focused on the most promising biomarker candidates and the development of a dedicated database for clinical and selected omics data of all BestAgeing studies. The first objective is the definition / selection of the most promising omics based biomarker candidates which had been initially identified by the consortium. This selection was based on proven value of the markers (statistical measures in profiling studies, biological replication), previously demonstrated analytical quality (technical reproducibility), diagnostic and prognostic power, IP protection, addressed disease cohorts of elderly patients including the clinical need in previous screening studies of the consortium partners and all related biomaterial and pre-analytical requirements. Based on first results of the validation studies the initial list has revised e.g. markers that were published for proven diagnostic or prognostic value have been added; and markers that turned out to be non-informative (no statistically significant (added) diagnostic or prognostic information) have been eliminated. The major changes are related to the selection of miRNA markers where several new potential markers were added and few deleted in the phenotypes acute coronary syndrome, coronary artery disease and heart failure. Besides the changes in miRNA marker candidates, RBM20 was added as an important and promising candidate in the revised list of protein markers.
Beside the revision of the biomarker candidates list, the selection and definition of reference marker for comparative analyses (objective two) was made. For comparison with the novel biomarkers, BestAgeing defined a panel of already established or currently investigated high sensitive cardiac biomarkers, clinical risk scores, and imaging modalities together with their associated phenotypes that are needed as best standard reference markers and phenotypes for comparative analyses. We have identified, based on current literature and clinical use in routine, three established biomarkers that will be primarily used as biomarker comparators: (1) high sensitivity cardiac Troponin (T and/or I); (2) B-type natriuretic peptides (BNP and/or NT-proBNP); and (3) high sensitivity C-reactive protein (CRP).
Complementary, the clinical characterisation for the cardiovascular phenotypes addressed in BestAgeing: Acute Coronary Syndrome (ACS), Coronary Artery Disease (CAD), Heart Failure with preserved ejection fraction (HFpEF), Dilated Cardiomyopathy (DCM) and Systolic Dysfunction as well as for “healthy” controls was re- evaluated and defined in the beginning of the project.
The third main objective aims to establish specific validation concepts for each omics-entity considering their specific needs in preanalytic and analytic processes of samples as well as statistics. Validation of the selected omics-panel in disease cohorts and controls will be performed at the responsible partner sites. Applied methods include qrtPCR, Luminex, next-generation sequencing, Fluidigm, Massarray, Infinium, mass-spectrometry, and immuno-assays. For all markers sensitivity, specificity, AUC and predictive power were assessed. Diagnostic and prognostic performance of novel markers has been benchmarked for diagnostic classification against current gold-standard (e.g. troponins for ACS, natriuretic peptides for HF, imaging phenotypes, etc.), for risk and outcome prediction against defined primary and secondary endpoints and for treatment monitoring against clinical scores and advanced clinical phenotyping/imaging. Another result generated in WP1 is the development of the basic requirements for validation concepts and for diagnostic and prognostic studies which are available for the entire consortium. WP1 will advise all project leaders in planning and implementing retrospective and prospective cohorts including basics of ethics requirements which need to be implemented at different BestAgeing partner sites.
Establishment of a consortium database for data integration and analyses (objective 4) is a pivotal objective for successful multi-center studies in BestAgeing. The database contains a minimum and extended dataset for the phenotypes and the application is deployed in a secure datacenter where all groups of the consortium have access to. In the first two periods the database was developed, tested and redefined and is from this time on fully operational. In the second reporting period the database could be finalized after the evaluation of the consortium. All groups of the consortium have access to the data base, which is accessible via the web: https://www.bestageing.org. This final version includes last revised definitions of the markers, diagnoses, studies, diagnostic complementary tests, events, etc. A regularly update is done over the entire project life-time and will be done beyond this. Up to date 2.393 patient with their respective clinical data are listed.
As this work package sets the basis for the collaborative work in WP2-8, all objectives and tasks were imbedded in the first two years of the project term. All objectives and tasks have been successfully fulfilled.
Work Package 2 - Standardization, quality control, exchange of biomaterials
In the field of omics-biomarker research, standardization and quality control (QC) are essential prerequisites for successful validation of biomarkers by the different technologies. The on-going quality control of the biomaterials and analytical procedures is central to obtain the best possible results. The BestAgeing validation concept consists of two main components: the retrospective (i.e. existing) cohorts and prospective (i.e. newly to be recruited) cohorts of the Beneficiaries. Open exchange between all partners of expertise, experimental results on pre-analytics and discussion led to the definition of a uniform procedure for sample collection, handling, processing and storage aimed to obtain the maximum number of –omics analyses with a minimum number of different matrices, aliquots and total volume for the prospective cohort. This guaranteed both clinical validation of a high number of new –omics biomarkers for cardiovascular diseases and the ethic requirement of a maximal amount of blood to be obtained from any single patient.
The first objective is the standardization of sampling and storage conditions as omics-biomarkers include fragile molecules such as RNAs or metabolites. In the first year Standard Operating Procedures (SOPs) for all processes including collection, preanalytics, shipping, storage and analysis of samples were defined. The second objective specifically aims to establish stable and robust clinical routines for RNA-based biomarker analysis from whole blood and serum. According to the working plan, the sample group investigates different sampling, extraction and storage protocols and established a SOP valid for the BestAgeing consortium. Objective 3 focuses on quality control of biomaterials and data integrity quality control of sampling, storage, and assessment of the condition of biomaterials. Therefore the samples were first checked centrally at the Heidelberg CardioBiobank upon arrival. To investigate center specific effects the prospectively collected samples have been analyzed with a metabolite based quality assay performed by partner MTXH. Beside that specific assessment of the quality of samples was also done at the different sites where the respective omics-technologies were applied. Results were integrated in the website (intranet) and regularly assessed for completeness and data integrity. Exchange of biomaterials represents objective four in WP2 to setup rules for data collection, pseudonymization of patient data and biosamples, and organization of sharing of biomaterials and data. In the course of the project the consortium decided that the Heidelberg CardioBiobank (HCB, part of the SMCU) takes over the responsibilities for the storage and shipping of all samples. Therefore the HCB developed SOPs for the on local storage of the samples and organized certified shipping processes to Heidelberg and later on to the analyzing partner sites. The consortium benefited from the long-term experience in handling and processing of biomaterial and of the basically existing SOPs and quality control mechanisms. Furthermore the HCB provides comprehensive and state-of-the-art infrastructure in Biobanking. This includes experienced study nurses, technicians, documentalists and scientific staff as well as cutting edge technical equipment such as custom specific robotics cryotank for >1 Mio samples, robotics for sample aliquoting, nucleic acid extraction etc.. As WP 2 is fundamental for standardization processes and therefore for the reproducibility of results, all objectives has been successfully fulfilled. All in all, the extensive work on the final definition of SOPs for standardized pre-analytics, a pre-requisite to ensure the highest quality for -omics based biomarkers discovery using prospective cohorts of patients leads to detailed insights of the consequences of preanalytical processes. This elaborately work have been published in 2017 in a peer review scientific journal thus reaching the objective to share our knowledge with the scientific community (Basso D, Padoan A, Laufer T, Aneloni V, Moz S, Schroers H, Pelloso M, Saiz A, Krapp M, Fogar P, Cornoldi P, Zambon CF, Rossi E, La Malfa M, Marotti A, Brefort T, Weis TM, Katus HA, Plebani M. Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling. Clin Biochem. 2017 Mar;50(4-5):186-193).
Work Package 3 - Assay-technology development
Usually for omics analyses highly expensive and complicated analytical methods and equipment is required. BestAgeing not only aims to validate novel omics based biomarkers in large multicenter studies but also to develop analytical tools and assays to ultimately asses these markers in a clinical setting. In WP3 SME and industrial partners have been working on assays for miRNAs, (epi-) genomics, and metabolomics assays.
In the first and second funding period the assessment of various technologies for miRNA validation and quantification have been made. As result of the testing, Multiplicom selected the FireFly Bioworks method as the most flexible and efficient method for quantitative analysis of miRNAs in a clinical setting. In collaboration with partner 11 (ACSB), a first custom Firefly panel has been composed and tested for miRNA biomarker validation. In addition, to the versatile and easy-to-use Firefly Bioworks the BestAgeing partner HBx (CBC) successfully implemented, validated and optimized the high-throughput qRT-PCR platform Fluidigm Biomark HD. Ideally suited for the high throughput validation studies this platform provides excellent complementation to the Firefly Bioworks technology and additionally facilitates the immediate translation of the microRNA biomarkers from the validation studies to installed qRT-PCR platforms in most (clinical) labs.
In the third funding period the above mentioned systems were used to analyse the first sets of retrospective and prospective patients samples. Thus HBx successfully implemented and validated Fluidigm’s high throughput qRT-PCR (Biomark HD) technology for microRNA biomarker measurements of liquid biopsy samples. Also optimized protocols (SOPs) for the high throughput qRT-PCR microRNA assessment of body fluid samples have been established to facilitate immediate engagement into the analyses of validation cohorts. Robust and reliable protocols for the processing of different sample materials (i.a. Serum, Plasma and PAXgene samples) and RNA input amounts have been developed and validated on the Fluidigm HT-qRT-PCR Platform.
In addition to the Firefly Platform and on-going in house developed technologies, Multiplicom has investigated the miRNA multiplex quantification capabilities of the MyCartis technology platform, a novel technology acquired in house during the first trimester of 2016.
Siemens developed a first high level extraction system concept for the isolation of miRNA: In order to provide rapid information on the miRNA status of AMI (Acute Myocardial Infarction) – suspected patients, a de-centralized and automated system has to be provided. Only a minimum of hands-on procedures would be acceptable for such a rapid test and the turnaround time should be in the range of one hour or even shorter. Siemens developed an automated sample preparation module with a disposable cartridge using Siemens Versant chemistry and an automated miRNA detection module based on a double strand ligation assay on a CMOS-chip. The feasibility test on a miRNA POC-test format was performed with both modules, respectively. The results showed the miRNA extraction took about 30 minutes and yield of miRNA was sufficient for the CMOS-chip based detection; the miRNA detection had a time to result of 30 minutes and detection limit was up to 1 pM with a dynamic range between 1 pM to 1 nM. The developed methods have been led to several patent applications.
Work Package 4 - Validation of protein and reference biomarkers in elderly
This WP focuses on the role of protein biomarkers in the diagnosis and prognosis of cardiovascular diseases. For many years proteins have been the major source for informative circulation biomarkers. They are generally easy to measure and are excellent tools for discriminating between healthy and diseased subjects and for risk prediction. In recent years many other molecules such as non-coding RNAs and metabolites have been identified as promising novel classes of biomarkers. Proteins however, are still the gold standard biomarkers for many diseases including cardiovascular diseases. Therefore the SME partner ACSB has validated the diagnostic and prognostic value of several novel proprietary single protein markers aimed at improving further stratification of heart failure patients into meaningful clinical entities. Before multimarker panels for diagnosis and prognosis of heart failure can be established, individual (novel) biomarkers have to be validated separately in discovery cohorts. We have validated formin-binding protein 1; amyloid precursor protein; collagen type IV alpha 1; and RNA binding motif protein 20 in two respective discovery cohorts, which allows the combination of these markers with established markers Galectin-3 (Gal-3) and NT-proBNP for the diagnosis and prognosis of heart failure. Individual biomarkers have been validated in larger patient cohorts but before a third discovery cohort was chosen from the available cohorts from the BestAgeing consortium to confirm previous protein biomarker measurements. To allow valid comparison between patient and controls, samples were chosen from partner institutions SERMAS (Madrid) and AMC (Amsterdam) that had both collected biosamples from both patients as well as control persons. In addition to the biomarkers Gal-3; FNBP1; APP; COL4A1 and RBM20, NT-proBNP have been measured if these data are unavailable from cohorts. In addition, a subset of controls from IMCB (Tartu) has been validated to be used as controls for larger patient cohorts.
A further task focused then on the generation of a combined protein and miRNA multi-marker biomarker panel. Predicting the prognosis of individual heart failure patients is vital to individualize treatment. At present, many classification models for heart failure have been proposed. In these models, demographics (age and sex), severity of the disease, renal insufficiency, blood pressure, diabetes, sodium are among the most frequently used parameters for HF risk stratification. However, these parameters, alone or combined, often fall short of accurate prediction or are too elaborate to be widely clinically implemented. Therefore, additional simple and objective measures to stratify HF patients according to their risk are needed. Biomarkers, such as natriuretic peptides (NT-proBNP) myocardial stretch) or high-sensitivity cardiac troponins (markers of myocyte injury) have shown value to refine risk prediction in heart failure. MicroRNAs (miRNAs) are small non-coding RNAs that influence protein translation and represent a level of biologic regulation that was essentially unknown a decade ago. To this end both previously identified and validated miRNAs and protein biomarkers were analyzed in two large prognostic heart failure cohorts.
Partner ACSB also used first cohorts to assess the importance of novel ASCB proprietary biomarkers to differentiate between controls and HF, and more importantly, to differentiate between different stages of HF. The group in Heidelberg has already addressed the effect of age on diagnostic and prognostic performance of high-sensitivity cardiac troponin T (hs-cTnT). Recently, the Katus group showed that elevated hs-cTnT is more common in the elderly due to higher prevalence of non-ACS conditions and significantly impairs diagnostic performance in discriminating non-ST-segment elevation myocardial infarction. In a recent study in 2015 the prognostic value of elevated hs-cTnT levels in a low risk outpatient population with cardiovascular disease was investigated. Also in this cohort, both in univariate and in multivariate analyses, hs-cTnT was independently associated with age and gender.
During the project, partner UNIPD established the reference limits of the two new protein markers TWEAK and HMGB1 and verified that in healthy subjects they are not influenced by variations in anthropometric parameters, gender, age or biochemical tests of liver and renal function, and of heart failure. These findings support further analysis in patients’ cohorts.
Work Package 5 - miRNA biomarkers for CAD, ACS, and HF in elderly
Main Focuses of WP5 was the validation of already identified single markers or miRNA signatures in appropriate cohorts and biomaterials from the BestAgeing consortium and compare these with established biomarkers and clinical variables.
One of the tasks in this this WP was the development and validation of miRNA biomarkers expressed in the blood cells of whole blood samples collected in PAXgene Blood RNA tubes. Thus, the miRNA-signatures derived from this approach relate to the expression of miRNAs in all blood cells, including erythrocytes, leukocytes and thrombocytes. Utilizing a first discovery cohort of 15 CAD-patients and 15 CAD-controls patient samples were analyzed on whole-miRNome Agilent microarrays resulting in 23 miRNA-biomarker signature candidates for diagnosis of CAD. HBDx validated these candidates on both Fluidigm high-throughput qPCR platform and standard StepOne 96-well qPCR platform for comparison using TaqMan chemistry and HBDx’s established qPCR SOPs. Unfortunately, 11 out of 23 microRNAs could not be detected on Fluidigm and 9 out of 23 microRNA could not be detected on StepOne platform. These results may be due to huge chemical, physical and technical differences between the microarray and qPCR based method. Based on this results and consideration of costs and benefits it was decided to generate genome-wide miRNA profiles using Agilent’s SurePrint microarray technique in the case study. HBDx has gained great expertise for Agilent’s microarray technology over the last decade and is repeatedly certified by Agilent for microRNA profiling using Agilent microarray slides. Additionally, HBDx is ISO 17025 certified by the DAkkS (Deutsche Akkreditierungsstelle GmbH) for the complete miRNA profiling process (RNA extraction, quality control and Agilent miRNA profiling). For miRNnome microarray screening study HBDx received PAXgene samples of 536 CAD-patients and 175 CAD-controls from BestAgeing partners in total. HBDx extracted total RNA from these samples and performed quantification and quality control of corresponding RNA eluates. Finally, 511 CAD-patient and 168 CAD-control samples (679 in total) were measured on Agilent microRNA microarrays (comprising 2,549 in miRBase v21 published microRNAs) for microRNA profiling. Raw data files of all measured samples were transferred to partner USAAR for bioinformatics analysis (WP8).
Using EDTA plasma blood taken from the coronary venous sinus (CVS) as miRNA-source, Partner GUF analyzed ACS samples on Agilent’s SurePrintG3 Human miRNA (8x60K) microarrays leading to a selection of a panel of 6 miRNA biomarker candidates for further validation. For validation CVS blood of a cohort of 17 patients suffering an ACS vs. 17 controls via TaqMan qRT-PCR resulted in a panel of 4 miRNAs with robust diagnostic information to identify ACS. To test the prognostic ability of this panel, the 4 miRNA were determined in EDTA plasma of 198 patients with ACS including 49 patients who died within 200 days in a case control setting. Herein, the panel showed a significant association with prognosis, amendatory to the clinically used TIMI risk score with comparable magnitude to the established biomarker BNP. For validation of the diagnostic potential of these 4 miRNA biomarker candidates will be carried out from the prospectively collected ACS patient samples within the BestAgeing consortium in further studies.
Partner ACSB focused on the validation of miRNA biomarkers for heart failure (HF). Twelve circulating miRNAs were previously either identified in plasma from patients with HF or selected based on their presence in the myocardium. These biomarkers have now been validated in plasma samples from a large cohort consisting of 1380 patients with chronic heart failure. To understand the extent of normal variation of circulating miRNA variation in general population, 268 healthy control individuals has been selected from the Estonian Genome Center Biobank (partner UT) and determined the expression levels as well as expression variability of circulating miRNAs by QIAGEN Human & Plasma miScript miRNA PCR Array.
Additionally Partners UKL-HD and HBx successfully fractionated whole blood samples into the cellular components most relevant for ACS and HF and analyzed the miRNA-profiles of these blood cell subfractions by next generation sequencing. Herein, a method for analyzing miRNA-profiles in 6 leukocyte blood cell subfractions by combining antibody-based microbead technology and next generation sequencing was established and respective miRNA biomarkers have been identified.
UKL-HD and HBDx established the study protocol for validating miRNA-biomarkers in ACS-patients. Herein the input from the other clinical recruitment centers were integrated, to result in a study protocol useable by all partner for sample collection.
The successful collaborative work in WP5 is proven by the seven publications in peer review scientific journals.
Work Package 6 - Validation of metabolomic biomarkers for HF in elderly
The main focus of Work Package 6 was the validation of previously identified and IP-protected biomarkers from metabolite profiling analyses in Heart Failure patients. Within BestAgeing these metabolomics biomarkers have been validated in large multicenter European cohorts in the group of elderly patients. Specific study synopses have been designed to address the most relevant clinical use cases by WP6 leader Metanomics Health (MTXH), University Hospital Heidelberg (UKL-HD) and the entire consortium.
The novel lipid biomarker panel (Cardiac Lipid Panel; CLP plus NT-proBNP), developed by Metanomics Health, in combination with the established marker NT-proBNP, together with the newly developed simplified analytical method has the potential to facilitate the early diagnosis of systolic heart failure in both outpatient and clinical settings, enabling a timely treatment of the patients. In total, MTXH has analyzed so far 4069 human plasma samples derived from various heart failure-associated cohorts, and present metabolomics data successfully confirm the added value of the novel metabolic biomarker panel called MxP® CLP for the improved diagnosis of heart failure with reduced ejection fraction. The added value of Metanomics Health’s MxP® CLP for the diagnosis of heart failure with reduced ejection fraction was also confirmed by our key opinion leaders. MxP® CLP biomarker is able to reliably diagnose heart failure with reduced ejection fraction at an earlier disease stage. As a consequence, four-year survival rates are expected to become significantly shifted upwards to nearly 90% in the future. Through the earlier detection and improved risk stratification, admission rates and secondary diseases might be reduced, leading to a considerable decline of associated healthcare costs in the future. From a cost perspective, the biomarker-based decision for patients at-risk with a subsequent echocardiography - confirming the diagnosis of a reduced left ventricular ejection fraction (<50%) - is advantageous in comparison to the currently prevailing diagnostic methods. Thus far, all available data/studies with regard to the MxP® CLP biomarker performance indicate that this will hold true for the combination of CLP and NT-proBNP.
Work Package 7 - Genetic and epigenetic markers for HF diagnosis and risk assessment
Heart Failure (HF) shows remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of HF, either by genetic or epigenetic factors that may interact with environmental stimuli. Work Package 7 aimed to validate genetic but also epigenetic biomarkers identified in previous studies and dissect their value for diagnosis, risk stratification and therapy monitoring. We previously detected DNA methylation differences and genetic variants in several pathways related to HF, but also in genes with yet unknown function in this context. Since DNA methylation was previously accessed in myocardial tissue, we also aimed to identify correlating markers (cell-free DNA, blood DNA) in peripheral blood. This is not an issue for genetic markers (rare and common variants) that are routinely assessed through peripheral blood.
To study the impact of common genetic modifiers on heart failure risk prediction two GWAS (Genome-wide association studies) have been performed by BestAgeing partners including n = 5279 patients with HF (non-ischemic) and n = 8708 controls. This represents to our knowledge the largest study population on the genetic factors in HF so far. By using Affymetrix and Illumina genotyping arrays, the individual substudies obtained genome-wide SNP data. Using Taqman genotyping, candidate loci were validated in several replication cohorts. We found out that over 3000 individual protein coding transcripts are influenced by genetic variation, which is a remarkable finding. Furthermore a set of new loci in exonic regions those contribute to DCM and serve as additional risk alleles in a combined score have been identified.
The process of identification of methylation markers was performed in several stages. On the one previously identified markers have been tested in fcDNA and whole-blood. On the other hand, we have added additional markers identified in a larger screening cohort and by high-resolution methylation measurement (450,000 vs. 27,000 methylation positions). Specific isolation protocols for DNA (and RNA in parallel) from biopsies and from whole blood were developed and established in cooperation with Qiagen (Germany). ). This approach provided us information about genome-wide methylation of CpG methylation sites in promoter, transcription factor binding sites and enhancers / Isolater regions. It was also possible to validate successfully a panel of epigenetic biomarkers in peripheral blood that show superior performance compared to natriuretic peptides.
The successful collaborative work within this WP is proven by more than ten published scientific articles within the project duration.
Work Package 8 - Development of multi-marker strategies and economic impact modelling
The main focus of Work Package 8 is to develop strategies and provide tools for analyzing multi-omics data sets for the consortium to establish standardized and easy-to-apply tools for clinicians related to the different clinical use cases. In the first part of the project WP8 focused on the stability of markers and translation towards clinics. Therefore the cell type specificity, the clinical application platform and the stability with respect to transportation and batch effects have been considered. The gained respective information has been added to our knowledge management system (miEAA) for assessing the validity of biomarkers. Besides confounding factors such as age and gender now also other disease associations, stability of the biomarkers with respect to transportation as well as cell type specificity can be assessed. miEAA is freely available to all consortium members as web service.
As a statistical and bioinformatics tool the WP 8 leader developed a software for evaluating biomarkers within the BestAgeing consortium. The available solution integrates simple basics such as power calculations for single and combined markers over standard diagnostic performance measures such as AUC values up to complex multi-marker classification systems. As decision trees are powerful models for classification tasks, WP8 designed a decision tree based approach. Besides a high performance of these classifiers they can displayed graphically as flow chart representing a set of rules on which basis the prediction should be done. This allows physicians to evaluate diagnostic multi marker approaches in much more detail as compared to e.g. SVMs. Our solution allows for handling mixed types of input data, i.e. the input may be numeric (e.g. gene expression) or categorical (e.g. presence or absence of specific single nucleotide polymorphisms). Beyond this, even image-based features can be integrated. Furthermore, the model handles missing values in case that a certain parameter is not available for a patient.
Another major goal of WP8 is the development of a concept for multi-marker strategies. While the task of combining quantitative (or pseudo-quantitative) markers to patterns has been already successfully achieved in earlier project phases partner USAAR focused in the last year on combination of different omics entities. This effort starts with a new approach for combining the information content of genetic aberrations on small scales (e.g. SNVs, small in/dels) but also considering larger genomic aberrations. The respective approach relies on decision trees (manuscript in preparation). Beyond, they implemented complex statistical models for inferring marker cascades, not only including genes but also messenger RNAs, miRNAs and proteins. A manuscript describing the stoichiometry-based approach is in review.
The software that has been implemented in the project is used by up to 800 individuals per week, on average over 2,000 researchers worldwide are working with our software per month. Altogether, over 1,000 research organizations, most of them in Germany, the UK and the USA are analyzing data or performing studies with the respective tools. This makes the dissemination of BestAgeing results a huge success and we will work further to ensure long time sustainability and availability of the implemented solutions.
During the project we already published 25 manuscripts with data from the BestAgeing project. 5 manuscripts with data and resources from BestAgeing are currently under review or under revision. Moreover, 3 manuscripts are currently finalized.
Work Package 9 - Project Management and Coordination
The main responsibility of Work Package 9 is the coordination and management of the consortium. In the implementation phase WP9 was responsible for the draft, negotiation and final agreement of the Consortium Agreement. A second achievement of the first funding period was the fast and coordinated distribution of community funds to the beneficiaries including some participants where special clause 10 had to be considered.
As an all-day available support service for all project partners, the SMCU coordinates and evaluates the scientific activities, interactions and progresses within the consortium. Furthermore the SMCU takes care of the administrative and legal issues of the consortium as a whole and also of the regulations / requirements of certain partner sites. This includes assistance and guidance on staff hiring regulations, financial controlling, reporting processes (deliverables, annual reporting), organization of meetings and telephone conferences and public relations.
The second and third funding period was focused on planning and starting prospective studies. Unfortunately the start of the recruitments at all clinical partner sites were delayed because the ethics committees from several partner sites came up with specific requests which were unforeseen as the studies are planned as observational studies. In order to fast and efficiently solve the various issues with the different local ethics application the Coordinator took the role as trial leader and supported the partners in their local implementation of ethics votes. The coordinator and her group then invested much effort to provide “master ethics application” documents for ethics application, patient informed consent, patient information etc. and to preparing partner specific drafts of the before mentioned documents and to support the partners in the negotiation process of all ethics related issues.
WP9 was also responsible for establishing the project website and web-based tools to support the scientific (periodic) report as well as the financial reports (form C). The dedicated website (www.bestageing.eu) is fully operational since June 2013 and is regularly updated by the SMCU. The website provides comprehensive information on structure, aims and current achievements of the project (including publications) for scientists, journalists, patient groups and the general public. Besides the public part the website also contains a secure internal part restricted to participants. This password protected intranet section provides a comprehensive document repository containing all relevant documents such as the DoW, the Grant Agreement, the Consortium Agreement, meeting protocols, agendas as well as the large number of specialized SOPs developed by the consortium, deliverables and the periodic reports.
During the project time a further task has been added: the organization of the biomaterial transport and storage, as well as the quality control became also part of the SMCU. The consortium decided to store the biomaterial at the Heidelberg CardioBiobank (HCB) which now operates as a trustee for the BestAgeing samples. As well as the SMCU the HCB is also headed by Dr. Tanja Weis and therefore the BestAgeing consortium decided to benefit from the knowledge and experience of the SMCU and the HCB. The HCB provides comprehensive and state-of-the-art infrastructure in Biobanking. This includes experienced study nurses, technicians, documentalists and scientific staff as well as cutting edge technical equipment such as custom specific robotics cryotank for >1 Mio samples, robotics for sample aliquoting, nucleic acid extraction etc. Within this additional duty, the scientific staff of the SMCU developed BestAgeing specific SOPs for all recruiting partner sites containing the handling and sampling of the biomaterial, the shipping and the labelling of the samples to ensure equalized and optimized standards for all partner sites.
Potential Impact:
The aim of BestAgeing was to use advanced biotechnology and the joint expertise of a consortium of expert clinicians and scientists to improve the health of elderly patients in Europe. Up to date, most efforts to translate omics-biomarker candidates into clinical practice have had little impact on patient care. This is partly explained by the small size of most studies, but also reflects a failure to integrate clinical, basic research, technological, and bioinformatics expertise from academia and industry into coherent and comprehensive biomarker development strategies. With its multi-disciplinary integrated translational approach, BestAgeing can overcome these limitations by 1) capitalizing on innovative omics candidates identified by the members of the alliance, 2) bringing together academic and industry partners with the necessary methodological expertise, 3) applying and validating the novel markers to population and patient cohorts all over Europe, and 4) accelerating bench-to-bedside translation by highly synergistic work packages.
1.4.1 Medical implications
BestAgeing impacts on the most prevalent disorders in the elderly. In industrialized countries more than one of three adults have one or more types of CVD and by 2030 it is for example projected that 40.5% of the US population will have some form of CVD. In male and female subjects aged 60-79 and 80+ prevalence of CVD in 2008 was at an impressive rate of 72.6/71.9% and 80.1/86.7%, respectively. Analyses of Framingham Health Study data among participants free of CVD at age 50 years furthermore showed a lifetime risk for developing CVD was 51.7% for men and 39.2% for women. It is in addition projected that heart failure will become an epidemic disorder in Europe due to the success of acute cardiac care and increased life expectancy following acute cardiac events. Thus CVD is a major threat to public health in ageing Europe and there is an urgent need to prevent cardiovascular events or to initiate effective treatment as early as possible. BestAgeing aimed to detect CVD earlier and more precisely for a better care of patients and to identify subjects at increased risk with a high predictive power to initiate preventive and treatment measures against acute and chronic CVD.
Furthermore the proper diagnosis of a disease is the prerequisite for any therapeutic intervention and thus the quality of diagnostic tools determines the success of preventive and curative therapies. For example after we introduced the cardiac troponin T (cTnT) assay, 25% of ACS patients had to be reclassified from unstable angina to NSTEMI. If these newly identified NSTEMI patients were then treated according to the guidelines, it is estimated that 17,000 lives are being saved per year in the US alone. The further refinement of the sensitivity of the troponin assays by us and others (high sensitivity troponin) enabled the detection of an additional 10% of patients with NSTEMI amongst patients with suspected ACS. Guideline adjusted treatment for these patients reduced death and MI rate from 39% to 21% within 1 year (Mills et al. JAMA 2011). Similar data are also observed in heart failure cohorts. Thus by use of the high sensitivity cTnT assay an additional 25% of high risk dyspnea patients can be detected, which are missed by the conventional cTnT assay (Jacobs et al. Clin Chem 2011). These findings highlight the indispensable value of precise diagnostic modalities for optimal patient care and a better patient outcome.
In the recent years substantial technical progress was achieved in the dissection of complex biological systems by omics technologies. The comprehensive analyses of e.g. the metabolome or miRNome in blood have provided unexpected clues to the existence of disorders and their functional implication. These novel biomarkers will expand the diagnostic armamentarium beyond conventional protein markers and hold the potential for added diagnostic value. The BestAgeing consortium was assembled to bring novel omics-based diagnostic modalities into clinical practice to better address the following clinical scenarios: First and most importantly we expected that the selected omics-markers will enable a more precise and more sensitive diagnosis of sub-clinical CVD as compared to available diagnostic tools. While measures for primary prevention are often ignored, diagnosis of affection status- even in early disease stages - is an accepted starting point for treatment and life style changes. Second we anticipated that the novel markers will enable an earlier diagnosis of disease progression and by that indicate an increased risk and urgent treatment. Thus for example plaque vulnerability may be detected before vessel occlusion by miRNAs from blood cells or heart failure may be identified by metabolic signatures in subjects with yet BNP negative myocardial dysfunction. Third it was estimated that the omics marker results will aid in the selection and intensity of treatment strategies since earlier detection of reversible ischemia or heart failure by genomic markers, epigenetic alterations, circulating proteins, metabolites or miRNAs will mandate a more aggressive diagnostic work up and optimization of treatment. Fourth, by analyses of multiple markers such as DNA, miRNA, metabolites and proteins reflecting different molecular disease signatures and cellular adaption processes, substantial information will be obtained on the underlying myocardial disease and its systemic impact, aiding risk stratification and therapeutic decision making.
The next aim was to improve risk stratification for heart failure in the ageing populations. Heart failure prevalence increases markedly in the elderly and at present is the most frequent admission diagnosis to internal medicine hospitals in subjects >65 years of age. Cardiomyopathies, which are genetic disorders in a large proportion of patients, account for 30-40% of all heart failure cases. In previous work, we could identify genetic variants associated with a markedly increased risk for progressive myocardial dysfunction, which may be relevant to the progression of any form of heart failure. Therefore in it was planned to use next generation and high throughput DNA sequencing to seek for mutations in known disease genes of heart failure and known genetic risk variants. It is predicted that the proper identification of subjects at high risk for heart failure and disease progression will lead to better patient care and improved outcome.
Despite the major contribution of evidence-based medicine derived from large randomized clinical trials for an improved patient outcome, it is important to better account for the individuality of the affected subjects and their specific treatment response. Thus, for example in oncology only a third of the patients do respond to a specific therapy. Importantly, in these patients cause specific treatment and monitoring with companion diagnostics proved to be a major step towards a better patient care with improved treatment response and outcome. In BestAgeing we tried to better describe the individual “molecular background” of subjects at risk for CVD and provide clues as to the underlying causes of the disorder. It should also be possible to better assess the individual (mal)-adaptive response to CVD and predict treatment response by omics technologies. Thus the multi-marker approach proposed in BestAgeing tends to enable a better description of disease processes as well as individual response to injury and treatment. BestAgeing thus aims to promote novel strategies for a more individualized therapy in the care of CVD and elderly subjects.
The analyses of disease associated patterns of different omics-markers which has been proposed by BestAgeing should provide novel strategies in biomarker validation and clinical chemistry testing, but also generate novel hypotheses on disease mechanisms, dys-regulated pathways and potentially useful therapeutic targets in CAD, ACS, HF, and healthy ageing. Therefore the academic groups within BestAgeing share the data generated within the consortium with each other and the scientific community and attempt to dissect novel disease pathways and treatment options under alternative research programs in cooperation with biomedical industry. The novel omics-based biomarkers should provide clues to the biology of ageing as exemplified by the age associated variation of expression of distinct miRNAs. The analysis of these molecular markers in large cohorts enables to compare patients of different ages and to reveal ageing-related physiological and pathophysiological changes. The omics profiles may also identify similar dysregulated pathways in clinically distinct syndromes such as heart failure and chronic coronary artery diseases but also non-cardiac disorders like inflammatory diseases as indicators of commonalities in disease mechanisms. Thus it is predicted that BestAgeing will deliver new targets for treatment of CVD and generate novel metabolomic, genomic, epigenomic and miRNomic data in ageing.
Systems biology approaches have gained particular interest in the last decade since they aid in the discrimination of relevant from irrelevant data obtained in complex biological systems. In many cases, comprehensive systems biology techniques require for profiling datasets from diverse omics-technologies, connecting the different systems of cellular regulation and identifying connected components with key biological relevance. The biomarker candidates validated in BestAgeing were in part retrieved by using such techniques, which lead to the identification of promising markers reflecting key disease processes in CAD, ACS and HF. Although the data we will assess in the validation experiments only represent a smaller subset of the complexity of the cardiovascular systems, by combining those already proven as relevant datasets we could address possible interactions between genomic, epigenomic and transcriptomic regulatory pathways derived from systems biology studies.
Individual genetic background and life style, which strongly affects epigenetic mechanisms and metabolism, is not taken into account for individualized treatment and therapy control for CVD. During the last years, significant insight into the role of genetics and gene regulation could be achieved. In parallel the role of genomics, epigenomics, transcriptomics, metabolomics and miRNomics for the individual disease has been recognized. Additionally, genome wide association studies and functional genomics analyses gained exciting insights into causes and mechanisms of individual susceptibility to therapy response or the range from non-response to hypersensitivity, respectively. Our scientific approach comprises different omics technologies that built the base for future achievements in companion diagnostics by biomarkers indicative for therapy response or failure. We are convinced that some of our omics based biomarkers or their combination will significantly contribute to improved clinical phenotyping, earlier diagnosis, improved individual prognosis and more precise outcome prediction of ACS, CAD and HF in elderly and ageing patients. We are also confident that this work will promote omics-technologies in general and systems biology studies of CVD in particular and underline their usability and value for translational research.
1.4.2 Structural impact
Concerted action against CVD: The BestAgeing consortium brought together scientists, physicians and businessmen from different European regions each experienced in distinct fields such as omics research, test development, epidemiology, and clinical research. Although bilateral cooperation amongst many members of BestAgeing has existed for many years, the consortial structure was unique and enabled not only to fulfil the goals of this project but also stimulated basic and translational science of each individual member. Thus we have a close interaction of the experts in genome, miRNom, metabolome and protein analysis and with this expertize we harmonized the sampling, storage, and analytical strategies across the different omics-fields. The interaction of SME with academia is crucial for a successful translation of a test development into clinical practice, which requires consideration of not only technical feasibilities and business options on one side but also medical needs and clinical workflow on the other side. We also experienced the incremental value of an early synergistic developmental approach of SME and big diagnostic industry as implemented in this consortium and the significance of detailed knowledge in the different fields by each of the academic member. Thus the concerted action of this consortium covered the entire spectrum of technologies, biomaterials, basic sciences, clinical medicine and business is not only benefitting research of each participant but is required for the translation of omics-biomarkers into clinical practice.
One goal of the BestAgeing project was to provide a unique collection of patient cohorts and omics-grade biomaterials. High-sensitive omics-technologies have enabled completely novel ways for biomarker identification, test development and validation. However, each omics-technology has specific pre-analytical requirements on sample quality, preparation, storage and processing. The required quality of input material is a limitation to the broader application of these technologies to disease cohorts. BestAgeing has long-standing experience in omics-technologies and hence each partner could contribute a high number of biosamples from retrospective cohorts with “omics-grade quality” to the project. However, we recognized that the pre-analytical requirements of most omics-methodologies needed to be standardized and adjusted for routine clinical use, which solved within this project. Hence, we also saw the need to recruit elderly patients prospectively to investigate under optimal pre-analytical conditions the different omics-markers in the same well-characterized individual. Therefore we recruited with low additional costs European elderly-cohorts with high quality clinical phenotyping and “omics-grade” biomaterials, which could be shared with others. The established SOPs and methodologies for pre-analytical sample management and quality control procedures was available for all BestAgeing partners and the scientific community to leverage the potential of omics-biomarkers for all disease entities.
During the last years, a wealth of high-throughput technologies has emerged allowing the rapid parallel analysis of a multitude of molecules in complex mixtures. Substantial work has led to the validation of distinct genomic, epigenomic, proteomic and metabolomic markers or signatures in proof-of-principle studies. However, despite substantial progress in precision and turnaround time of high-throughput technologies the translation of omics into clinical practice was far from reality. For clinical use it is required to reduce omics results to a manageable number disease specific or risk predictive variables or to provide convenient algorithms for rapid analyses of complex data. Furthermore in clinical care robust and optimally standardized analytical test systems are needed, which provide the relevant results in a clinically useful turnaround time. In the WP2 and 3 our consortium focused on the optimization of available analytical techniques and on the development of novel assay formats for omics-derived biomarkers and their validation. This is an important part of our translational research, which benefits from the close interaction not only of the SMEs and the academic groups with long lasting experience in test development and validation but also from the ongoing interaction with big diagnostic industry, which is a prerequisite for a successful implementation of omics biomarkers in clinical chemistry laboratories. Thus our work in test development and optimization of analytical hardware is crucial for omics based diagnostics in other disease areas as well and will be an important step towards personalized medicine.
1.4.3 Socioeconomic impact
European lead in innovative diagnostics and creation of a network of excellence:
When planning the composition the BestAgeing consortium, we were aware that for a successful translation of novel biomarkers we need to include not only strong, technology driven SME partners, but also incorporate bigger players in the diagnostic industry. As such, we were able to enforce our consortium with Metanomics Health and Siemens Healthcare as active partners that added not only regulatory but also worldwide marketing expertise to the project. This should allow successful translation of diagnostic assays all the way through to implementation into the clinical routine, supported by the networks of a global player. The collaboration between our well-connected European partners also resultes in additional benefits for the consortium and the individual partners. Each of our partners has an outstanding track record in his field and is involved in several European and other international networks. We envisaged active networking inside and outside of BestAgeing to promote best scientific quality and excellence. Such a network of excellence enables a truly translational research and technology driven innovation in Europe. The joining of clinical expertise from different countries and partners dedicated to research for CVD, the identification of novel biomarkers, development and validation of diagnostic modalities, experience in omics technologies and ageing research resulted in a unique research environment.
Healthy ageing is a critical determinant for economic growth and prosperity in Europe. The economical productivity for instance will increasingly depend on the contribution of elderly workers. Already today however CVD accounts for 20.4% of disability adjusted life years (DALY) in developed countries and for 32.8% of all deaths (Murray et al. Lancet 1997). Not surprisingly due to the trend towards demographic ageing in Europe and the increasing prevalence of CVD with age, it is predicted that CVD will remain the leading cause of death in industrialized countries and continue to contribute markedly to morbidity and health care costs. It is therefore paramount to detect affected subjects early, to identify those at high risk for cardiac events properly, and to provide sensitive diagnostic tools for immediate therapeutic interventions.
Considering the rising expenses for health-care, results from health economic research will likely influence medical decision-making in the future. Hence, payers and health care providers in Europe need to be provided with robust information on optimal resource allocation to support healthy-ageing. This ambitious aim can be achieved in BestAgeing, since we have longstanding experience and insights into relevant national and international data sources and economic measures of diagnostic and therapeutic procedures. With our concomitant activities for public outreach, we plan to communicate our scientific work, the results of biomarker development, and the potential medical and economic consequences.
1.4.3. Main Dissemination activities and exploitation of results
The dissemination activities were one major part of WP9 (Project Management and Coordination) beside the task to offer a central project management facility for supporting the interdisciplinary European consortium in various administrative and scientific businesses. This comprehensive work is realized by the existing “Scientific Management and Coordination Unit (SMCU)” at the Department of Internal Medicine III in Heidelberg. At the beginning of the project the SMCU developed a dedicated webpage (www.bestageing.eu) which serves as professional platform for internal and external sharing of information. The website provides a frequently updated, large variety of up to date information on structure, aims and current achievements (including publications) to scientists, journalists, patient groups and the general public. Furthermore a professional information desk and information brochures (figure 1) were designed and realized. The information desk has been presented at least twice per year at the meetings of the German Society of Cardiology. At this meetings clinicians, scientists, nurses and technical assistants as well as patients were informed and had the chance to discuss directly the aims/results and challenges of the BestAgeing project.
Furthermore the SMCU organizes special press releases and printed information brochures for different target groups. To do so, the connection to all established information channels via the press offices of the partners is given. One other important information source for the public community and physicians and scientists is the BestAgeing webpage www.bestageung.eu (Fig.2) . Beside a secure intranet area for BestAgeing partners the website contains a public accessible area providing target specific information including the latest news and research releases. Beside the dissemination activities of the SMCU, several partners presented their results originated within the frame of the BestAgeing project with a poster or an oral presentation at high-ranking international meetings. The successful, constructive and productive execution of the BestAgeing project is also reflected by the numerous (> 37) scientific publications generated within the last years. And it is expected that several publications will follow as the generated data serves as the base for further research activities.
One main goal was the successful identification of omics-based biomarker and the translation of their application in the daily clinical routine. To proof the effective approach of the BestAgeing project, we can present eight patent applications up to date.
List of Websites:
www.bestageing.eu
