The primary aim of this Work Package is to bundle the efforts of the work packages on the European side of the consortium, and, together with WP11 on the Russian side, to provide a platform for research that makes use of large blood sample banks into type 2 diabetes, cachexia, and obesity as co-morbidities in heart failure. Due to the large number of patients (estimated more than 1000 patients with heart failure) that will be enrolled into the project, the Central Blood and DNA Bank will by use of innovative and collaborative approaches enable research to investigate and establish novel biomarkers or panels of biomarkers to better characterize heart failure patients with one or more of the aforementioned co-morbidities in terms of their prognosis, their clinical course (e.g., imminent decompensation of heart failure), and the verification of their clinical diagnosis. Genetic samples will, together with WP11, enable us to identify candidate genes in this regard.
For use in the project, a biomarker is any quantitatively or qualitatively measurable parameter that is able to help in clinical decision making. As mentioned above, estimation of the patients' prognosis and their clinical course are important for this purpose, because patients with more advanced disease or at risk of imminent decompensation may benefit from more aggressive therapies. Therefore, close collaboration with other WPs (especially WP03, WP04, WP05, WP06, WP07) that collect detailed clinical information about the patients and their clinical course over time, is necessary. The relationship between pathophysiological changes in the blood and on the tissue level has been little investigated in obesity, cachexia, and type 2 diabetes in heart failure. Hence, another aim of this WP is to make progress into this area by interaction with WP 08 and WP09. However, several studies over the last years have shown that different co-morbidities affect the reliability of biomarkers in heart failure. The plasma values of the current gold standards for diagnostic assessment, prognosis estimation and treatment guidance, BNP and NT-proBNP, appear to be 2-3 times lower in obese patients with heart failure than in those with a BMI <25 kg/m2 (Krauser 2005, Januzzi 2006). Furthermore, we found that in cachectic patients the values of these markers are raised more than is appropriate for these patients' degree of cardiac dysfunction (Rauchhaus, submitted). However, no study has prospectively tested the long-term effect of obesity or cachexia in this regard. It also remains unclear whether biomarkers that reflect the amount of adipose tissue may be able to serve as a correction factor for the natriuretic peptides. Early evidence supports the hypothesis that the assessment of MR-proANP may be superior to NT-proBNP for the subgroups of obese patients with heart failure (von Haehling 2007). Data to support the assessment of the natriuretic peptides (or any other biomarker) in patients with type 2 diabetes and heart failure is scarce.
The Central Blood and DNA Bank of the consortium and the assessment of different biomarkers will be led by the Center for Cardiovascular Research of the Charité Medical School. The participating members in Berlin and Zabrze (Poland), Hull (England), Rome (Italy), Wroclaw (Poland) have a long-standing expertise in biomarker research, which includes the shipment, storage, and analysis of different types of biomedical specimens as well as an extensive publication record.
Objective 1 involves the setting up of a standardised -80°C freezer system using existing freezers at each site that allows safe and reliable storage of biomedical samples under standardised conditions until shipment to the consortium's Central Blood and DNA Bank. Upon arrival in the Central Blood and DNA Bank, all samples will be stored in freezers that are connected to an alarm system (internet-based) to alert staff in case of machine break-down. Further, air-conditioning is required in all freezer-rooms to prevent machine break-down by emission of their own heat. All samples will be stored in screw-top aliquots at a standardised volume of 500 microlitres for serum and EDTA plasma in 500 microlitre Eppendorf tubes that help keep the amount of trapped air at a minimum. This procedure allows safe long-term storage. Colour-coded aluminium racks can hold up to 2000 samples each. Labelling is supposed to make use of two different labels for each sample: (1) a bar-code on the lid to make easy identification possible without the necessity to remove the sample from the rack, (2) a real-text label on the sample front with the patient's surname, given name, date of birth, study date, and study site. Labels are frost-resistant and can be printed on standard laser printers. Shipment of samples from different sites to the Central Blood and DNA Bank (objective 2) will be carried out on dry ice using an experienced professional carrier (TNT Clinical Express, Berlin, Germany). EDTA full blood samples will be shipped at room temperature. Upon arrival in the Central Blood and DNA Bank, DNA will be isolated using a DNA isolation kit (for example, GNOME® DNA Isolation Kit, Qbiogene Incorporated). Storage of DNA biospecimen is possible at -20°C for many years. All samples will be coded in a central database. If necessary, this database can be easily anonymised in order to share such information with third parties. All procedures will be held within the tight borders of the ethics approval. Depending on the consent provided by the respective subject, he/she will take part in serum biomarker analysis only of in additional genetic testing. We are currently seeking partners for this project, particularly for the analysis of genetic test, because these tests are not covered by the SICA-HF budget. Potential partners come particularly from the ADAMS and the BIOSTAT-CHF project, both of which are also funded by the European Commission. However, we cannot promise at this point that genetic testing will be performed, because the funds for this project have not been clarified so far. Genetic tests, if performed, will particularly focus on inflammatory pathways, apoptosis, the rennin-angiotensin-aldosterone system, and pathways deemed to be related to cachexia-development.
Biomaterial in the Central Blood and DNA Bank will enable research into the impact of type 2 diabetes, obesity, and cachexia on established biomarkers of heart failure prognosis, severity, and the clinical course of the disease (objective 3). This is of particular importance for both cachexia and type 2 diabetes whose influence on classical biomarkers has not been investigated in heart failure so far. Even investigations into the effect of obesity on markers like BNP or NT-proBNP remain in their early phases. Of particular importance in this context is whether or not such markers remain reliable in patients with the above-mentioned co-morbidities. Thus, a large-scale prospective study such as the proposed research can help to establish known markers in important subgroups of patients with heart failure for whom prospective (and sometimes even retrospective) data are lacking. It is well possible, however, that the cut-offs for the known markers may be different in these subgroups than in non-diabetics or patients with normal weight. Receiver operator characteristic curve analysis with which the team leader of WP 10 has a long-standing experience (von Haehling et al., 2007, Kempf & von Haehling et al., 2007), will help to identify such cut-off values for different "classical" end points such as cardiovascular/all-cause mortality and hospitalization. Novel end points will include weight gain or weight loss and the development of type 2 diabetes during follow-up. Receiver operator characteristic curve analysis will also help to establish sensitivity and specificity data for established and candidate biomarkers. Multivariable Cox regression analysis (visualized by Kaplan-Meier survival/event rate plots) will help to verify the prognositc applicability of these markers. If known markers fail to demonstrate their usefulness in clinical decision making at established cut-off points, the consortium hopes to establish novel, yet unidentified cut-offs that still enable the clinician to use known markers after consideration of, for example, body weight or presdence of type 2 diabetes, using the aforementioned statistical approaches. Such established markers include haemoglobin (Sharma et al., 2004, Komajda et al., 2006), creatinine, uric acid (Anker et al., 2003), and NT-proBNP (von Haehling et al., 2007).
Samples from the Central Blood and DNA Bank will be enable the identification of new biomarkers that can be of use in the characterisation of patients with heart failure and type 2 diabetes, cachexia, or obesity (objective 4). Candidate biomarkers include MR-proANP (von Haehling et al., 2007), MR-proADM, high sensitivity troponins, and GDF-15 (Kempf et al., 2007), biomarkers of apoptosis, catabolism, inflammation, catabolic/anabolic imbalance, however, a vast array of potentially interesting other candidates has probably not even been identified at the time of this writing. We currently have an agreement with BRAHMS AG (Henningsdorf, Germany) that the following markers will be analysed as part of SICA-HF: mid-regional pro-atrial natriuretic peptide, mid-regional pro-adrenomedullin, C-terminal pro endothelin-1, C-terminal pro-endothelin-1. Biosite (San Diego, CA, USA) has shown an interest in a collaboration with regards to the analysis of B-type natriuretic peptide. BG Medicine (Boston, MA, USA) agreed to perform analysis of galactin-3. The analysis of biomarkers usually requires different types of samples, depending on the type of assay. In most cases, EDTA samples are required; some assays require usage of serum. For example, whilst MR-proANP (immunoluminometric assay) can be assessed from serum or EDTA plasma, MR-proADM (immunoluminometric assay) can only be assessed from plasma samples (EDTA or lithium heparin). However, both can be analysed by the same company (BRAHMS Aktiengesellschaft, Hennigsdorf, Germany). The typical volume required for one test is between 20 and 100 microlitres. In most cases, more than one biomarker can be assessed in one sitting. In addition, most assays require a dead volume of 50-100 microlitres. Therefore, as mentioned above, samples will be stored in 500 microlitre tubes. The consortium agreed to study candidate molecules that may help in the characterization of patients with heart failure and obesity/cachexia and/or type 2 diabetes in the clinical and the preclinical setting both on the European and on the Russian side of the consortium (objective 5). Biomarker approaches to heart failure and its co-morbidities will also include multimarker strategies, as such panels with different powerful biomarkers appear to be superior to the assessment of single markers (Zethelius et al., 2008). Such a panel of biomarkers may also serve to identify patients with heart failure at risk of developing co-morbidities (objective 4). In this context it is of particular importance to study changes of biomarker panels over time.