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Novel Diagnostics and Biomarkers for Early Identification of Chronic Inflammatory Joint Diseases

Final Report Summary - D-BOARD (Novel Diagnostics and Biomarkers for Early Identification of Chronic Inflammatory Joint Diseases)

Executive Summary:
The aim of this research programme was to bring together a consortium of specialist academic institutions and leading SMEs from strategically important geographical and economic regions of Europe to focus on the identification of new biomarkers and development of diagnostic tests capable of subclinical disease diagnosis for degenerative and inflammatory diseases of joints, specifically focusing on osteoarthritis (OA). OA is the most common form of arthritis and a major cause of joint pain, inflammation and loss of mobility. It is a leading cause of chronic disability and one of the top five health care costs in Europe and across developed industrialised countries. It is also a major burden to developing nations. The disease is characterized by progressive deterioration and loss of articular cartilage. It primarily affects load-bearing synovial joints (knees, hips) but other joints may be affected (hands). When this consortium was formed, the members of this consortium recognized the fact that uniform definitions of joint disease are essential for biomarker validation and comparison between studies. Understanding the tissue source, formation, and clearance of biomarkers is important for correctly interpreting data from biomarker assays. Some biomarkers will reflect catabolic processes resulting in tissue degradation whereas others will be indicators of tissue repair, turnover, extracellular matrix (ECM) remodelling. Therefore, our definition of “biomarker” has reflected these different processes and the validation process for new biomarkers must depend on the context and specific purpose of their intended use. To achieve the stated aim above, we have achieved the following objectives:
• We have identified novel biomarkers of early disease onset and progression using patient samples with validation and cross-validation in animal models and ubiquitously adopted in vitro culture systems. The consortium used proteomic, metabolomic, genomic, transcriptomic and bioinformatic approaches to identify new biomarkers (or combinations thereof) using in vitro models of chondrocytes and cartilage inflammation as well as animal models of OA and clinical materials from OA patients (urine, serum and synovial fluid).
• We developed cutting edge molecular and immunological assays for the multiplex detection of panels of existing and novel biomarkers. The consortium had significant industrial involvement and was SME led. We focused on the identification and selection of biomarkers that can be used to develop new biochemical and immunological assays for the early diagnosis of OA. The academic partners collaborated with the leading SMEs to develop and test sensitive non-radioactive single and multiplex diagnostic assays.
• We have carried out validation and qualification of combinations of the identified biomarkers in animal models and cohorts of affected and “at risk” patients from the Rotterdam Study and its sub-groups. The assays developed were used to validate and qualify the combination biomarkers in animal models of OA (including rodent and guinea pig models) and in cohorts of human patients. We used patient subclassification/stratification strategies to increase homogeneity in study and treatment groups. The “combination biomarkers” developed were biochemical, immunologic, genetic and image based and it is anticipated that they will have future diagnostic and prognostic value with the capacity to predict responses to pharmaceutical agents and non-pharmacological therapies.
• We developed bioassays and customized kits for diagnostic and prognostic investigation of OA. The biomarker combinations that were validated and qualified in “at risk” human patients and animal models of OA were used by collaborating SMEs with research intensive R&D capabilities to produce customized prototype kits for diagnostic and prognostic investigation of OA in human patients and experimental models.
• We have established a pan European network of scientists and industrialists engaged in biomarker research and commercialization. We integrated our consortium deeper in Osteoarthritis Research Society International (OARSI) and the Arthritis Foundation in the United States and we have regular dialogue with these organisations.

Project Context and Objectives:
During the first 18 months of the project in the proteomics work package (WP1) we identified new protein biomarker targets in the secretome of culture models of cartilage inflammation exposed to pro-inflammatory cytokines. We successfully extended these studies to the membranome of chondrocytes. We refined existing culture models to include other inflammatory mediators but we did not manage to incorporate mechanical factors. We also studied SIRT1 fragments and shed syndecan-4 in culture systems and models of OA, focusing on their expression profiles and relation to severity of disease. In WP2 we used a combination of metabolomics and lipidomics to analyze synovial fluid and serum from animal models (and OA patients) by correlating the serum lipid profiles to structural changes in the joint and symptomatic features such as pain. In WP3 we used genetic, epigenetic and genomic tools and technologies to identify biochemical pathways and genes relevant to OA. We also published some high-quality papers in the genomics area. In WP4 we used MRI and advanced cell imaging to determine the relationship between inflammatory markers and joint tissue alterations. We also analyzed different aspects of MRI “scan quality” to better establish our expectations for the resulting quantifications in different MRI collections, focusing on signal-to-noise, contrast-to-noise, and position variation. In WP5, we integrated the data generated in WP1-4 into a unified framework that helped us generate new hypotheses and research questions. In WP6 we developed draft guidelines for OA biomarker development and validation. We have published some relevant papers in this area. In WP7 we developed the project website and established all the necessary communication and social media channels.
During the second 18 months of the project in we continued using high-throughput proteomics and mass spectrometry (MS) to identify new protein biomarkers of cartilage metabolism and degradation. We studied the secretome of cartilage explants exposed to pro-inflammatory and anti-inflammatory stimuli and successfully developed proteomic techniques to study proteins in membrane fractions from normal and OA chondrocytes. We also explored the potential of SirT1 and syndecan-4 as novel biomarkers of OA using a combination of immunoassays, molecular biology and protein biochemistry. In WP2 we quantified levels of bioactive lipids in the synovial fluid from OA and RA patients and found evidence for involvement of lipid mediators in OA. We demonstrated that metabolic stress-induced systemic inflammation contributes to the onset and progression of OA in a high fat diet-induced OA mouse model. In WP3 we carried out a comparative analysis of the transcriptome of cartilage from OA patients who have undergone elective surgery and from age-matched control individuals, using microarray and next generation sequencing (NGS) and completed a high-throughput genome-wide assessment of DNA methylation from healthy and OA cartilage. WP4 examined the correlation between macrophage activation and other biomarkers and determined bone shape and appearance in the cohorts. In WP5 we integrated the data generated in WP1-4 into a unified framework and applied Big Data analysis methods to develop robust prediction models of OA, identifying combinations of biomarkers and building OA disease pathways. In WP6 we selected the best biomarkers, which are partly diagnostic, prognostic and efficacious with special focus on inflammatory diseases and achieved preproduction of selected biomarkers. In WP7 we facilitated dissemination and developed the DELF system which was successfully implemented and tested. In WP8 handover of Co-ordinator role from UNOTT to SURREY was successfully achieved and the Project Manager at UNOTT (Hammond) handed over to the Project Manager at SURREY (Wright).
Work in D-BOARD progressed satisfactorily in the third period. In the final period in WP1 we examined the role of LRP-1, chondroadherin and lumican as novel biomarkers and examined metabolic markers of OA using metabolomics and the Agilent Seahorse instrument. We found that pro-inflammatory cytokines, particularly IL-1β, induce a dramatic metabolic switch in primary chondrocytes. In WP2 we carried out metabolomics and lipidomics on synovial fluid and serum samples from animal models of OA and human subjects and in WP4 we attempted to image inflammation and inflammatory lipids in the infrapatellar fat pad (IPFP). We also studied the role of macrophages in OA-induced changes in the synovium and IPFP. IN WP3 we attempted chondrogenic differentiation of human iPSC lines but did not achieve this task. In WP6 we completed all tasks of the work package and introduced the BEST resources as a suitable enhancement to the BIPEDs criteria. We initiated biomarker development plans that should ultimately lead to approval of the biomarkers as diagnostics. We continued data analysis and modelling in WP5 and dissemination in WP7. We struggled to meet deadlines in WP8 due to changes in Project Manager (Wright departed and Ramzan and Dodd helped since December 2016).
Project Results:
The main impact of our work in D-BOARD has been the provision of sophisticated new OMICs methods, assays and biomarker technologies for the early detection and monitoring of OA. The technologies have been predominantly proteomics and bio(immuno)assay based. However, significant progress has also been made in the areas of genomics, lipidomics and bioinformatics. The new biomarkers discovered and delivered by the D-BOARD consortium will hopefully be incorporated into new affordable diagnostic and prognostic that are easy and straightforward to setup, standardize and carry out. Although many of the biomarkers that we have discovered will be classified as exploratory, their identification has increased the throughput within the OA biomarker pipeline. The new technologies and novel biomarkers developed will make it easier for clinicians to diagnose the disease and for pharmaceutical companies to develop more effective and better targeted medications for OA patients from a wider cross-section of the population. Novel OA biomarkers can provide an early warning of joint degeneration during the molecular (asymptomatic) phase of the disease, prompting earlier and more targeted treatment. This consortium’s innovative approach to the development of new early prognostic biomarker assays will help to identify the disease early and facilitate earlier and more ‘personalised’ treatment. This approach will significantly improve the quality of life of elderly Europeans.

This project has delivered lists of new genes, biomarker proteins and metabolites that may facilitate early diagnosis of chronic and inflammatory joint disease, especially when used in combination with the currently available imaging technologies. We have also made progress in MRI image quantification. The combination of imaging, “omic” and bioinformatic approaches originally proposed in this project has begun the new trend of integrating diverse data generated from patients and models into a unified framework that can identify previously unknown combinations of biomarkers, build molecular and cellular pathways of OA disease that can effectively predict OA early. The remaining challenge is to use this approach in a healthy population, from individuals who are not yes showing any of the clinical signs and symptoms of the disease. This approach is new being tested in the Innovative Medicines Initiative (IMI) funded project APPROACH (Applied Public-Private Research enabling OsteoArthritis Clinical Headway).

The project aimed to deliver new lists of biomarkers capable of indicating the onset of pre-radiographic inflammatory changes in the joint so that established therapies may be more effectively directed and new strategies for therapeutic intervention developed for treating more established forms of the disease. This aspect of the project will need a considerable amount of additional effort. The project has already delivered new prototype biomarkers for bioassays of disease and/or joint tissue specific protein products, which after being released from the affected tissue(s) into the circulation and excreted in urine can be measured. New urinary biomarkers will support non-invasive bioassays and novel serological assays for disease diagnosis and monitoring will enhance blood tests. The project has also attempted to validate and qualify existing biomarkers that have not yet been tested in the appropriate settings and select the most promising ones for inclusion in the “biomarker pipeline”.

The incidence of OA is rising, seriously affecting the wellbeing of millions of EU citizens. OA is expected to place a heavy economic burden on European healthcare systems and community services. The end stage treatment for OA is surgery, either to modify or replace the joint. With increasing life expectancy, growth in the elderly population and an alarming escalation of chronic, inflammatory and age-related conditions (such as OA), there is increased demand for new diagnostic tests and prognostic and therapeutic indicators for OA.
Potential Impact:
The main impact of our work in D-BOARD has been the provision of sophisticated new OMICs methods, assays and biomarker technologies for the early detection and monitoring of OA. The technologies have been predominantly proteomics and bio(immuno)assay based. However, significant progress has also been made in the areas of genomics, lipidomics and bioinformatics. The new biomarkers discovered and delivered by the D-BOARD consortium will hopefully be incorporated into new affordable diagnostic and prognostic that are easy and straightforward to setup, standardize and carry out. Although many of the biomarkers that we have discovered will be classified as exploratory, their identification has increased the throughput within the OA biomarker pipeline. The new technologies and novel biomarkers developed will make it easier for clinicians to diagnose the disease and for pharmaceutical companies to develop more effective and better targeted medications for OA patients from a wider cross-section of the population. Novel OA biomarkers can provide an early warning of joint degeneration during the molecular (asymptomatic) phase of the disease, prompting earlier and more targeted treatment. This consortium’s innovative approach to the development of new early prognostic biomarker assays will help to identify the disease early and facilitate earlier and more ‘personalised’ treatment. This approach will significantly improve the quality of life of elderly Europeans.

This project has delivered lists of new genes, biomarker proteins and metabolites that may facilitate early diagnosis of chronic and inflammatory joint disease, especially when used in combination with the currently available imaging technologies. We have also made progress in MRI image quantification. The combination of imaging, “omic” and bioinformatic approaches originally proposed in this project has begun the new trend of integrating diverse data generated from patients and models into a unified framework that can identify previously unknown combinations of biomarkers, build molecular and cellular pathways of OA disease that can effectively predict OA early. The remaining challenge is to use this approach in a healthy population, from individuals who are not yes showing any of the clinical signs and symptoms of the disease. This approach is new being tested in the Innovative Medicines Initiative (IMI) funded project APPROACH (Applied Public-Private Research enabling OsteoArthritis Clinical Headway).

The project aimed to deliver new lists of biomarkers capable of indicating the onset of pre-radiographic inflammatory changes in the joint so that established therapies may be more effectively directed and new strategies for therapeutic intervention developed for treating more established forms of the disease. This aspect of the project will need a considerable amount of additional effort. The project has already delivered new prototype biomarkers for bioassays of disease and/or joint tissue specific protein products, which after being released from the affected tissue(s) into the circulation and excreted in urine can be measured. New urinary biomarkers will support non-invasive bioassays and novel serological assays for disease diagnosis and monitoring will enhance blood tests. The project has also attempted to validate and qualify existing biomarkers that have not yet been tested in the appropriate settings and select the most promising ones for inclusion in the “biomarker pipeline”.
The incidence of OA is rising, seriously affecting the wellbeing of millions of EU citizens. OA is expected to place a heavy economic burden on European healthcare systems and community services. The end stage treatment for OA is surgery, either to modify or replace the joint. With increasing life expectancy, growth in the elderly population and an alarming escalation of chronic, inflammatory and age-related conditions (such as OA), there is increased demand for new diagnostic tests and prognostic and therapeutic indicators for OA.

D-BOARD has addressed this global issue in a new and innovative way, providing real solutions to a real-life problem. D-BOARD has allowed the leading academic groups and SMEs working on OA biomarkers in Europe to join forces in order to tackle this problem. We have been successful with several subsequent EU grant applications and D-BOARD consortium partners will continue to pursue commercial opportunities within Europe, giving the EU an advantage over our competitors. Delivery “combination biomarkers” and early diagnostic tests and products for OA is an ongoing effort.

The biomarker discovery strategy proposed in D-BOARD has also facilitated the drug discovery process for OA by enhancing the ability to detect the disease process prior to the onset of the main symptoms (i.e. pain and loss of joint function) and supporting therapeutic interventions to slow or stop the disease process and allow the joint to heal and repair

Linking new biomarkers to clinical endpoints will support their use as surrogate biomarkers. Such markers will facilitate, drug discovery and development, post approval and regulatory decision-making. This area needs additional support from the European Commission and other funding agencies to synergise the activities of the European Medicines Agency (EMA), the US Food and Drug Administration (FDA) and the US National Institutes of Health (NIH).

In the meantime, OA and joint damage following injuries are still diagnosed radiographically when the clinical signs of pain and loss of mobility have already appeared. This is unfortunate and outdated. Current management strategies for OA primarily involve symptomatic pain relief but there is emerging evidence to suggest that many painkillers are ineffective and cause additional harm by increasing the chances of adverse effects in the cardiovascular system. Pharmacotherapy with conventional drugs does not influence disease progression and can be associated with significant side effects. Therefore, the acute need for alternative remedies for OA and novel biomarkers is still strongly felt. New OA biomarkers will help us diagnose the disease earlier and more effectively, understand the different phenotypes of OA and make drug discovery more efficient. We would like to thank the European Commission for their financial support and for making the D-BOARD project possible.


List of Websites:
http://www.d-board.eu/