Final Report Summary - MIAMI (Monitoring innate immunity in arthritis and mucosal inflammation)
Executive Summary:
In ‚inflammatory arthritis’, only a subgroup of patients has clear features of autoimmunity with classical autoantibodies (exemplified by rheumatoid factor-positive rheumatoid arthritis, RF+ RA), while the other variants are ‘seronegative’ arthritis syndromes (i.e. spondylarthritis, SpA; psoriatic arthritis, PsA; juvenile idiopathic arthritis, JIA) with a predominance of innate immune disturbances. These seronegative forms of joint diseases frequently show extra-articular manifestations of epithelial tissues like skin and gut. MIAMI’s main goal was to consolidate existing and develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis.
1) Mechanisms of initiation and progression of the disease. MIAMI researchers investigated the role of proinflammatory innate immune mechanisms in early inflammatory arthritis and bowel disease, aiming for translation into improved/novel diagnostics (short-term) and therapeutics (long-term). From the results we concluded that S100-DAMPs are important in the development of disease in different animal models of seronegative arthritis and in the acute state of bowel inflammation in human SpA patients. Overall, our results pave the way for further investigations that will be key to understand the biology of these S100-DAMPs in different disease settings.
2) Biomarkers for patient identification and stratification. MIAMI partners deliver improved/novel methodologies to enable early diagnosis of chronic joint and gut inflammation. In particular, the early identification of the disease in at-risk populations as well as disease extension (e.g. from the joint to the gut or skin) has been addressed in WP3 and WP6. In different clinically relevant diseases presenting with seronegative arthritis like JIA, SpA and PsA we have defined novel cut-offs for S100-proteins defining stages of active and inactive disease which are innovative and useful parameters for prediction of relapses in clinical settings. We provide evidence that phagocyte specific S100-proteins can be used for prediction of systemic dissemination of primarily local inflammatory processes, e.g. intestinal inflammation in SpA or systemic flares in SJIA in remission on medication.
3) Identification of novel disease markers for monitoring activity. To develop improved methodologies to support early diagnosis of chronic joint and gut inflammation, we aimed for the discovery of novel markers and innovative means of monitoring inflammation. In vivo optical imaging of S100A8 was found suitable for monitoring inflammation in IL-1Ra-/- mice, but not in SCW induced arthritis. We defined the innate inflammatory response patterns in joint and gut by cytokinomic, transcriptomic and proteomic analysis. Statistical analysis has been undertaken on the discovery data obtained independently from all three omics approaches. This will be used to generate a ‘megaplex’ assay for each of the clinical question raised in all MIAMI patient sample cohorts. We focused on monitoring of disease activity by biomarkers to support individualised medicine. We demonstrated the performance of biomarkers when used in therapeutic decisions concerning medication use in JIA.
4) Monitoring local disease activity and extension. For the diagnosis and therapy follow-up we have technically and clinically validated the S100A8/A9 ELISA as well as the lateral flow test, which are now ready to come as CE marked IVDs on the market. The MIAMI SME partner Bühlmann has developed and validated the lateral flow test system for S100A8/9 in stool extract samples and in serum. As there is a need for an easy detection system of lateral flow tests the project included the development of an App to be run on a series of different smartphones.
The MIAMI project has lead to significantly improved understanding of seronegative arthritis and also an excellent transfer of knowledge for innovative solutions that will be available for clinical applications in the monitoring of disease activity and prediction of complications. The results lead to numerous scientific publications in peer-reviewed high-impact journals as well as data presentations at major congresses. In light of the proceedings we are confident that MIAMI will continue to have significant impact and use when the final results become available after publishing further manuscripts that are already in preparation. MIAMI will facilitate the quick translation of significant innovations into clinical practice, with the aim to directly improve patient care.
In conclusion, the MIAMI project has delivered improved and/or novel methodology for early diagnosis of disease in people at risk, who don’t exhibit clinically relevant conventional indicators (yet). We could establish a list of biomarkers indicating onset and course of inflammation. With the help of SMEs that have a strong R&D commitment to biomarkers and personalized medicine solutions, assays have been developed that go beyond academic research formats. This lead to the development of proprietary point-of-care tests for easy and unrestricted use in the clinic or at the bedside. These tests will now be available for further validation. Lateral-flow immunoassays have been developed for simultaneous detection of a variety of proteins and evaluated in a prospective setting. Novel targets such as microRNA have been addressed. Finally, pilot data on the applicability of novel identified biomarker targets to be used in cutting-edge molecular imaging approaches have been provided, which will foster future validation studies. The latter holds great potential of further innovations going beyond the scope of in vitro biomarker determination.
Project Context and Objectives:
Despite enormous efforts in translating findings on autoantibodies and genetic susceptibility loci into diagnostic tools, biomarkers for individualized care of patients who suffer from chronic arthritis or bowel disease are still lacking. Over the past decade the perceived exclusive role of autoimmunity in chronic inflammatory diseases of the joint and gut has been challenged by the realisation that pure autoimmune processes are the exception rather than the rule. For example, in inflammatory bowel disease (IBD) key insights into mucosal immunity and genetic variants have underlined the additional importance of innate immune mechanisms. In joint disease, only a subgroup of patients has classical autoantibodies (exemplified by rheumatoid factor-positive rheumatoid arthritis or RF+ RA), while the other variants are ‘seronegative’ arthritis syndromes (i.e. spondylarthritis, SpA; psoriatic arthritis, PsA; juvenile idiopathic arthritis, JIA) with a predominance of innate immune disturbances.
It is of particular interest that these seronegative forms of joint diseases frequently show extra-articular manifestations of epithelial tissues like skin and gut. At the molecular level, we have shown that innate immune activation and the release of damage-associated molecular pattern proteins (DAMPs) of the S100 family are important mechanisms of disease initiation and progression. Despite extensive and impressive genetic studies by international consortia, the early diagnosis/identification of seronegative arthritis remains a significant problem, and prediction of disease progression is still extremely challenging. As a consequence, although our therapeutic arsenal is growing, it is currently not possible to optimally adapt the therapeutic regimen to the individual needs of a patient. We believe that the identification of specific inflammatory mechanisms that correlate to patterns of disease characteristics would allow more effective targeted therapeutic approaches.
MIAMI’s main goal was to consolidate existing and develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis.
Seronegative arthritis is an example of a multi-factorial disease with certain known genetic associations conferring overall susceptibility to the disease. A variety of environmental triggers may ultimately activate the disease in genetically susceptible individuals. It is established that following initial activation of both innate and adaptive immunity, various effector mechanisms contribute to the on-going inflammation. These mechanisms include contributions from immune cells and also soluble factors that act as mediators of the immune response. Two types of soluble factors, cytokines and DAMP molecules, have been identified as being of critical importance - they serve as immune amplifiers and have an impact on innate and adaptive immune responses.
Classically, the hallmark of ‘seropositive’ RA is the presence of rheumatoid factor and autoantibodies against cyclic citrullinated peptides. The fact that autoantibodies can be detected in RA has lead to attempts to define serological disease patterns based on these autoimmune features. In sharp contrast to RA however, other significant arthritis syndromes subsumed under the umbrella description of seronegative joint diseases are characterised by the absence of autoantibodies. By definition, disease marker patterns established on the basis of autoantibodies cannot be transferred from RA to seronegative arthritis. While this is undoubtedly a drawback for the development of biomarkers in arthritis in general, we are positive that recognizing the differences rather than emphasizing similarities are required for a better understanding of the disease processes, identification of biomarkers and therapeutic targets. In the long-term each of these will lead to better individually-tailored treatment options for patients with seronegative arthritis.
It is important to stratify patients according to disease extension and prognosis. It is a key task to identify patients who will develop complications such as the extension of the disease, which is a hallmark of patients who suffer from the progression of chronic inflammation from one organ system to another (e.g. from the joint to the gut or vice versa). In particular monitoring response to treatment, which is a keystone to develop truly individualized therapies, benefit from transfer of new insights into diagnostic technologies.
The diagnosis of chronic joint and gut disease is extremely challenging, and so is medical care for affected patients. To effectively treat patients, the early recognition of the disease is essential. The exact nature of the underlying inflammatory process – if clinically established at all – needs to be deciphered in order to start an anti-inflammatory therapy. At the same time, as a measure of secondary prevention in heterogeneous arthritis syndromes, the progression of joint disease, the extension of the disease towards complications in other organ systems such as the intestine, and the perpetuation of the inflammatory process should be identified. Further, the early identification of disease in vulnerable populations is an important objective for primary prevention.
Even after diagnosis has been achieved, more challenges for individualised clinical decisions - personalised medicine - await. Personalised medicine must meet three important objectives: 1. optimize efficacy of treatment for the individual patient, 2. avoid use of ineffective or unnecessary therapeutics and, 3. minimize healthcare costs. Although significant improvements have been made in clinical management, such patient-tailored therapy is still not a reality in rheumatology. Notably, the use of biomarkers that would be predictive of treatment response is not yet a part of clinical decision-making in daily practice. It seems apparent that in seronegative arthritis especially (a) the multifactorial, and only partly understood nature of disease pathogenesis, (b) the various predisposing genetic variants with modest effect sizes, and (c) a long list of factors influencing disease outcome are all significant barriers to the development of new effective molecular biomarkers.
MIAMI followed a translational concept: We analysed the mechanisms of disease initiation and progression with a special focus on innate immune activation that connects the joint and the gut as well as the skin. This basic research, involving experimental models of the disease in the murine system, were linked to clinical studies in which we used markers of innate immune activation for patient identification and stratification. We combined the translation of our molecular insights into clinical practise with a biomarker discovery effort to identify novel markers that enable monitoring of disease activity which a prerequisite for individualised therapeutic strategies. Biomarkers that indicate the extension of disease were addressed. Finally, we explored how we can further translate our findings into novel technologies including imaging tools, for which the translation process is bidirectional. So validated markers have been investigated initially by molecular imaging in mice with the longer-term goal of achieving a biomarker imaging strategy in humans.
To achieve our goals, we have systematically identified the most significant limitations of current knowledge and used this to identify the main activities that had to be undertaken to overcome the pre-existing paucity of molecular markers. The scientific focus of MIAMI - directed to achieving better diagnostic tools and novel targets for pharmaceutical therapies to significantly improve patient care in seronegative arthritis - can be summarized into four main objectives, for which special work packages (WPs) had been designed:
Objective 1: WP1&2 provided, via deep analyses of disease mechanisms, details on the expression and pro-inflammatory function of S100-DAMPs in arthritis and mucosal inflammation. Apart from adding further knowledge on these biomarkers in vitro, molecular imaging tools for in vivo diagnostics have been delivered.
Objective 2: WP3&6 validated S100-DAMP biomarkers for patient identification and stratification. This hypothesis-driven approach offered the potential for early diagnosis of disease, comorbidities or complications in populations at risk, and concluded close to commercial use.
Objective 3: WP4&5 provided comprehensive unbiased research to establish a list of biomarkers indicating onset and course of inflammation, allowing improved strategies for therapy. Major staff efforts allocated to them demonstrating the role of ‘omics’.
Objective 4: WP2&5 overcame the lack of in vitro and in vivo diagnostics for monitoring local inflammation and disease extension or progression. Scientific coordination, dissemination, and project management were major tasks of WP7-9.
As our ultimate goal was the translation of knowledge into clinical tools, we co-initiated the project with industry partners who guaranteed the delivery of novel/improved technologies. It was a particular strength of MIAMI to have Bühlmann in a leading position (WP6, WP8). Since many years, this company develops and refines products on exactly those biomarkers, which were in the focus of our concept. Bühlmann was Work Package Leader of horizontal WP6 ‘Technical Innovation’, thus ideally contributing to the expected impact of MIAMI.
The choice of the scientific as well as industry partners was entirely based on excellence. Thus, MIAMI brought together the leading teams that were working on mechanisms of innate immunity in arthritis and IBD. The available technologies, cohorts, and biobanks were linked and centrally coordinated to assure that our deliverables could be accomplished within the project’s timeframe. The success of MIAMI and a number of links to other initiatives, including other EU-projects, will offer sustainability of MIAMI’s progress.
Project Results:
As outlined above, on objective of MIAMI was to investigate the role of proinflammatory innate immune mechanisms in early inflammatory arthritis and bowel disease, aiming for translation into improved/novel diagnostics (short-term) and therapeutics (long-term).
The basic research in WP1 and WP2 was performed with the following Tasks and deliverables:
• To analyse of the expression of S100-DAMPs in arthritis and mucosal inflammation, using synovial fluid, articular and intestinal biopsy tissue. Special attention was given to compare their role in systemic immune activation (by analysing S100-DAMPs on human monocyte-macrophage functions) as opposed to their role at sites of inflammation (gut or/and joint compartments).
• To investigate S100-DAMPs as bystanders, amplifiers, or as disease-driving forces in cellular assays and mouse models of experimental arthritis and colitis, with a focus on the proinflammatory function of S100-DAMPs in arthritis and mucosal inflammation).
WP1 – Deep analysis of molecular mechanisms
WP 1 focussed on S100A8, S100A9 and S100A12, pro-inflammatory members of the DAMP-family, which are released by activated phagocytes. It provided a framework for evaluating S100 proteins as therapeutic targets in chronic inflammatory diseases of gut and joint.
Spondyloarthritis (SpA) is a multifactorial disease that consists of a group of chronic inflammatory diseases that primarily affect the musculoskeletal system and share common clinical features, genetic susceptibility and pathophysiological mechanisms. Subtypes of SpA include ankylosing spondylitis (AS), psoriatic arthritis (PsA), some juvenile forms, reactive arthritis (ReA) and inflammatory bowel disease (IBD) associated arthritis. Interestingly, about 50% of SpA-patients develop subclinical bowel inflammation, a trait that is correlated to disease severity. Furthermore, SpA is typically characterized by inflammatory stiffness, swelling and/or loss of function of the axial skeleton (spine and sacroiliac joints) or peripheral joints which may lead to irreversible structural and functional impairment and loss in quality of life.
Since SpA is not defined by the presence of auto-antibodies, it used to be classified as ‘seronegative’ arthritis. Discussion can be raised if SpA is an auto-immune disease or rather an auto-inflammatory disease, driven by danger associated molecular patterns (DAMPs). DAMPs are typically released upon cell stress and bind pattern recognition receptors (PRR) such as Toll like receptor (TLR)-4 and receptor for advanced glycosylation end products (RAGE). These molecules are known to have intracellular homeostatic but also extracellular pro-inflammatory functions. DAMPs known to be upregulated in rheumatoid arthritis (RA) and SpA are S100A8, S100A9, S100A8/A9 (calprotectin) and S100A12. They can steer immune responses by facilitating leucocyte extravasation, modulation of monocyte/macrophage function and differentiation and activation of adaptive immunity in general. While expression levels of S100A8/A9 correlate well with clinical arthritic disease activity in RA and SpA, surprisingly little is known about their role in the pathophysiology of these diseases.
In WP1 we focused on the role of these S100-DAMPs in the development of SpA, more specifically their link to gut-inflammation and cartilage/bone destruction, two common characteristics of SpA. Both in vitro and in vivo approaches were used to elucidate the role these S100-DAMPs can play in disease initiation and progression. Our data show that S100-DAMPs are very interesting markers to stratify patients, but that they do not always play a mechanistic role in the pathogenesis of SpA.
Analyses on serum and fecal calprotectin-levels from the GIANT-cohort showed that these DAMPs can be used to predict – with a certain level of certitude - if a patient has subclinical gut inflammation or not. Immunohistochemical stains were performed on gut biopsies from this cohort, indicating that S100-DAMPs were mostly present when an acute inflammatory infiltrate was present. To date, we have limited results on the impact of S100-DAMPs on monocyte/macrophage function due to some technical issues combined with recent advances and progressions in the innate immune field that changed our vision on the polarisation of macrophages.
Different mousemodels were used to assess the role of S100-DAMPs in TNF-dependent and TNF-independent models, as well as models used to evaluate the impact of S100-DAMPs on cartilage and bone destruction. Interestingly, not all animal models responded equally to depletion of S100A8/A9. We hypothesize that in some models calprotectin might be a contributing factor to disease development - especially when disease is in an early stage - but not the driver of disease. In contrast, some TNF-dependent models had worsened disease upon deletion of S100A9. Also, the IL1ra-/- - an IL-1 dependent model – showed amelioration of disease upon deletion of S100A9. These results are very interesting and need further investigation as they might unravel a dual role for S100A8 in the presence or absence of S100A9 and different effects of S100A9-depletion depending on the cytokine-environment.
It can be cocluded from WP1 that S100-DAMPs are interesting biomarkers in both human SpA and for some SpA-models. However, their role as initiators and drivers of disease needs further investigation as divergent results were obtained from different animal models. These data also implicate that depending on the cytokine-environment; S100-DAMPs and especially S100A8 might have different effector-functions. This is an interesting observation, since not all patients react similarly to anti-cytokine therapy. Overall, further investigation is needed and it will be key to understand the biology of these S100-DAMPs in different disease settings.
WP2 – Molecular Imaging of S100-damps in experimental arthritis
All forms of seronegative arthritis are characterized by inflamed joints and cartilage/bone destruction without interference of autoantibodies and pro-inflammatory S100-DAMPs are crucial in mediating synovial activation and joint destruction during experimental arthritis. The objective of WP2 was to image macrophages and S100-DAMPs in mouse models of seronegative arthritis and correlate this to joint destruction.
To monitor monocyte migration in vivo, immortalized murine monocyte ER-Hoxb8 cells from wildtype (WT) and S100A9-/- mice were successfully labelled with the fluorescent dye DIR, which allowed for in vivo tracking of monocyte migration by optical imaging. Migration of DIR-ER-Hoxb8 cells were investigated in two experimental models for seronegative arthritis; the Streptococcus pyogeneses cell wall (SCW) fragment induced arthritis and IL-1Ra-/- mice. Specific migration of both WT and S100A9-/- DIR-ER-Hoxb8 cells was observed in inflamed SCW injected ankle joints compared to contra-lateral saline injected ankle joints at 24 hour p.i. At 48 hours p.i. the signal of WT Hoxb8 cells further increased in SCW injected ankle joints, however the signal of S100A9-/- Hoxb8 cells remained similar. Also in IL-1Ra-/- mice a specific signal of WT and S100A9-/- DIR-ER-Hoxb8 cells was observed in the inflamed ankle joints, which correlated to the degree of joint swelling and the signal did not differ between WT and S100A9-/- DIR-ER-Hoxb8 cells. To investigate localisation of DIR-ER-Hoxb8 cells within the IL-1Ra-/- ankle joint, cells were transfected with GFP-lentivirus and GFP immunohistochemistry revealed similar distribution of WT and S100A9-/- ER-Hoxb8 cells in the inflamed synovium and bone marrow of the IL-1Ra-/- ankle joint.
To monitor local levels of S100-proteins in the inflamed joints, mice with SCW induced arthritis and IL-1Ra-/- mice were injected with anti-S100A8-Cy7 or irrelevant rabbit-IgG-Cy7. A significant increased signal in inflamed ankle joints of IL-1Ra-/- mice was observed for anti-S100A8-Cy7 compared to rabbit-IgG-Cy7 using optical imaging, indicating specific targeting of anti-S100A8-Cy7 to S100A8 in vivo. Indeed, targeting of anti-S100A8-Cy7 correlated to joint swelling in IL-1Ra-/- mice. However, in SCW induced arthritic ankle joints no specific targeting of anti-S100A8-Cy7 could be observed.
To further explore the relation of the observed increased S100-protein levels to joint destruction, we investigated MMP mediated cartilage damage and MMP activity using optical imaging in SCW induced arthritis and IL-1Ra-/- mice Increased VDIPEN neoepitopes in articular cartilage was associated with increased S100A9 expression in arthritic ankle joints of IL-1Ra-/- mice and increased MMP activity was monitored in vivo by molecular imaging of AF489-Cy5.5 a small molecule inhibitor of activated MMPs. To investigate the role of MMP in SCW induced arthritis, arthritis was induced in S100A9-/- mice. No change in inflammation or joint destruction could be observed between WT and S100A9-/- mice. Moreover, although increased MMP activity could be detected upon SCW induced arthritis using optical imaging of MMPSense680 (a probe which becomes fluorescent after cleavage by MMPs), no difference in signal was detected between WT and S100A9-/- mice.
It can be concluded from WP2 that migration of DIR labeled ER-Hoxb8 cells to inflamed joints in experimental models of seronegative arthritis can be monitored by in vivo optical imaging. No difference in signal between WT and S100A9-/- ER-HoxB8 cells was observed 24 hours p.i. but 48 hours p.i. WT ER-Hoxb8 cell infiltration further increased over time while S100A9-/- ER-HoxB8 signal remained unchanged, indicating that intracellular S100A9 is involved in long term migration of monocytes to inflamed joints. I.v. injected WT and S100A9-/- ER-HoxB8 cells were detected equally by immunohistochemistry in the synovium and bone marrow of the inflamed joint, indicating that intracellular S100A9 is not involved in the localisation of the monocyte within the inflamed joint.
Furthermore, local expression of S100A8 in IL-1Ra-/- mice can be monitored by in vivo optical imaging using anti-S100A8-Cy7, but not in SCW induced arthritis. A continuous expression of S100-proteins, as in IL-1Ra-/- mice, is necessary for sufficient targeting of anti-S100A8-Cy7 in the inflamed joint, while a short increase in expression of S100-proteins, as in SCW induced arthritis, is not adequate for in vivo imaging.
Finally, increased expression of S100-proteins in inflamed joints of IL-1Ra-/- mice is associated with increased MMP activity which can be monitored by in vivo optical imaging. Increased MMP activity in SCW induced arthritis, as assessed by optical imaging, is not changed in S100A9-/- mice compared to WT mice, indicating that S100-proteins need to be released continuously in high amounts (IL-1Ra-/- mice) in order to contribute to MMP mediated cartilage destruction.
The translational research in WP3 and WP6 was performed with the following Tasks and deliverables:
• DAMPs have been used as biomarkers for an early identification of patients (e.g. systemic JIA) or groups at special risk (e.g. mucosal or articular inflammation in relatives of patients with SpA, IBD, or Psoriasis).
• The performance of different assays for S100A8/A9 and S100A12 regarding disease-specific cut-offs in the different forms of seronegative arthritis and bowel disease was optimized. In addition, and as a strong translational link to objective 1, a quantitative assay for murine S100A8/A9 was established (in murine models of arthritis) to monitor disease activity.
• For a major technical innovation, we translated our findings from objective 1 into clinical application by addressing specific targets (monomers of S100A8 and S100A9, complex forms, other S100-DAMPS); specific formats (semi-quantitative bedside tests, stool assays, blood, synovial fluid, imaging); or specific indications (screening of risk populations, detection of organ-involvement, prediction of response and outcome, confirmation of disease remission). Novel point-of-care tests were developed and validated, e.g. lateral-flow immunoassays.
WP3 – S100-DAMPs as early disease markers in preclinical disease stages and risk populations for disease extension
In WP3, by using ELISA technology we established S100A8/S100A9 as the first reliable biomarker for monitoring disease activity in various murine models of arthritis and analysed samples from different MIAMI partners with this newly developed ELISA. We found that complexes of S100A8/S100A9 are highly expressed in synovial fluid and tissue in inflammatory arthritis in mice and serum concentrations are reliable biomarkers for quantification of local disease activity. We analysed patient samples for S100-concentrations and defined cut-off levels for residual disease activity and clinical remission. Combining CRP and serum S100A8/S100A9 provided added value for detection of bowel inflammation in SpA patients, which is probably explained by the fact that serum S100A8/S100A9 was linked especially with bowel inflammation of the acute type, whereas CRP was rather linked with chronic bowel inflammation.
In SpA, microscopic bowel inflammation is present in up to 50% patients without associated gastrointestinal symptoms. Histologically it can be indistinguishable from early Crohn’s disease and the presence of this inflammation entails an increased risk of developing full blown Crohn’s disease. Hence, it can be seen as a preclinical form of Crohn’s disease. It has also been shown to be linked with a more severe SpA phenotype. Two types of inflammation can be distinguished based on histology (not disease duration): an acute type showing mainly granulocytic inflammation, and an chronic type showing disturbance of mucosal architecture and a chronic lymphoplasmacytic cellular infiltrate. Since it is generally asymptomatic, screening based on symptoms is not possible, and diagnosis is based on endoscopy, which is an invasive procedure. Therefore, there is a great need for reliable biomarkers.
S100A8/S100A9 was evaluated as a marker for microscopic bowel inflammation in SpA patients from the GIANT cohort. Combining CRP and serum S100A8/S100A9 provided added value for detection of bowel inflammation, which is probably explained by the fact that serum S100A8/S100A9 was linked especially with bowel inflammation of the acute type, whereas CRP was rather linked with chronic bowel inflammation. Faecal S100A8/S100A9 was also significantly higher in SpA patients with microscopic bowel inflammation and could provide further differentiation when only CRP or only serum S100A8/S100A9 was elevated. A cut-off for serum and faecal S100A8/S100A9 was defined using ROC analysis, and a screening approach for microscopic bowel inflammation in SpA was developed based on combinations of CRP, serum and faecal S100A8/S100A9, which provided an AUC of 74.4%. These results were published in 2015.
WP6 – Technical Innovations
In WP6 existing immunological test systems have been optimized, and they were tested them in different body fluids (e.g. blood, synovial fluids, stool). Existing tests were brought on new, more precise (desirably multiplexed) platforms for bedside tests.
During the MIAMI project a series of peer-reviewed articles have been published reporting on either the value in following inflammatory status or monitoring the therapeutic efficiency of DMARDs. At the start of the MIAMI project, BÜHLMANN could already provide both a CE/IVD marked ELISA and a quantitative lateral flow rapid test for stool applications, fCAL® ELISA and Quantum Blue® Calprotectin.
In the first phase of the project we have technically validated both assays for serum application according to the European IVD directive and the CLSI guidelines. The clinical validation using over 500 patient samples was performed with the serum calprotectin (sCAL) ELISA and the serum MRP8/14 Quantum Blue® lateral flow assay. The correlation of the sCAL ELISA and the Quantum Blue® MRP8/14 was performed with a total of 451 serum samples and yielded following results: r = 0.911 and a slight bias of 7% for the ELISA. We have achieved a reliable, reproducible and robust standardisation for both test systems. These two tests have been launched as CE/IVD marked products within the time frame of the MIAMI project.
Although S100A8/A9 seems to be a more promising and more specific marker for the therapy follow-up than the currently used C-reactive protein (CRP), CRP remains a useful marker to determine inflammation status of RA patients and may also be predictive for a successful DMARD therapy. In this context, BÜHLMANN and the consortium decided to develop and validate a quantitative rapid test, the Quantum Blue® hsCRP, as a near patient solution. The test is as sensitive as laboratory methods and exhibits a clinical valuable measuring range from 1 to 25 mg/L. The correlation to a clinical chemistry method running on the Konelab™ system (Thermo Scientific) is so excellent (r = 0.972; bias -3.6%) that it is not necessarily required to perform a lot of clinical studies, but the results and conclusions of hundreds of such clinical studies conducted with precise and accurate laboratory methods can be translated to our hsCRP point-of-care test. The serum Quantum Blue® hsCRP has been launched as CE/IVD marked product within the time frame of the MIAMI project. Furthermore, we have also validated the hsCRP test for capillary blood. We have developed a tailor-made capillary blood collection and processing tool set which can be used together with the Quantum Blue® hsCRP.
Within the time frame of the MIAMI project, a third point-of-care test could be launched as CE/IVD marked product, namely the Quantum Blue® Infliximab test for serum. This assay shall be the first in a series of assays to be developed beyond the time frame of the MIAMI project to assess the trough levels of biologics (particularly anti-TNF monoclonal antibody drugs) which are of most importance in the therapy monitoring of chronic inflammatory diseases (such as RA) treated with bDMARDs. The quantitative measuring range of the rapid Infliximab test covers well the therapeutically important decision range between 3 and 7 µg/mL. It exhibits excellent correlations to the two “gold standard” ELISAs in the field (r = 0.933 and 0.938 respectively). The most attractive feature of our new assay is that the therapeutic trough levels can be determined on-site in the presence of the patient (or ultimately by the patient her/himself; cf. task 6.3) just before (within 15 minutes) the next drug infusion is applied what is not possible with alternative and time-consuming methods involving a laboratory infrastructure.
The project was also used to develop of a dedicated App which should turn a smartphone into a reading system together with a concomitant web portal for the storage of all test, batch, patient and physician/clinic specific data. Apart from turning the smartphone into a reading device three particular challenges were to solve: the perfect and frictionless communication between app and web portal, the structuration of the web portal into and different user access levels each containing very different information and their interconnections, and last but not at least the data security. At the end of the MIAMI project we can report that i) we could solve all the technical and regulatory challenges satisfactorily, ii) the system was rated as very useful by patients as well as treating clinicians or nurses in a series of pilot studies, and iii) we achieved end of March 2015 the CE/IVD mark issued by the TÜV Süd for the first self testing (home testing) system of its kind. The system was branded as “IBDoc®” (as the first application of this system is the monitoring of faecal calprotectin in diagnosed IBD patients under treatment and under control of healthcare professionals) and released to the market in summer 2015. In the meantime approximately a dozen of exploratory, but also clinical studies have started all around the globe and more than 600 real patients are using the IBDoc® regularly at home to monitor the efficiency of the treatment of their IBD diseases.
Another main objective of the MIAMI project was the unbiased identification of novel biomarkers. For several reasons, RNA has great biomarker potential: RNA is dynamic, diverse, measurement technologies are ready and RNA data outpace other –omics levels for clinical decision making. Non-coding RNA like microRNAs have some additional advantages as biomarkers including their tissue, cell type and disease specific expression patterns and the wide variety expressed in body fluids, showing their potential as minimal-invasive biomarkers. Within WP6 of the MIAMI project, Biogazelle has further developed and optimized the technologies and workflows for microRNA biomarker discovery and development with a clear focus on body fluids (serum, stool supernatans and synovial fluid). First of all, a modified RNA isolation protocol to increase yield, purity and concentration of the RNA extracted from body fluids was extensively validated and approved. Further, a qPCR-based platform for miRNA expression profiling allowing expression profiling of all miRNA entries in miRBase 20 (+2400) was introduced and validated. In addition to a new qPCR workflow, Biogazelle also introduced small RNA sequencing as a workflow for biomarker discovery. Finally, we developed an end-to-end solution for biomarker development, including analytical and clinical validation of a qPCR-based clinical grade test.
It can be concluded from WP6 that the “highlight” out of BÜHLMANN’s MIAMI development pipeline is the first CE/IVD marked home test ever to monitor the treatment of a chronic inflammatory disease by the patient her/himself using the own smartphone out of a current selection of up to 21 different Android and iPhone models. Biogazelle has successfully developed and implemented a microRNA biomarker discovery workflow to identify miRNA biomarker signatures in body fluids and to further develop the signatures in a qPCR-based clinical grade test.
The translational research in WP4 and WP5 was performed with the following Tasks and deliverables:
• To develop new improved methodologies to support early diagnosis of chronic joint and gut inflammation, we discovered novel markers. In WP4, we defined the innate inflammatory response patterns in joint and gut inflammation by cytokinomic, transcriptomic and proteomic analysis. In WP5, we focused on monitoring of disease activity by biomarkers to support individualised medicine.
• We delivered a panel of biomarkers, which indicate onset and severity of disease and which are the foundation for biomarker-assays (in patient material ex vivo) for therapeutic intervention. The further validation of the list of biomarkers delivered is important for future approaches to monitor local inflammatory activity and disease extension.
WP 4 – Unbiased identification of novel biomarkers
In WP4, we identified novel markers for risk for disease extension using unbiased ‘omics’ approaches to define the inflammatory expression patterns in joint and gut. Rigorous and integrated application of cytokinomic, transcriptomic and proteomic strategies was employed to support: i) mechanistic insight into disease pathogenesis, ii) novel target discovery and, iii) identification of potential early biomarkers. Therefire, we used advanced unbiased protein expression profiling strategies to comprehensively examine diseases induced changes in protein expression. There was a focus on highly multiplexed and sensitive measurement of cytokines, chemokines and other significant inflammation-related proteins by Multiplex Immuno Assay/xMAP technology. In addition, microRNA expression profiling using the state-of-the-art stem-loop RT-PCR was analysed.
Novel biomarkers for disease diagnosis and extension using unbiased ‘omics’ approaches to define the inflammatory expression patterns in joint and gut have been identified using rigorous and integrated application of cytokinomic (Luminex), transcriptomic (miRNA) and proteomic (LC/MS/MS) strategies. Statistical analysis has been undertaken on the discovery data obtained independently from all three omics approaches. This omics (cytokinomic, transcriptomic and proteomic) discovery data for all four patient sample cohorts (UCD, Ghent, Muenster, and Utrecht) have now been collated onto a shared (within MIAMI consortium members) Google drive folder and is being analysed by collaborating statisticians at UCD, who are applying various methods of multivariate analysis to the combined data. This might potentially generate a ‘megaplex’ assay for each of the clinical question raised in the four patient sample cohorts. Additionally, the identified multi-biomarker panels for some of the raised clinical questions are being further evaluated/verified on additional clinical patient samples beyond the MIAMI project for which additional external funding have been obtained.
WP 5 – Monitoring disease activity by biomarkers for individualized medicine
In WP5, we focused on monitoring of disease activity using biomarkers for individualized medicine in order to use the identified biomarker panels as tools to improve outcome measures in chronic joint and gut disease. After providing the first proof-of-principle, we designed prospective clinical studies to implement stratified therapeutic approaches based upon these biomarker panels. This was used to translate the findings of the other WP’s into potential applications. In particular, longitudinal determination of biomarkers in clinical studies implementing stratified therapeutic approaches in PsA, SpA and/or IBD has been performed. Longitudinal determination of biomarkers in clinical studies implementing stratified therapeutic approaches in SpA and/or IBD. The performance of biomarkers in guiding therapeutic decisions in JIA has also been evaluated.
The main goal of WP5 was to translate biomarker findings from other work packages, and from previously observed biomarkers into potential diagnostic tools. In order to achieve this, new cohorts were started in which the performance of biomarkers during therapeutic intervention was monitored. Cohorts of PSA patients, SpA patients and systemic JIA patients were successfully initiated within this work package. Important questions that were addressed are whether we can predict sustained remission or flares on therapy in PsA and sJIA and whether subclinical disease activity can be followed in SpA patients. Results show that S100A8/A9 levels correlate with PsA disease activity. A cohort with of 80 PsA patients has been formed to prospectively follow up on these data and to set cut off values of S100A8/A9 and S100A12 for prediction of flares.
Results also show that indeed subclinical disease activity can be followed by faecal S100A8/A9 levels. For the cohort in which this data will further be validated after stopping early anti-TNFa treatment follow up data are still being collected. WP5 has resulted cut off values of S100-proteins and IL-18 for prediction of flares after treatment stop, which can be validated in a new cohort.
In coclusion, WP5 has resulted in the translation of biomarkers into potential clinical tools for prediction of disease course and therapy responses. With the cohorts started in this work package, potential biomarkers that were described years ago are now finally being validated and prospectively followed in PsA, SpA and sJIA. Moreover, the biomarkers that were discovered in an unbiased manner in WP4 were further investigated in WP5 cohorts, thereby successfully providing an immediate platform for further development of clinical and diagnostic applications.
Potential Impact:
Overall contribution of MIAMI to expected impacts
The MIAMI project has provided a number of significant innovations and facilitated their quick translation into clinical practice, with the aim to directly improve patient care.
MIAMI delivered improved and/or novel methodology for early diagnosis of disease in people at risk, who don’t exhibit clinically relevant conventional indicators (yet). We established a list of biomarkers indicating onset and course of inflammation, and we devised potential strategies for therapeutic intervention, including identification of cellular and molecular targets for treatment of the disease.
With the help of SMEs that have a strong R&D commitment to biomarkers and personalized medicine solutions, assays were developed that go beyond academic research formats. BÜHLMANN developed proprietary point-of-care tests for easy and unrestricted use in the clinic or at the bedside, and validated them. Multiplex assays or lateral-flow immunoassays were developed for simultaneous detection of a variety of proteins, and they were evaluated in a prospective setting. Novel targets such as microRNA were addressed in innovative assay formats and validated prospectively as well. Finally, pilot data on the applicability of the identified biomarker targets to be used in cutting-edge molecular imaging approaches were provided. The latter holds great potential of further innovations going beyond the scope of in vitro biomarker determination.
Dissemination to the scientific community started already at the early beginning of the project by the implementation of a website (www.miamiproject.eu) to share the goal and the activities of the MIAMI project. Throughout the course of the MIAMI project, the website was at regular time points updated with relevant publications, news items and event announcements. Furthermore, the MIAMI partners attended major conferences which gave them the opportunity for scientific discussions and published in peer-reviewed journals. Finally, at the end of the project, the MIAMI International Symposium was organized, with more than 80 participants attending. This was the final communication event for the MIAMI consortium. The consortium members presented the work done during the 39 months of project duration, focussing on translational approaches in seronegative arthritis and juvenile rheumatic diseases and looking at new aspects of innate immune dysregulation while employing new means for monitoring inflammation. The program was organized into six scientific and one technical innovation session, which was included to showcase the translation of the MIAMI consortium scientific findings into the clinical market.
Dissemination of the results to the industry, the clinicians, and the end users was mainly done by the two SMEs part of the MIAMI consortium (BÜHLMANN Laboratories and Biogazelle). They presented their products at a large number of scientific and commercial conferences and exhibitions. Finally, the two SME’s established a roadmap for the further use and exploitation of the results. Biogazelle, responsible for the unbiased identification of miRNA biomarker signatures, has an established workflow for the further development of the biomarker signature into a qPCR-based clinical grade test, including analytical and clinical validation. BÜHLMANN has launched an improved MRP8/14 ELISA and a quantitative MRP8/14 rapid test. Furthermore, they were also able to launch two novel quantitative rapid tests. Finally, the IBDoc® home monitoring testing system has gained CE/IVD mark in March 2015. It is worldwide the first (patient) self-testing tool of its kind certified by a notified body (TÜV Süd). Today, it is operating in 26 countries in 16 languages on 21 different types of iPhones and Android phones and used by more than 700 patients all around Europe, Middle East, New Zealand and Australia. It is planned to expand the use of the IBDoc® system for a series of other chronic inflammatory diseases and new biomarker tests.
As a major impact of the project, MIAMI thus delivered improved methodology to enable early diagnosis of chronic inflammatory diseases and establishment of a list of biomarkers indicating onset of inflammation. It was our main goal to develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis with and without mucosal inflammation. It is obvious that better diagnostic tools are urgently required for both objective diagnosis and optimal management. Reliable biomarkers should indicate the onset of inflammation and thereby enable a timely diagnosis in the early disease phase. This is a key task to prevent long-term sequela of the disabling inflammatory diseases subsumed under the umbrella of ‘seronegative arthritis’. It is important to search for novel biomarkers and diagnostic tools that go beyond our current clinical practise primarily based upon clinical impression or secondary laboratory phenomena. Instead we need biomarkers that are sensitive enough to provide information on the immunological status of the patient including subclinical inflammation, which is present at disease onset but “under the surface” and not initially leading to overt symptoms as chronic inflammatory disorders of the joint and gut are progressing variably.
After having accomplished MIAMI we are able to translate novel biomarkers into improved methodology based on profound knowledge on the pathophysiology of chronic inflammation. The focus of biomarker development was on those factors that clearly have a molecular role in the initiation, progression or modulation of the disease processes in arthritis and/or mucosal inflammation. We applied novel methodologies such as point-of-care tests and rapid detection assays. S100-DAMPs fulfil all prerequisites. Furthermore, novel methodologies using the potential of S100-DAMPs in molecular imaging were established in preclinical research, which are translated into clinical tools quickly as promising molecules that target S100-DAMPs are already approved for the use in humans and can therefore be adapted for the use as molecular imaging probes visualizing S100-DAMPs. MIAMI also went beyond the hypothesis-driven by providing novel markers and further applications. In unbiased ‘omics’ approaches the innate inflammatory response patterns in joint and gut was analysed by rigorous application of cytokinomic, transcriptomic and proteomic technology. This comprehensive approach, combining data from different cutting-edge research technologies, was unique in the field and thus provided for the first time a list of biomarker candidates in hand that can be used in clinical practice.
As another major impact of the project, MIAMI lead to the identification of molecular and cellular pathways involved in initiation of the diseases, which allow for prediction of potential development of the disease in yet healthy population. It is of significant socio-economic importance to have biomarkers that not only indicate the onset of inflammation but also allow identification of patients who will develop a disease or specific phenotype in the preclinical phase. In order to end up with meaningful biomarker candidates for this purpose it is mandatory to focus on inflammatory processes and to identify factors that are involved in initiating, amplifying and perpetuating the disease. To contribute to this expected impact we therefore followed a translational concept in MIAMI and brought together leading experts in the field into a multidisciplinary team of rheumatologists, gastroenterologist, immunologists and industry partners.
After having accomplished MIAMI we are able to predict the development of major disease manifestation in yet unaffected population based upon biomarkers. Mechanisms of disease initiation and extension in seronegative arthritis and/or IBD characterised by a dominant activation of innate immune mechanisms compared to autoimmune features were established. We took advantage of the expertise of three of our partners working on animal models of arthritis and colitis. By using diverse disease models with varying involvement of different effector pathways and by employing genetically modified animals available in our hands to modulate specific components relevant to innate immune activation we provided deep analyses of molecular mechanisms. We analysed arthritis and colitis in the various mouse models with a special focus on S100-DAMPs, which will also serve as targets for imaging. We also provided a better understand of the development of complications or specific features in human seronegative arthritis. As an example, we addressed the extension of arthritis into gut by comparing our data with IBD cohorts that were also included. We now have a better understanding of the molecular basis of the heterogeneity, but we also better know how to use this understanding for diagnostic purposes. This allows individualised therapeutic interventions. We were able to predict of the development of these disease manifestation in yet unaffected population by using biomarkers. Patients with arthritis have a risk to develop gut disease, patients with psoriasis of the skin to develop arthritis, children with JIA to develop systemic features, and patients with IBD to develop joint disease. The MIAMI results showed that we can identify patients at risk, including the prediction of disease flares.
A socio-economic impact with wider societal implications was due to the development of potential strategies for therapeutic intervention including identification of cellular and molecular targets for treatment of the disease. The development of novel strategies for therapeutic intervention was impacted by MIAMI in two ways: 1) Modern therapeutic strategies shall be personalised instead of generalised (as they are today); they should take the individual immunological predisposition and risk-profiles into account and allow to adapt medication to the actual need of a given patient at a particular time or stage of the disease. 2) Novel therapeutic strategies shall include new drugs that intelligently target specific molecules and factors directly involved in the inflammatory processes; they should allow more specific interventions, thus minimizing adverse unspecific effects while at the same time enhancing effectiveness.
After having accomplished MIAMI we are able to steer therapeutic strategies by addressing both of the above-mentioned ways. We implemented biomarker-driven therapeutic strategies beyond proof-of-concept studies. During the long-term management of patients, therapies constantly need to be adjusted. The monitoring of therapy responses and the detection of subclinical inflammation in patients in disease remission was thus analysed for the use of biomarkers in the further management of patients. To this end, a study on therapy withdrawal in patients with JIA based upon the remission status as defined by S100-DAMPs was already initiated. Thus, MIAMI contributed to the expected impact by promoting personalised medicine with the aim to have improved strategies for therapeutic interventions that take the individual patients’ condition into account. MIAMI also contributed to the expected impact of providing novel molecular targets for direct therapeutic interventions.
The goals of MIAMI were ambitious and could only be reached with cutting-edge research performed by the most excellent researchers in the field. In addition to providing experience and technologies, well-characterised cohorts and sample collections were available through the combination in a strong and comprehensive consortium. In addition, combining the expertise already present in different centres in the different EU countries helped to establish new lines of cooperative research. MIAMI was also well-connected with other relevant European initiatives. This ensures quick uptake of our results by the clinical and scientific community. D. Foell, J. Roth, and B. Prakken contribute to the European training network ‘EUTRAIN’ and ‘PHARMACHILD’. D. Elewaut is the Co-director of the EULAR and FOCIS Centre of Excellence at UGent, a leading centre of SpA research. O. Fitzgerald is a founding member of the Psoriatic Arthritis Genetics in Europe (PAGE) and on the co-ordinating group within the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) organization for both the GRACE and PsA BioDAM projects. S. Pennington is a committee member of the British Society for Proteome Research (BSPR) and is in the General Council of the European Proteomics Association (EuPA). P. van Lent also participates in other European consortia like NanoDiaRA and BT-cure.
Similar to the issue above, the correlation of biomarker results to disease activity relies on valid clinical disease activity scores. In fact, disease activity measurements have been changing over the past years, and the definition of status dimensions such as ‘active disease’, ‘inactive disease’, ‘clinical remission on medication’, ‘clinical remission off medication’, or ‘response to therapy’ is far from being definite. Some of the MIAMI partners are actively involved in defining these criteria (O. FitzGerald, D. Elewaut, and D. Foell). As outlined above, classifying the status of a patient regarding clinical impression on inflammatory disease activity will be performed on the basis of current standards, which will not necessarily hold true forever. The results of MIAMI turned out to be very useful for the definition of improved disease activity measurements.
Main dissemination activities and exploitation of results
We are confident that MIAMI produced significant applicable knowledge. We identified four main target groups, which need to be addressed using different strategies:
1. The general public, in particular patients
2. The scientific community, including clinicians
3. Industry, on a precompetitive base
4. “End users” of biomarker assays such as clinical routine and University Hospital laboratories.
We have very rigorously disseminated our project results by setting up different activities and addressing the target groups. MIAMI raised awareness about the project and beyond MIAMI about chronic inflammation, including the general public and patients. The results were presented in lay summaries via the project's website and distributed among patient organisations, to which the consortium has excellent relations. Press releases have been used to inform a broader audience.
Chronic inflammatory joint and gut diseases are among the most significant challenges for our health care system, but also our whole society. Up to 8 million European citizens suffer from seronegative arthritis alone. Approximately 2 million European citizens have chronic IBD. Moreover, about 200,000 European children are affected by JIA. With the aging of the population, the disease burden is expected to increase over the coming decades. These diseases often take a disabling course and are among the health care problems with the highest social burden worldwide. In addition, the treatment of the disease and associated complications mean a substantial limitation of the quality of life to the patients and their families. It is challenging to determine whether, and in which subgroup of patients, specific therapeutic interventions offer additional benefit. Therefore, the major challenges with direct relevance for clinical practice are currently the development of techniques for early diagnosis, monitoring of inflammation and prediction of complications or manifestations of the disease, e.g. the parallel existence of joint and gut inflammation.
We took all efforts to reach the public and to inform not only about MIAMI, but also about the often neglected chronic inflammatory diseases of the joints and gut. We addressed the patients, but also the general public and also key opinion leaders. Our project contributed to the improvement of patient care in chronic inflammatory joint and bowel disease. It had a major impact on the development of therapeutic approaches not only in seronegative arthritis syndromes, but also in other inflammatory diseases. As chronic arthritis confers an enormous economical burden to the society, the dissemination of our results was very important. On the other hand, the distribution among economy and stakeholders was also important to attract industry to the field of biomarker discovery and assay development. With emphasizing the relevance of biomarker-driven therapies for the general health-care system, research and development in this area was encouraged.
The regular way to reach the scientific community including clinicians is often relatively slow. Of course, the partners of MIAMI used their results in lecturing and editorial activities, and naturally results will be presented at conferences, such as: the American College of Rheumatology conference (ACR), the annual meetings of the European League Against Rheumatism (EULAR) and the Paediatric Rheumatology European Society (PRES). The results were further distributed by publications in peer reviewed journals. The project's webpage informed about the diseases, the research activities, biomarker lists and proceedings. Workshops are more interactive, direct dissemination activity that proved very effective in the past: we performed workshops for clinical labs and practitioners. An activity which went considerably beyond usual dissemination plans is the offer for biomarker assays free of charge, which addresses a major drawback in biomarker research: the lack of translation into clinical applications that can be used in daily clinical practise. As the development of novel technologies takes time and the availability on the market is not easy to predict, we believe that a great way of dissemination was to offer valid biomarker tests to clinicians and scientist who are not partners of MIAMI. We invited centers in Europe to use biomarker analyses free of charge.
The results of MIAMI also had a significant impact on industry. Both the ability to provide biomarker candidates ready for being applied and the ability to bring up targets for future successful therapies beared enormous potential for industry. Directly within MIAMI already there was significant innovation, and the project results clearly benefit the SME-partners and lead to improved technologies and applications for diagnostic purposes. MIAMI added further application fields, improved quality and standardisation, and also lead to new technologies and markers. Bühlmann was co-developer, manufacturer, and markets the products directly and worldwide. The developed assays are easy to perform, efficient and not costly. Another impact was the identification of novel therapeutic targets, which could be licensed by the SMEs and universities to pharmaceutical companies. The influence on industry was strong as the partners of MIAMI had links to larger pharmaceutical companies who were also informed about biomarker-driven measurements of therapeutic responses. This already successfully led to cooperation (e.g. with Novartis, Pfizer). On the other hand, links to pharmaceutical companies also attracted their attention to new molecular targets that could be used for the development of therapies.
The SMEs clearly benefited from MIAMI and also focus on end users. The finding of potential new markers of the S100 superfamily and others lead to new research into, and characterization of these markers. They were used for the development of new immunological tests. Bühlmann introduced new quantitative point-of-care tests (POC, lateral flow). The experience gained in this project by expanding Biogazelle's qPCR based microRNA gene expression platform content by 30% wase useful for future content expansions as well. It allowed Biogazelle to develop high quality custom multiplex primer pools better and faster for other future applications. The data established with S100A8/A9 test formats of Bühlmann (ELISA and lateral flow POC) in the seronegative inflammatory diseases were of outmost importance so that the test can be used as clinically validated test for a precise diagnosis and for a proper patient treatment with the very expensive new biologics such as anti-TNFα drugs (e.g. therapy control). Bühlmann made use of a proof-of-concept study on a new, more robust and quantitative POC platform with several markers on it (multiplexing capability of the new platform is a further asset and superior than the existing one). Biogazelle will obtained critical insights in and hands-on experience with the various RNA extraction procedures tested for the different tissue types or body fluids. Bühlmann submitted patents of new assays and/or applications and will manufacture and distribute existing and new products over its own channels.
In summary, MIAMI significantly contributed to progress on the key questions: Who will be affected by which disease manifestation or complication, how can we use this knowledge to identify the disease, and what will be a meaningful target to treat or even prevent deleterious outcome. In MIAMI, an expert team successfully performed a joint enterprise with the goal of understanding the disease and translating it into personalised medicine. Novel biomarkers, improved technologies, and promising targets for therapeutic interventions were established. We provided profound and focussed trainings, both to the members of the consortium and to clinicians and clinical laboratories. Results were extensively disseminated to the scientific community and the end users (clinicians and clinical laboratories). The MIAMI international symposium, organized at the end of the project, was a huge success and the perfect platform for interesting scientific discussions. Both SME partners in the consortium have established a robust plan for the further exploitation of the obtained results. In the case of BÜHLMANN, the newly developed diagnostic and therapy monitoring tests have already been released to the market as CE/IVD labeled products and some of them showed considerable turnover numbers and are well accepted at various customers’ sites.
List of Websites:
Public MIAMI website:
http://www.miamiproject.eu
Contact:
Prof. Dirk Foell, MD
University Hospital Muenster
Pediatric Rheumatology and Immunology
Domagkstr. 3
D-48149 Muenster
Phone ++49-251-8358178
Fax ++49-251-8358104
Email dfoell@uni-muenster.de
In ‚inflammatory arthritis’, only a subgroup of patients has clear features of autoimmunity with classical autoantibodies (exemplified by rheumatoid factor-positive rheumatoid arthritis, RF+ RA), while the other variants are ‘seronegative’ arthritis syndromes (i.e. spondylarthritis, SpA; psoriatic arthritis, PsA; juvenile idiopathic arthritis, JIA) with a predominance of innate immune disturbances. These seronegative forms of joint diseases frequently show extra-articular manifestations of epithelial tissues like skin and gut. MIAMI’s main goal was to consolidate existing and develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis.
1) Mechanisms of initiation and progression of the disease. MIAMI researchers investigated the role of proinflammatory innate immune mechanisms in early inflammatory arthritis and bowel disease, aiming for translation into improved/novel diagnostics (short-term) and therapeutics (long-term). From the results we concluded that S100-DAMPs are important in the development of disease in different animal models of seronegative arthritis and in the acute state of bowel inflammation in human SpA patients. Overall, our results pave the way for further investigations that will be key to understand the biology of these S100-DAMPs in different disease settings.
2) Biomarkers for patient identification and stratification. MIAMI partners deliver improved/novel methodologies to enable early diagnosis of chronic joint and gut inflammation. In particular, the early identification of the disease in at-risk populations as well as disease extension (e.g. from the joint to the gut or skin) has been addressed in WP3 and WP6. In different clinically relevant diseases presenting with seronegative arthritis like JIA, SpA and PsA we have defined novel cut-offs for S100-proteins defining stages of active and inactive disease which are innovative and useful parameters for prediction of relapses in clinical settings. We provide evidence that phagocyte specific S100-proteins can be used for prediction of systemic dissemination of primarily local inflammatory processes, e.g. intestinal inflammation in SpA or systemic flares in SJIA in remission on medication.
3) Identification of novel disease markers for monitoring activity. To develop improved methodologies to support early diagnosis of chronic joint and gut inflammation, we aimed for the discovery of novel markers and innovative means of monitoring inflammation. In vivo optical imaging of S100A8 was found suitable for monitoring inflammation in IL-1Ra-/- mice, but not in SCW induced arthritis. We defined the innate inflammatory response patterns in joint and gut by cytokinomic, transcriptomic and proteomic analysis. Statistical analysis has been undertaken on the discovery data obtained independently from all three omics approaches. This will be used to generate a ‘megaplex’ assay for each of the clinical question raised in all MIAMI patient sample cohorts. We focused on monitoring of disease activity by biomarkers to support individualised medicine. We demonstrated the performance of biomarkers when used in therapeutic decisions concerning medication use in JIA.
4) Monitoring local disease activity and extension. For the diagnosis and therapy follow-up we have technically and clinically validated the S100A8/A9 ELISA as well as the lateral flow test, which are now ready to come as CE marked IVDs on the market. The MIAMI SME partner Bühlmann has developed and validated the lateral flow test system for S100A8/9 in stool extract samples and in serum. As there is a need for an easy detection system of lateral flow tests the project included the development of an App to be run on a series of different smartphones.
The MIAMI project has lead to significantly improved understanding of seronegative arthritis and also an excellent transfer of knowledge for innovative solutions that will be available for clinical applications in the monitoring of disease activity and prediction of complications. The results lead to numerous scientific publications in peer-reviewed high-impact journals as well as data presentations at major congresses. In light of the proceedings we are confident that MIAMI will continue to have significant impact and use when the final results become available after publishing further manuscripts that are already in preparation. MIAMI will facilitate the quick translation of significant innovations into clinical practice, with the aim to directly improve patient care.
In conclusion, the MIAMI project has delivered improved and/or novel methodology for early diagnosis of disease in people at risk, who don’t exhibit clinically relevant conventional indicators (yet). We could establish a list of biomarkers indicating onset and course of inflammation. With the help of SMEs that have a strong R&D commitment to biomarkers and personalized medicine solutions, assays have been developed that go beyond academic research formats. This lead to the development of proprietary point-of-care tests for easy and unrestricted use in the clinic or at the bedside. These tests will now be available for further validation. Lateral-flow immunoassays have been developed for simultaneous detection of a variety of proteins and evaluated in a prospective setting. Novel targets such as microRNA have been addressed. Finally, pilot data on the applicability of novel identified biomarker targets to be used in cutting-edge molecular imaging approaches have been provided, which will foster future validation studies. The latter holds great potential of further innovations going beyond the scope of in vitro biomarker determination.
Project Context and Objectives:
Despite enormous efforts in translating findings on autoantibodies and genetic susceptibility loci into diagnostic tools, biomarkers for individualized care of patients who suffer from chronic arthritis or bowel disease are still lacking. Over the past decade the perceived exclusive role of autoimmunity in chronic inflammatory diseases of the joint and gut has been challenged by the realisation that pure autoimmune processes are the exception rather than the rule. For example, in inflammatory bowel disease (IBD) key insights into mucosal immunity and genetic variants have underlined the additional importance of innate immune mechanisms. In joint disease, only a subgroup of patients has classical autoantibodies (exemplified by rheumatoid factor-positive rheumatoid arthritis or RF+ RA), while the other variants are ‘seronegative’ arthritis syndromes (i.e. spondylarthritis, SpA; psoriatic arthritis, PsA; juvenile idiopathic arthritis, JIA) with a predominance of innate immune disturbances.
It is of particular interest that these seronegative forms of joint diseases frequently show extra-articular manifestations of epithelial tissues like skin and gut. At the molecular level, we have shown that innate immune activation and the release of damage-associated molecular pattern proteins (DAMPs) of the S100 family are important mechanisms of disease initiation and progression. Despite extensive and impressive genetic studies by international consortia, the early diagnosis/identification of seronegative arthritis remains a significant problem, and prediction of disease progression is still extremely challenging. As a consequence, although our therapeutic arsenal is growing, it is currently not possible to optimally adapt the therapeutic regimen to the individual needs of a patient. We believe that the identification of specific inflammatory mechanisms that correlate to patterns of disease characteristics would allow more effective targeted therapeutic approaches.
MIAMI’s main goal was to consolidate existing and develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis.
Seronegative arthritis is an example of a multi-factorial disease with certain known genetic associations conferring overall susceptibility to the disease. A variety of environmental triggers may ultimately activate the disease in genetically susceptible individuals. It is established that following initial activation of both innate and adaptive immunity, various effector mechanisms contribute to the on-going inflammation. These mechanisms include contributions from immune cells and also soluble factors that act as mediators of the immune response. Two types of soluble factors, cytokines and DAMP molecules, have been identified as being of critical importance - they serve as immune amplifiers and have an impact on innate and adaptive immune responses.
Classically, the hallmark of ‘seropositive’ RA is the presence of rheumatoid factor and autoantibodies against cyclic citrullinated peptides. The fact that autoantibodies can be detected in RA has lead to attempts to define serological disease patterns based on these autoimmune features. In sharp contrast to RA however, other significant arthritis syndromes subsumed under the umbrella description of seronegative joint diseases are characterised by the absence of autoantibodies. By definition, disease marker patterns established on the basis of autoantibodies cannot be transferred from RA to seronegative arthritis. While this is undoubtedly a drawback for the development of biomarkers in arthritis in general, we are positive that recognizing the differences rather than emphasizing similarities are required for a better understanding of the disease processes, identification of biomarkers and therapeutic targets. In the long-term each of these will lead to better individually-tailored treatment options for patients with seronegative arthritis.
It is important to stratify patients according to disease extension and prognosis. It is a key task to identify patients who will develop complications such as the extension of the disease, which is a hallmark of patients who suffer from the progression of chronic inflammation from one organ system to another (e.g. from the joint to the gut or vice versa). In particular monitoring response to treatment, which is a keystone to develop truly individualized therapies, benefit from transfer of new insights into diagnostic technologies.
The diagnosis of chronic joint and gut disease is extremely challenging, and so is medical care for affected patients. To effectively treat patients, the early recognition of the disease is essential. The exact nature of the underlying inflammatory process – if clinically established at all – needs to be deciphered in order to start an anti-inflammatory therapy. At the same time, as a measure of secondary prevention in heterogeneous arthritis syndromes, the progression of joint disease, the extension of the disease towards complications in other organ systems such as the intestine, and the perpetuation of the inflammatory process should be identified. Further, the early identification of disease in vulnerable populations is an important objective for primary prevention.
Even after diagnosis has been achieved, more challenges for individualised clinical decisions - personalised medicine - await. Personalised medicine must meet three important objectives: 1. optimize efficacy of treatment for the individual patient, 2. avoid use of ineffective or unnecessary therapeutics and, 3. minimize healthcare costs. Although significant improvements have been made in clinical management, such patient-tailored therapy is still not a reality in rheumatology. Notably, the use of biomarkers that would be predictive of treatment response is not yet a part of clinical decision-making in daily practice. It seems apparent that in seronegative arthritis especially (a) the multifactorial, and only partly understood nature of disease pathogenesis, (b) the various predisposing genetic variants with modest effect sizes, and (c) a long list of factors influencing disease outcome are all significant barriers to the development of new effective molecular biomarkers.
MIAMI followed a translational concept: We analysed the mechanisms of disease initiation and progression with a special focus on innate immune activation that connects the joint and the gut as well as the skin. This basic research, involving experimental models of the disease in the murine system, were linked to clinical studies in which we used markers of innate immune activation for patient identification and stratification. We combined the translation of our molecular insights into clinical practise with a biomarker discovery effort to identify novel markers that enable monitoring of disease activity which a prerequisite for individualised therapeutic strategies. Biomarkers that indicate the extension of disease were addressed. Finally, we explored how we can further translate our findings into novel technologies including imaging tools, for which the translation process is bidirectional. So validated markers have been investigated initially by molecular imaging in mice with the longer-term goal of achieving a biomarker imaging strategy in humans.
To achieve our goals, we have systematically identified the most significant limitations of current knowledge and used this to identify the main activities that had to be undertaken to overcome the pre-existing paucity of molecular markers. The scientific focus of MIAMI - directed to achieving better diagnostic tools and novel targets for pharmaceutical therapies to significantly improve patient care in seronegative arthritis - can be summarized into four main objectives, for which special work packages (WPs) had been designed:
Objective 1: WP1&2 provided, via deep analyses of disease mechanisms, details on the expression and pro-inflammatory function of S100-DAMPs in arthritis and mucosal inflammation. Apart from adding further knowledge on these biomarkers in vitro, molecular imaging tools for in vivo diagnostics have been delivered.
Objective 2: WP3&6 validated S100-DAMP biomarkers for patient identification and stratification. This hypothesis-driven approach offered the potential for early diagnosis of disease, comorbidities or complications in populations at risk, and concluded close to commercial use.
Objective 3: WP4&5 provided comprehensive unbiased research to establish a list of biomarkers indicating onset and course of inflammation, allowing improved strategies for therapy. Major staff efforts allocated to them demonstrating the role of ‘omics’.
Objective 4: WP2&5 overcame the lack of in vitro and in vivo diagnostics for monitoring local inflammation and disease extension or progression. Scientific coordination, dissemination, and project management were major tasks of WP7-9.
As our ultimate goal was the translation of knowledge into clinical tools, we co-initiated the project with industry partners who guaranteed the delivery of novel/improved technologies. It was a particular strength of MIAMI to have Bühlmann in a leading position (WP6, WP8). Since many years, this company develops and refines products on exactly those biomarkers, which were in the focus of our concept. Bühlmann was Work Package Leader of horizontal WP6 ‘Technical Innovation’, thus ideally contributing to the expected impact of MIAMI.
The choice of the scientific as well as industry partners was entirely based on excellence. Thus, MIAMI brought together the leading teams that were working on mechanisms of innate immunity in arthritis and IBD. The available technologies, cohorts, and biobanks were linked and centrally coordinated to assure that our deliverables could be accomplished within the project’s timeframe. The success of MIAMI and a number of links to other initiatives, including other EU-projects, will offer sustainability of MIAMI’s progress.
Project Results:
As outlined above, on objective of MIAMI was to investigate the role of proinflammatory innate immune mechanisms in early inflammatory arthritis and bowel disease, aiming for translation into improved/novel diagnostics (short-term) and therapeutics (long-term).
The basic research in WP1 and WP2 was performed with the following Tasks and deliverables:
• To analyse of the expression of S100-DAMPs in arthritis and mucosal inflammation, using synovial fluid, articular and intestinal biopsy tissue. Special attention was given to compare their role in systemic immune activation (by analysing S100-DAMPs on human monocyte-macrophage functions) as opposed to their role at sites of inflammation (gut or/and joint compartments).
• To investigate S100-DAMPs as bystanders, amplifiers, or as disease-driving forces in cellular assays and mouse models of experimental arthritis and colitis, with a focus on the proinflammatory function of S100-DAMPs in arthritis and mucosal inflammation).
WP1 – Deep analysis of molecular mechanisms
WP 1 focussed on S100A8, S100A9 and S100A12, pro-inflammatory members of the DAMP-family, which are released by activated phagocytes. It provided a framework for evaluating S100 proteins as therapeutic targets in chronic inflammatory diseases of gut and joint.
Spondyloarthritis (SpA) is a multifactorial disease that consists of a group of chronic inflammatory diseases that primarily affect the musculoskeletal system and share common clinical features, genetic susceptibility and pathophysiological mechanisms. Subtypes of SpA include ankylosing spondylitis (AS), psoriatic arthritis (PsA), some juvenile forms, reactive arthritis (ReA) and inflammatory bowel disease (IBD) associated arthritis. Interestingly, about 50% of SpA-patients develop subclinical bowel inflammation, a trait that is correlated to disease severity. Furthermore, SpA is typically characterized by inflammatory stiffness, swelling and/or loss of function of the axial skeleton (spine and sacroiliac joints) or peripheral joints which may lead to irreversible structural and functional impairment and loss in quality of life.
Since SpA is not defined by the presence of auto-antibodies, it used to be classified as ‘seronegative’ arthritis. Discussion can be raised if SpA is an auto-immune disease or rather an auto-inflammatory disease, driven by danger associated molecular patterns (DAMPs). DAMPs are typically released upon cell stress and bind pattern recognition receptors (PRR) such as Toll like receptor (TLR)-4 and receptor for advanced glycosylation end products (RAGE). These molecules are known to have intracellular homeostatic but also extracellular pro-inflammatory functions. DAMPs known to be upregulated in rheumatoid arthritis (RA) and SpA are S100A8, S100A9, S100A8/A9 (calprotectin) and S100A12. They can steer immune responses by facilitating leucocyte extravasation, modulation of monocyte/macrophage function and differentiation and activation of adaptive immunity in general. While expression levels of S100A8/A9 correlate well with clinical arthritic disease activity in RA and SpA, surprisingly little is known about their role in the pathophysiology of these diseases.
In WP1 we focused on the role of these S100-DAMPs in the development of SpA, more specifically their link to gut-inflammation and cartilage/bone destruction, two common characteristics of SpA. Both in vitro and in vivo approaches were used to elucidate the role these S100-DAMPs can play in disease initiation and progression. Our data show that S100-DAMPs are very interesting markers to stratify patients, but that they do not always play a mechanistic role in the pathogenesis of SpA.
Analyses on serum and fecal calprotectin-levels from the GIANT-cohort showed that these DAMPs can be used to predict – with a certain level of certitude - if a patient has subclinical gut inflammation or not. Immunohistochemical stains were performed on gut biopsies from this cohort, indicating that S100-DAMPs were mostly present when an acute inflammatory infiltrate was present. To date, we have limited results on the impact of S100-DAMPs on monocyte/macrophage function due to some technical issues combined with recent advances and progressions in the innate immune field that changed our vision on the polarisation of macrophages.
Different mousemodels were used to assess the role of S100-DAMPs in TNF-dependent and TNF-independent models, as well as models used to evaluate the impact of S100-DAMPs on cartilage and bone destruction. Interestingly, not all animal models responded equally to depletion of S100A8/A9. We hypothesize that in some models calprotectin might be a contributing factor to disease development - especially when disease is in an early stage - but not the driver of disease. In contrast, some TNF-dependent models had worsened disease upon deletion of S100A9. Also, the IL1ra-/- - an IL-1 dependent model – showed amelioration of disease upon deletion of S100A9. These results are very interesting and need further investigation as they might unravel a dual role for S100A8 in the presence or absence of S100A9 and different effects of S100A9-depletion depending on the cytokine-environment.
It can be cocluded from WP1 that S100-DAMPs are interesting biomarkers in both human SpA and for some SpA-models. However, their role as initiators and drivers of disease needs further investigation as divergent results were obtained from different animal models. These data also implicate that depending on the cytokine-environment; S100-DAMPs and especially S100A8 might have different effector-functions. This is an interesting observation, since not all patients react similarly to anti-cytokine therapy. Overall, further investigation is needed and it will be key to understand the biology of these S100-DAMPs in different disease settings.
WP2 – Molecular Imaging of S100-damps in experimental arthritis
All forms of seronegative arthritis are characterized by inflamed joints and cartilage/bone destruction without interference of autoantibodies and pro-inflammatory S100-DAMPs are crucial in mediating synovial activation and joint destruction during experimental arthritis. The objective of WP2 was to image macrophages and S100-DAMPs in mouse models of seronegative arthritis and correlate this to joint destruction.
To monitor monocyte migration in vivo, immortalized murine monocyte ER-Hoxb8 cells from wildtype (WT) and S100A9-/- mice were successfully labelled with the fluorescent dye DIR, which allowed for in vivo tracking of monocyte migration by optical imaging. Migration of DIR-ER-Hoxb8 cells were investigated in two experimental models for seronegative arthritis; the Streptococcus pyogeneses cell wall (SCW) fragment induced arthritis and IL-1Ra-/- mice. Specific migration of both WT and S100A9-/- DIR-ER-Hoxb8 cells was observed in inflamed SCW injected ankle joints compared to contra-lateral saline injected ankle joints at 24 hour p.i. At 48 hours p.i. the signal of WT Hoxb8 cells further increased in SCW injected ankle joints, however the signal of S100A9-/- Hoxb8 cells remained similar. Also in IL-1Ra-/- mice a specific signal of WT and S100A9-/- DIR-ER-Hoxb8 cells was observed in the inflamed ankle joints, which correlated to the degree of joint swelling and the signal did not differ between WT and S100A9-/- DIR-ER-Hoxb8 cells. To investigate localisation of DIR-ER-Hoxb8 cells within the IL-1Ra-/- ankle joint, cells were transfected with GFP-lentivirus and GFP immunohistochemistry revealed similar distribution of WT and S100A9-/- ER-Hoxb8 cells in the inflamed synovium and bone marrow of the IL-1Ra-/- ankle joint.
To monitor local levels of S100-proteins in the inflamed joints, mice with SCW induced arthritis and IL-1Ra-/- mice were injected with anti-S100A8-Cy7 or irrelevant rabbit-IgG-Cy7. A significant increased signal in inflamed ankle joints of IL-1Ra-/- mice was observed for anti-S100A8-Cy7 compared to rabbit-IgG-Cy7 using optical imaging, indicating specific targeting of anti-S100A8-Cy7 to S100A8 in vivo. Indeed, targeting of anti-S100A8-Cy7 correlated to joint swelling in IL-1Ra-/- mice. However, in SCW induced arthritic ankle joints no specific targeting of anti-S100A8-Cy7 could be observed.
To further explore the relation of the observed increased S100-protein levels to joint destruction, we investigated MMP mediated cartilage damage and MMP activity using optical imaging in SCW induced arthritis and IL-1Ra-/- mice Increased VDIPEN neoepitopes in articular cartilage was associated with increased S100A9 expression in arthritic ankle joints of IL-1Ra-/- mice and increased MMP activity was monitored in vivo by molecular imaging of AF489-Cy5.5 a small molecule inhibitor of activated MMPs. To investigate the role of MMP in SCW induced arthritis, arthritis was induced in S100A9-/- mice. No change in inflammation or joint destruction could be observed between WT and S100A9-/- mice. Moreover, although increased MMP activity could be detected upon SCW induced arthritis using optical imaging of MMPSense680 (a probe which becomes fluorescent after cleavage by MMPs), no difference in signal was detected between WT and S100A9-/- mice.
It can be concluded from WP2 that migration of DIR labeled ER-Hoxb8 cells to inflamed joints in experimental models of seronegative arthritis can be monitored by in vivo optical imaging. No difference in signal between WT and S100A9-/- ER-HoxB8 cells was observed 24 hours p.i. but 48 hours p.i. WT ER-Hoxb8 cell infiltration further increased over time while S100A9-/- ER-HoxB8 signal remained unchanged, indicating that intracellular S100A9 is involved in long term migration of monocytes to inflamed joints. I.v. injected WT and S100A9-/- ER-HoxB8 cells were detected equally by immunohistochemistry in the synovium and bone marrow of the inflamed joint, indicating that intracellular S100A9 is not involved in the localisation of the monocyte within the inflamed joint.
Furthermore, local expression of S100A8 in IL-1Ra-/- mice can be monitored by in vivo optical imaging using anti-S100A8-Cy7, but not in SCW induced arthritis. A continuous expression of S100-proteins, as in IL-1Ra-/- mice, is necessary for sufficient targeting of anti-S100A8-Cy7 in the inflamed joint, while a short increase in expression of S100-proteins, as in SCW induced arthritis, is not adequate for in vivo imaging.
Finally, increased expression of S100-proteins in inflamed joints of IL-1Ra-/- mice is associated with increased MMP activity which can be monitored by in vivo optical imaging. Increased MMP activity in SCW induced arthritis, as assessed by optical imaging, is not changed in S100A9-/- mice compared to WT mice, indicating that S100-proteins need to be released continuously in high amounts (IL-1Ra-/- mice) in order to contribute to MMP mediated cartilage destruction.
The translational research in WP3 and WP6 was performed with the following Tasks and deliverables:
• DAMPs have been used as biomarkers for an early identification of patients (e.g. systemic JIA) or groups at special risk (e.g. mucosal or articular inflammation in relatives of patients with SpA, IBD, or Psoriasis).
• The performance of different assays for S100A8/A9 and S100A12 regarding disease-specific cut-offs in the different forms of seronegative arthritis and bowel disease was optimized. In addition, and as a strong translational link to objective 1, a quantitative assay for murine S100A8/A9 was established (in murine models of arthritis) to monitor disease activity.
• For a major technical innovation, we translated our findings from objective 1 into clinical application by addressing specific targets (monomers of S100A8 and S100A9, complex forms, other S100-DAMPS); specific formats (semi-quantitative bedside tests, stool assays, blood, synovial fluid, imaging); or specific indications (screening of risk populations, detection of organ-involvement, prediction of response and outcome, confirmation of disease remission). Novel point-of-care tests were developed and validated, e.g. lateral-flow immunoassays.
WP3 – S100-DAMPs as early disease markers in preclinical disease stages and risk populations for disease extension
In WP3, by using ELISA technology we established S100A8/S100A9 as the first reliable biomarker for monitoring disease activity in various murine models of arthritis and analysed samples from different MIAMI partners with this newly developed ELISA. We found that complexes of S100A8/S100A9 are highly expressed in synovial fluid and tissue in inflammatory arthritis in mice and serum concentrations are reliable biomarkers for quantification of local disease activity. We analysed patient samples for S100-concentrations and defined cut-off levels for residual disease activity and clinical remission. Combining CRP and serum S100A8/S100A9 provided added value for detection of bowel inflammation in SpA patients, which is probably explained by the fact that serum S100A8/S100A9 was linked especially with bowel inflammation of the acute type, whereas CRP was rather linked with chronic bowel inflammation.
In SpA, microscopic bowel inflammation is present in up to 50% patients without associated gastrointestinal symptoms. Histologically it can be indistinguishable from early Crohn’s disease and the presence of this inflammation entails an increased risk of developing full blown Crohn’s disease. Hence, it can be seen as a preclinical form of Crohn’s disease. It has also been shown to be linked with a more severe SpA phenotype. Two types of inflammation can be distinguished based on histology (not disease duration): an acute type showing mainly granulocytic inflammation, and an chronic type showing disturbance of mucosal architecture and a chronic lymphoplasmacytic cellular infiltrate. Since it is generally asymptomatic, screening based on symptoms is not possible, and diagnosis is based on endoscopy, which is an invasive procedure. Therefore, there is a great need for reliable biomarkers.
S100A8/S100A9 was evaluated as a marker for microscopic bowel inflammation in SpA patients from the GIANT cohort. Combining CRP and serum S100A8/S100A9 provided added value for detection of bowel inflammation, which is probably explained by the fact that serum S100A8/S100A9 was linked especially with bowel inflammation of the acute type, whereas CRP was rather linked with chronic bowel inflammation. Faecal S100A8/S100A9 was also significantly higher in SpA patients with microscopic bowel inflammation and could provide further differentiation when only CRP or only serum S100A8/S100A9 was elevated. A cut-off for serum and faecal S100A8/S100A9 was defined using ROC analysis, and a screening approach for microscopic bowel inflammation in SpA was developed based on combinations of CRP, serum and faecal S100A8/S100A9, which provided an AUC of 74.4%. These results were published in 2015.
WP6 – Technical Innovations
In WP6 existing immunological test systems have been optimized, and they were tested them in different body fluids (e.g. blood, synovial fluids, stool). Existing tests were brought on new, more precise (desirably multiplexed) platforms for bedside tests.
During the MIAMI project a series of peer-reviewed articles have been published reporting on either the value in following inflammatory status or monitoring the therapeutic efficiency of DMARDs. At the start of the MIAMI project, BÜHLMANN could already provide both a CE/IVD marked ELISA and a quantitative lateral flow rapid test for stool applications, fCAL® ELISA and Quantum Blue® Calprotectin.
In the first phase of the project we have technically validated both assays for serum application according to the European IVD directive and the CLSI guidelines. The clinical validation using over 500 patient samples was performed with the serum calprotectin (sCAL) ELISA and the serum MRP8/14 Quantum Blue® lateral flow assay. The correlation of the sCAL ELISA and the Quantum Blue® MRP8/14 was performed with a total of 451 serum samples and yielded following results: r = 0.911 and a slight bias of 7% for the ELISA. We have achieved a reliable, reproducible and robust standardisation for both test systems. These two tests have been launched as CE/IVD marked products within the time frame of the MIAMI project.
Although S100A8/A9 seems to be a more promising and more specific marker for the therapy follow-up than the currently used C-reactive protein (CRP), CRP remains a useful marker to determine inflammation status of RA patients and may also be predictive for a successful DMARD therapy. In this context, BÜHLMANN and the consortium decided to develop and validate a quantitative rapid test, the Quantum Blue® hsCRP, as a near patient solution. The test is as sensitive as laboratory methods and exhibits a clinical valuable measuring range from 1 to 25 mg/L. The correlation to a clinical chemistry method running on the Konelab™ system (Thermo Scientific) is so excellent (r = 0.972; bias -3.6%) that it is not necessarily required to perform a lot of clinical studies, but the results and conclusions of hundreds of such clinical studies conducted with precise and accurate laboratory methods can be translated to our hsCRP point-of-care test. The serum Quantum Blue® hsCRP has been launched as CE/IVD marked product within the time frame of the MIAMI project. Furthermore, we have also validated the hsCRP test for capillary blood. We have developed a tailor-made capillary blood collection and processing tool set which can be used together with the Quantum Blue® hsCRP.
Within the time frame of the MIAMI project, a third point-of-care test could be launched as CE/IVD marked product, namely the Quantum Blue® Infliximab test for serum. This assay shall be the first in a series of assays to be developed beyond the time frame of the MIAMI project to assess the trough levels of biologics (particularly anti-TNF monoclonal antibody drugs) which are of most importance in the therapy monitoring of chronic inflammatory diseases (such as RA) treated with bDMARDs. The quantitative measuring range of the rapid Infliximab test covers well the therapeutically important decision range between 3 and 7 µg/mL. It exhibits excellent correlations to the two “gold standard” ELISAs in the field (r = 0.933 and 0.938 respectively). The most attractive feature of our new assay is that the therapeutic trough levels can be determined on-site in the presence of the patient (or ultimately by the patient her/himself; cf. task 6.3) just before (within 15 minutes) the next drug infusion is applied what is not possible with alternative and time-consuming methods involving a laboratory infrastructure.
The project was also used to develop of a dedicated App which should turn a smartphone into a reading system together with a concomitant web portal for the storage of all test, batch, patient and physician/clinic specific data. Apart from turning the smartphone into a reading device three particular challenges were to solve: the perfect and frictionless communication between app and web portal, the structuration of the web portal into and different user access levels each containing very different information and their interconnections, and last but not at least the data security. At the end of the MIAMI project we can report that i) we could solve all the technical and regulatory challenges satisfactorily, ii) the system was rated as very useful by patients as well as treating clinicians or nurses in a series of pilot studies, and iii) we achieved end of March 2015 the CE/IVD mark issued by the TÜV Süd for the first self testing (home testing) system of its kind. The system was branded as “IBDoc®” (as the first application of this system is the monitoring of faecal calprotectin in diagnosed IBD patients under treatment and under control of healthcare professionals) and released to the market in summer 2015. In the meantime approximately a dozen of exploratory, but also clinical studies have started all around the globe and more than 600 real patients are using the IBDoc® regularly at home to monitor the efficiency of the treatment of their IBD diseases.
Another main objective of the MIAMI project was the unbiased identification of novel biomarkers. For several reasons, RNA has great biomarker potential: RNA is dynamic, diverse, measurement technologies are ready and RNA data outpace other –omics levels for clinical decision making. Non-coding RNA like microRNAs have some additional advantages as biomarkers including their tissue, cell type and disease specific expression patterns and the wide variety expressed in body fluids, showing their potential as minimal-invasive biomarkers. Within WP6 of the MIAMI project, Biogazelle has further developed and optimized the technologies and workflows for microRNA biomarker discovery and development with a clear focus on body fluids (serum, stool supernatans and synovial fluid). First of all, a modified RNA isolation protocol to increase yield, purity and concentration of the RNA extracted from body fluids was extensively validated and approved. Further, a qPCR-based platform for miRNA expression profiling allowing expression profiling of all miRNA entries in miRBase 20 (+2400) was introduced and validated. In addition to a new qPCR workflow, Biogazelle also introduced small RNA sequencing as a workflow for biomarker discovery. Finally, we developed an end-to-end solution for biomarker development, including analytical and clinical validation of a qPCR-based clinical grade test.
It can be concluded from WP6 that the “highlight” out of BÜHLMANN’s MIAMI development pipeline is the first CE/IVD marked home test ever to monitor the treatment of a chronic inflammatory disease by the patient her/himself using the own smartphone out of a current selection of up to 21 different Android and iPhone models. Biogazelle has successfully developed and implemented a microRNA biomarker discovery workflow to identify miRNA biomarker signatures in body fluids and to further develop the signatures in a qPCR-based clinical grade test.
The translational research in WP4 and WP5 was performed with the following Tasks and deliverables:
• To develop new improved methodologies to support early diagnosis of chronic joint and gut inflammation, we discovered novel markers. In WP4, we defined the innate inflammatory response patterns in joint and gut inflammation by cytokinomic, transcriptomic and proteomic analysis. In WP5, we focused on monitoring of disease activity by biomarkers to support individualised medicine.
• We delivered a panel of biomarkers, which indicate onset and severity of disease and which are the foundation for biomarker-assays (in patient material ex vivo) for therapeutic intervention. The further validation of the list of biomarkers delivered is important for future approaches to monitor local inflammatory activity and disease extension.
WP 4 – Unbiased identification of novel biomarkers
In WP4, we identified novel markers for risk for disease extension using unbiased ‘omics’ approaches to define the inflammatory expression patterns in joint and gut. Rigorous and integrated application of cytokinomic, transcriptomic and proteomic strategies was employed to support: i) mechanistic insight into disease pathogenesis, ii) novel target discovery and, iii) identification of potential early biomarkers. Therefire, we used advanced unbiased protein expression profiling strategies to comprehensively examine diseases induced changes in protein expression. There was a focus on highly multiplexed and sensitive measurement of cytokines, chemokines and other significant inflammation-related proteins by Multiplex Immuno Assay/xMAP technology. In addition, microRNA expression profiling using the state-of-the-art stem-loop RT-PCR was analysed.
Novel biomarkers for disease diagnosis and extension using unbiased ‘omics’ approaches to define the inflammatory expression patterns in joint and gut have been identified using rigorous and integrated application of cytokinomic (Luminex), transcriptomic (miRNA) and proteomic (LC/MS/MS) strategies. Statistical analysis has been undertaken on the discovery data obtained independently from all three omics approaches. This omics (cytokinomic, transcriptomic and proteomic) discovery data for all four patient sample cohorts (UCD, Ghent, Muenster, and Utrecht) have now been collated onto a shared (within MIAMI consortium members) Google drive folder and is being analysed by collaborating statisticians at UCD, who are applying various methods of multivariate analysis to the combined data. This might potentially generate a ‘megaplex’ assay for each of the clinical question raised in the four patient sample cohorts. Additionally, the identified multi-biomarker panels for some of the raised clinical questions are being further evaluated/verified on additional clinical patient samples beyond the MIAMI project for which additional external funding have been obtained.
WP 5 – Monitoring disease activity by biomarkers for individualized medicine
In WP5, we focused on monitoring of disease activity using biomarkers for individualized medicine in order to use the identified biomarker panels as tools to improve outcome measures in chronic joint and gut disease. After providing the first proof-of-principle, we designed prospective clinical studies to implement stratified therapeutic approaches based upon these biomarker panels. This was used to translate the findings of the other WP’s into potential applications. In particular, longitudinal determination of biomarkers in clinical studies implementing stratified therapeutic approaches in PsA, SpA and/or IBD has been performed. Longitudinal determination of biomarkers in clinical studies implementing stratified therapeutic approaches in SpA and/or IBD. The performance of biomarkers in guiding therapeutic decisions in JIA has also been evaluated.
The main goal of WP5 was to translate biomarker findings from other work packages, and from previously observed biomarkers into potential diagnostic tools. In order to achieve this, new cohorts were started in which the performance of biomarkers during therapeutic intervention was monitored. Cohorts of PSA patients, SpA patients and systemic JIA patients were successfully initiated within this work package. Important questions that were addressed are whether we can predict sustained remission or flares on therapy in PsA and sJIA and whether subclinical disease activity can be followed in SpA patients. Results show that S100A8/A9 levels correlate with PsA disease activity. A cohort with of 80 PsA patients has been formed to prospectively follow up on these data and to set cut off values of S100A8/A9 and S100A12 for prediction of flares.
Results also show that indeed subclinical disease activity can be followed by faecal S100A8/A9 levels. For the cohort in which this data will further be validated after stopping early anti-TNFa treatment follow up data are still being collected. WP5 has resulted cut off values of S100-proteins and IL-18 for prediction of flares after treatment stop, which can be validated in a new cohort.
In coclusion, WP5 has resulted in the translation of biomarkers into potential clinical tools for prediction of disease course and therapy responses. With the cohorts started in this work package, potential biomarkers that were described years ago are now finally being validated and prospectively followed in PsA, SpA and sJIA. Moreover, the biomarkers that were discovered in an unbiased manner in WP4 were further investigated in WP5 cohorts, thereby successfully providing an immediate platform for further development of clinical and diagnostic applications.
Potential Impact:
Overall contribution of MIAMI to expected impacts
The MIAMI project has provided a number of significant innovations and facilitated their quick translation into clinical practice, with the aim to directly improve patient care.
MIAMI delivered improved and/or novel methodology for early diagnosis of disease in people at risk, who don’t exhibit clinically relevant conventional indicators (yet). We established a list of biomarkers indicating onset and course of inflammation, and we devised potential strategies for therapeutic intervention, including identification of cellular and molecular targets for treatment of the disease.
With the help of SMEs that have a strong R&D commitment to biomarkers and personalized medicine solutions, assays were developed that go beyond academic research formats. BÜHLMANN developed proprietary point-of-care tests for easy and unrestricted use in the clinic or at the bedside, and validated them. Multiplex assays or lateral-flow immunoassays were developed for simultaneous detection of a variety of proteins, and they were evaluated in a prospective setting. Novel targets such as microRNA were addressed in innovative assay formats and validated prospectively as well. Finally, pilot data on the applicability of the identified biomarker targets to be used in cutting-edge molecular imaging approaches were provided. The latter holds great potential of further innovations going beyond the scope of in vitro biomarker determination.
Dissemination to the scientific community started already at the early beginning of the project by the implementation of a website (www.miamiproject.eu) to share the goal and the activities of the MIAMI project. Throughout the course of the MIAMI project, the website was at regular time points updated with relevant publications, news items and event announcements. Furthermore, the MIAMI partners attended major conferences which gave them the opportunity for scientific discussions and published in peer-reviewed journals. Finally, at the end of the project, the MIAMI International Symposium was organized, with more than 80 participants attending. This was the final communication event for the MIAMI consortium. The consortium members presented the work done during the 39 months of project duration, focussing on translational approaches in seronegative arthritis and juvenile rheumatic diseases and looking at new aspects of innate immune dysregulation while employing new means for monitoring inflammation. The program was organized into six scientific and one technical innovation session, which was included to showcase the translation of the MIAMI consortium scientific findings into the clinical market.
Dissemination of the results to the industry, the clinicians, and the end users was mainly done by the two SMEs part of the MIAMI consortium (BÜHLMANN Laboratories and Biogazelle). They presented their products at a large number of scientific and commercial conferences and exhibitions. Finally, the two SME’s established a roadmap for the further use and exploitation of the results. Biogazelle, responsible for the unbiased identification of miRNA biomarker signatures, has an established workflow for the further development of the biomarker signature into a qPCR-based clinical grade test, including analytical and clinical validation. BÜHLMANN has launched an improved MRP8/14 ELISA and a quantitative MRP8/14 rapid test. Furthermore, they were also able to launch two novel quantitative rapid tests. Finally, the IBDoc® home monitoring testing system has gained CE/IVD mark in March 2015. It is worldwide the first (patient) self-testing tool of its kind certified by a notified body (TÜV Süd). Today, it is operating in 26 countries in 16 languages on 21 different types of iPhones and Android phones and used by more than 700 patients all around Europe, Middle East, New Zealand and Australia. It is planned to expand the use of the IBDoc® system for a series of other chronic inflammatory diseases and new biomarker tests.
As a major impact of the project, MIAMI thus delivered improved methodology to enable early diagnosis of chronic inflammatory diseases and establishment of a list of biomarkers indicating onset of inflammation. It was our main goal to develop novel biomarkers for individual adaptation of therapies (personalized medicine) for seronegative arthritis with and without mucosal inflammation. It is obvious that better diagnostic tools are urgently required for both objective diagnosis and optimal management. Reliable biomarkers should indicate the onset of inflammation and thereby enable a timely diagnosis in the early disease phase. This is a key task to prevent long-term sequela of the disabling inflammatory diseases subsumed under the umbrella of ‘seronegative arthritis’. It is important to search for novel biomarkers and diagnostic tools that go beyond our current clinical practise primarily based upon clinical impression or secondary laboratory phenomena. Instead we need biomarkers that are sensitive enough to provide information on the immunological status of the patient including subclinical inflammation, which is present at disease onset but “under the surface” and not initially leading to overt symptoms as chronic inflammatory disorders of the joint and gut are progressing variably.
After having accomplished MIAMI we are able to translate novel biomarkers into improved methodology based on profound knowledge on the pathophysiology of chronic inflammation. The focus of biomarker development was on those factors that clearly have a molecular role in the initiation, progression or modulation of the disease processes in arthritis and/or mucosal inflammation. We applied novel methodologies such as point-of-care tests and rapid detection assays. S100-DAMPs fulfil all prerequisites. Furthermore, novel methodologies using the potential of S100-DAMPs in molecular imaging were established in preclinical research, which are translated into clinical tools quickly as promising molecules that target S100-DAMPs are already approved for the use in humans and can therefore be adapted for the use as molecular imaging probes visualizing S100-DAMPs. MIAMI also went beyond the hypothesis-driven by providing novel markers and further applications. In unbiased ‘omics’ approaches the innate inflammatory response patterns in joint and gut was analysed by rigorous application of cytokinomic, transcriptomic and proteomic technology. This comprehensive approach, combining data from different cutting-edge research technologies, was unique in the field and thus provided for the first time a list of biomarker candidates in hand that can be used in clinical practice.
As another major impact of the project, MIAMI lead to the identification of molecular and cellular pathways involved in initiation of the diseases, which allow for prediction of potential development of the disease in yet healthy population. It is of significant socio-economic importance to have biomarkers that not only indicate the onset of inflammation but also allow identification of patients who will develop a disease or specific phenotype in the preclinical phase. In order to end up with meaningful biomarker candidates for this purpose it is mandatory to focus on inflammatory processes and to identify factors that are involved in initiating, amplifying and perpetuating the disease. To contribute to this expected impact we therefore followed a translational concept in MIAMI and brought together leading experts in the field into a multidisciplinary team of rheumatologists, gastroenterologist, immunologists and industry partners.
After having accomplished MIAMI we are able to predict the development of major disease manifestation in yet unaffected population based upon biomarkers. Mechanisms of disease initiation and extension in seronegative arthritis and/or IBD characterised by a dominant activation of innate immune mechanisms compared to autoimmune features were established. We took advantage of the expertise of three of our partners working on animal models of arthritis and colitis. By using diverse disease models with varying involvement of different effector pathways and by employing genetically modified animals available in our hands to modulate specific components relevant to innate immune activation we provided deep analyses of molecular mechanisms. We analysed arthritis and colitis in the various mouse models with a special focus on S100-DAMPs, which will also serve as targets for imaging. We also provided a better understand of the development of complications or specific features in human seronegative arthritis. As an example, we addressed the extension of arthritis into gut by comparing our data with IBD cohorts that were also included. We now have a better understanding of the molecular basis of the heterogeneity, but we also better know how to use this understanding for diagnostic purposes. This allows individualised therapeutic interventions. We were able to predict of the development of these disease manifestation in yet unaffected population by using biomarkers. Patients with arthritis have a risk to develop gut disease, patients with psoriasis of the skin to develop arthritis, children with JIA to develop systemic features, and patients with IBD to develop joint disease. The MIAMI results showed that we can identify patients at risk, including the prediction of disease flares.
A socio-economic impact with wider societal implications was due to the development of potential strategies for therapeutic intervention including identification of cellular and molecular targets for treatment of the disease. The development of novel strategies for therapeutic intervention was impacted by MIAMI in two ways: 1) Modern therapeutic strategies shall be personalised instead of generalised (as they are today); they should take the individual immunological predisposition and risk-profiles into account and allow to adapt medication to the actual need of a given patient at a particular time or stage of the disease. 2) Novel therapeutic strategies shall include new drugs that intelligently target specific molecules and factors directly involved in the inflammatory processes; they should allow more specific interventions, thus minimizing adverse unspecific effects while at the same time enhancing effectiveness.
After having accomplished MIAMI we are able to steer therapeutic strategies by addressing both of the above-mentioned ways. We implemented biomarker-driven therapeutic strategies beyond proof-of-concept studies. During the long-term management of patients, therapies constantly need to be adjusted. The monitoring of therapy responses and the detection of subclinical inflammation in patients in disease remission was thus analysed for the use of biomarkers in the further management of patients. To this end, a study on therapy withdrawal in patients with JIA based upon the remission status as defined by S100-DAMPs was already initiated. Thus, MIAMI contributed to the expected impact by promoting personalised medicine with the aim to have improved strategies for therapeutic interventions that take the individual patients’ condition into account. MIAMI also contributed to the expected impact of providing novel molecular targets for direct therapeutic interventions.
The goals of MIAMI were ambitious and could only be reached with cutting-edge research performed by the most excellent researchers in the field. In addition to providing experience and technologies, well-characterised cohorts and sample collections were available through the combination in a strong and comprehensive consortium. In addition, combining the expertise already present in different centres in the different EU countries helped to establish new lines of cooperative research. MIAMI was also well-connected with other relevant European initiatives. This ensures quick uptake of our results by the clinical and scientific community. D. Foell, J. Roth, and B. Prakken contribute to the European training network ‘EUTRAIN’ and ‘PHARMACHILD’. D. Elewaut is the Co-director of the EULAR and FOCIS Centre of Excellence at UGent, a leading centre of SpA research. O. Fitzgerald is a founding member of the Psoriatic Arthritis Genetics in Europe (PAGE) and on the co-ordinating group within the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) organization for both the GRACE and PsA BioDAM projects. S. Pennington is a committee member of the British Society for Proteome Research (BSPR) and is in the General Council of the European Proteomics Association (EuPA). P. van Lent also participates in other European consortia like NanoDiaRA and BT-cure.
Similar to the issue above, the correlation of biomarker results to disease activity relies on valid clinical disease activity scores. In fact, disease activity measurements have been changing over the past years, and the definition of status dimensions such as ‘active disease’, ‘inactive disease’, ‘clinical remission on medication’, ‘clinical remission off medication’, or ‘response to therapy’ is far from being definite. Some of the MIAMI partners are actively involved in defining these criteria (O. FitzGerald, D. Elewaut, and D. Foell). As outlined above, classifying the status of a patient regarding clinical impression on inflammatory disease activity will be performed on the basis of current standards, which will not necessarily hold true forever. The results of MIAMI turned out to be very useful for the definition of improved disease activity measurements.
Main dissemination activities and exploitation of results
We are confident that MIAMI produced significant applicable knowledge. We identified four main target groups, which need to be addressed using different strategies:
1. The general public, in particular patients
2. The scientific community, including clinicians
3. Industry, on a precompetitive base
4. “End users” of biomarker assays such as clinical routine and University Hospital laboratories.
We have very rigorously disseminated our project results by setting up different activities and addressing the target groups. MIAMI raised awareness about the project and beyond MIAMI about chronic inflammation, including the general public and patients. The results were presented in lay summaries via the project's website and distributed among patient organisations, to which the consortium has excellent relations. Press releases have been used to inform a broader audience.
Chronic inflammatory joint and gut diseases are among the most significant challenges for our health care system, but also our whole society. Up to 8 million European citizens suffer from seronegative arthritis alone. Approximately 2 million European citizens have chronic IBD. Moreover, about 200,000 European children are affected by JIA. With the aging of the population, the disease burden is expected to increase over the coming decades. These diseases often take a disabling course and are among the health care problems with the highest social burden worldwide. In addition, the treatment of the disease and associated complications mean a substantial limitation of the quality of life to the patients and their families. It is challenging to determine whether, and in which subgroup of patients, specific therapeutic interventions offer additional benefit. Therefore, the major challenges with direct relevance for clinical practice are currently the development of techniques for early diagnosis, monitoring of inflammation and prediction of complications or manifestations of the disease, e.g. the parallel existence of joint and gut inflammation.
We took all efforts to reach the public and to inform not only about MIAMI, but also about the often neglected chronic inflammatory diseases of the joints and gut. We addressed the patients, but also the general public and also key opinion leaders. Our project contributed to the improvement of patient care in chronic inflammatory joint and bowel disease. It had a major impact on the development of therapeutic approaches not only in seronegative arthritis syndromes, but also in other inflammatory diseases. As chronic arthritis confers an enormous economical burden to the society, the dissemination of our results was very important. On the other hand, the distribution among economy and stakeholders was also important to attract industry to the field of biomarker discovery and assay development. With emphasizing the relevance of biomarker-driven therapies for the general health-care system, research and development in this area was encouraged.
The regular way to reach the scientific community including clinicians is often relatively slow. Of course, the partners of MIAMI used their results in lecturing and editorial activities, and naturally results will be presented at conferences, such as: the American College of Rheumatology conference (ACR), the annual meetings of the European League Against Rheumatism (EULAR) and the Paediatric Rheumatology European Society (PRES). The results were further distributed by publications in peer reviewed journals. The project's webpage informed about the diseases, the research activities, biomarker lists and proceedings. Workshops are more interactive, direct dissemination activity that proved very effective in the past: we performed workshops for clinical labs and practitioners. An activity which went considerably beyond usual dissemination plans is the offer for biomarker assays free of charge, which addresses a major drawback in biomarker research: the lack of translation into clinical applications that can be used in daily clinical practise. As the development of novel technologies takes time and the availability on the market is not easy to predict, we believe that a great way of dissemination was to offer valid biomarker tests to clinicians and scientist who are not partners of MIAMI. We invited centers in Europe to use biomarker analyses free of charge.
The results of MIAMI also had a significant impact on industry. Both the ability to provide biomarker candidates ready for being applied and the ability to bring up targets for future successful therapies beared enormous potential for industry. Directly within MIAMI already there was significant innovation, and the project results clearly benefit the SME-partners and lead to improved technologies and applications for diagnostic purposes. MIAMI added further application fields, improved quality and standardisation, and also lead to new technologies and markers. Bühlmann was co-developer, manufacturer, and markets the products directly and worldwide. The developed assays are easy to perform, efficient and not costly. Another impact was the identification of novel therapeutic targets, which could be licensed by the SMEs and universities to pharmaceutical companies. The influence on industry was strong as the partners of MIAMI had links to larger pharmaceutical companies who were also informed about biomarker-driven measurements of therapeutic responses. This already successfully led to cooperation (e.g. with Novartis, Pfizer). On the other hand, links to pharmaceutical companies also attracted their attention to new molecular targets that could be used for the development of therapies.
The SMEs clearly benefited from MIAMI and also focus on end users. The finding of potential new markers of the S100 superfamily and others lead to new research into, and characterization of these markers. They were used for the development of new immunological tests. Bühlmann introduced new quantitative point-of-care tests (POC, lateral flow). The experience gained in this project by expanding Biogazelle's qPCR based microRNA gene expression platform content by 30% wase useful for future content expansions as well. It allowed Biogazelle to develop high quality custom multiplex primer pools better and faster for other future applications. The data established with S100A8/A9 test formats of Bühlmann (ELISA and lateral flow POC) in the seronegative inflammatory diseases were of outmost importance so that the test can be used as clinically validated test for a precise diagnosis and for a proper patient treatment with the very expensive new biologics such as anti-TNFα drugs (e.g. therapy control). Bühlmann made use of a proof-of-concept study on a new, more robust and quantitative POC platform with several markers on it (multiplexing capability of the new platform is a further asset and superior than the existing one). Biogazelle will obtained critical insights in and hands-on experience with the various RNA extraction procedures tested for the different tissue types or body fluids. Bühlmann submitted patents of new assays and/or applications and will manufacture and distribute existing and new products over its own channels.
In summary, MIAMI significantly contributed to progress on the key questions: Who will be affected by which disease manifestation or complication, how can we use this knowledge to identify the disease, and what will be a meaningful target to treat or even prevent deleterious outcome. In MIAMI, an expert team successfully performed a joint enterprise with the goal of understanding the disease and translating it into personalised medicine. Novel biomarkers, improved technologies, and promising targets for therapeutic interventions were established. We provided profound and focussed trainings, both to the members of the consortium and to clinicians and clinical laboratories. Results were extensively disseminated to the scientific community and the end users (clinicians and clinical laboratories). The MIAMI international symposium, organized at the end of the project, was a huge success and the perfect platform for interesting scientific discussions. Both SME partners in the consortium have established a robust plan for the further exploitation of the obtained results. In the case of BÜHLMANN, the newly developed diagnostic and therapy monitoring tests have already been released to the market as CE/IVD labeled products and some of them showed considerable turnover numbers and are well accepted at various customers’ sites.
List of Websites:
Public MIAMI website:
http://www.miamiproject.eu
Contact:
Prof. Dirk Foell, MD
University Hospital Muenster
Pediatric Rheumatology and Immunology
Domagkstr. 3
D-48149 Muenster
Phone ++49-251-8358178
Fax ++49-251-8358104
Email dfoell@uni-muenster.de
