Periodic Reporting for period 2 - GlySign (Exploitation of Glycosylation Signatures for Precision Medicine) Reporting period: 2018-10-01 to 2020-09-30 Summary of the context and overall objectives of the project During the progression of many chronic diseases including various inflammatory conditions and cancer complex, distinctive changes occur in the carbohydrates (glycans) that are attached to various proteins. These changes can be observed when measuring the glycomic profiles or – Glycan Signatures – of human glycoproteins by using appropriate analytical platforms. Within the GlySign research and training network a group of young researchers engaged in developing disease glycomic markers together with the suitable analytical technology for clinical translation in the context of Precision Medicine (PM). Glycan signatures have great potential for adding useful diagnostic and prognostic information in PM. However, advancement of this field is slow because (a) glycans have immense structural complexity resulting in major technical challenges for their analysis and (b) there is a lack of experts with required glycoanalytical skills. GlySign addressed this gap by training six early stage researchers (ESRs) within an innovative training-by-research programme with high industrial-academic mobility thereby contributing to the translation of novel glycomics-based diagnostic tools and markers into clinical practice. This was achieved by developing a range of selective and sensitive glycomics technologies for the analysis of samples from patients and healthy controls in close collaboration between industry and academia as well as clinicians. The training focused on clinical glycomics applied to different model diseases assessing changes in the glycosylation of circulating proteins specific to disease progression or subtype: auto- and alloimmune diseases, including rheumatoid arthritis, and diabetes and prostate cancer. With its strong industrial and translational focus, GlySign filled a gap in the healthcare market by contributing to the development of new in-vitro diagnostic platforms for clinical exploitation of glycomic biomarkers for PM.More information on the project and scientists in charge as well as descriptions of individual ESR projects can be found on: www.glysign.eu. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The ESRs involved in WP1 Auto- and alloimmune diseases established multi-platform assays for IgG-based patient stratification. ESR 1 set up a middle-up approach with orthogonal techniques to simultaneously analyse IgG N-glycosylation and other post-translational modifications in patient samples. ESR 2 has applied two different techniques for IgG glycosylation analysis on two cohorts of patients suffering from i) the autoimmune disease ANCA-Associated Vasculitis (AAV) and ii) Clostridium difficile infection. The longitudinal and pre- and post-treatment design of these studies allowed the (i) validation and (ii) discovery of predictive IgG glycobiomarkers. A ready-to-use microtitre-plate enzyme-based method for robust quantitation of IgG glycosylation was developed by ESR 3. Due to its simplicity, this assay has high potential for clinical translation.The focus of WP2 was the development of enhanced assays for diabetes and prostate cancer patient stratification. ESR 4 developed a mass spectrometry assay detecting different glyco- and other isoforms of prostate specific antigen (PSA) from urine. Two orthogonal methods for the analysis of antennary fucosylation of plasma N-glycans were developed by ESR 5 and applied to a clinical cohort including Maturity-Onset Diabetes of the Young (MODY) and other diabetes patients. The data allowed to validate findings from literature and to assess additional, novel glycomic markers to distinguish the different diabetes types. ESR 5 and 6 developed methods for specific plasma protein glycosylation analysis, i.e. apolipoprotein C3 and IgG, respectively. The assays were applied on a large type 2 diabetes cohort for glycobiomarker development to improve the management of diabetic complications. All six ESRs presented and disseminated their work, GlySign objectives and results, at multiple scientific conferences. In that way, they interacted with researchers from academia and healthcare industry as well as clinicians. Moreover, the ESRs were active in communicating their research to the general public via their Facebook page as well as via a short movie https://www.glysign.eu/glysign-video-august-2020/. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) GlySign focused on achieving research excellence, on exploiting research results for the benefit of the patients as well as for increasing the innovation capacity of the European scientific community, and finally, on the training of its ESRs to provide them with the necessary skills for their future career prospects. The GlySign project focused on developing analytical and commercially complete glycomic and glycoproteomic services and kits for rapid, high‐throughput patient stratification methods for complex diseases, which will ultimately help in matching therapies to patient populations, paving the way towards tailor-made therapeutically successful outcomes. Within the GlySign programme, great attention was given to ensuring intersectoral and multidisciplinary training including business skills, thereby enhancing the ESRs’ career perspectives and employability. To achieve this, next to providing them with network-wide training events that were tailor-made according to their needs, all ESRs had a minimum industrial placement of 18 months, with one industrial and one academic supervisor. With experience in diverse research and commercial areas, e.g. biopharmaceutical services and kit development, and translation of clinical markers, the involved SMEs significantly contributed to research training and career perspectives.