Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Background: Neuropilin (NRP) is a transmembrane protein, which has been shown to be a pro-angiogenic mediator and implicated as a potential driver of cancer progression. NRP-1 up-regulation in ovarian cancer tissue predicts poor prognosis. However, the clinical relevance of the soluble form of NRP-1 (sNRP-1) as a circulating biomarker in ovarian cancer patients is unknown. - Methods/patients cohort: sNRP-1 levels were quantified in a cohort of 88 clinically documented ovarian cancer patients by a commercially available sNRP-1 enzyme-linked immunosorbent assay (ELISA) kit (Biomedica, Vienna, Austria). Patients (81.8% with FIGOIII/IV) received primary cytoreductive surgery with the aim of macroscopic complete resection (achieved in 55.7% of patients) and the recommendation of adjuvant chemotherapy in line with national guidelines. - Results: Higher levels of sNRP-1 reflected more advanced disease (FIGO III/IV) and indicated a trend towards suboptimal surgical outcome, i.e. any residual tumor. sNRP-1 was neither related to the patients’ age nor the BRCA1/2 mutational status. Patients with higher sNRP-1 levels at primary diagnosis had a significantly reduced progression-free survival (PFS) (HR = 0.541, 95%CI: 0.304 - 0.963; p = 0.037) and overall survival (OS) (HR = 0.459, 95%CI: 0.225 - 0.936; p = 0.032). Principal component analysis showed that sNRP-1 levels were unrelated to the circulating hepatocyte growth factor (HGF) and the soluble ectodomain of its receptor the tyrosine kinase mesenchymal–epithelial transition (c-MET), suggesting that there is no proportional serological concentration gradient of soluble components of the NRP-1/HGF/c-MET signaling axis. - Conclusions: In line with the previously shown tissue-based prognostic role, we demonstrated for the first time that sNRP-1 can also act as a readily accessible, prognostic biomarker in the circulation of patients with ovarian cancer at primary diagnosis. Given its known role in angiogenesis and conferring resistance to the poly ADP-ribose polymerase (PARP) inhibitor olaparib in vitro, our results encourage more detailed investigation into sNRP-1 as a potential predictive biomarker for bevacizumab and/or PARP-inhibitor treatment.