Author: Billy Karnaros
Karnaros, Billy, 2025 The discovery and validation of biomarker candidates of immune dysfunction in Motor Neuron Disease, Flinders University, College of Medicine and Public Health
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Urine shows potential as a source of biomarkers, offering opportunities to reduce patient heterogeneity in clinical trials for Motor Neuron Disease (MND). To reduce heterogeneity, multiple biomarkers are needed to reflect disease pathology (Rogers et al., 2023), complementing serum neurofilament light. Our laboratory has identified urinary neopterin as a pro-inflammatory biomarker that increases over disease progression (Shepheard et al., 2022) which might have value as a predictive marker of response to anti-inflammatory therapy; however, improved analytical methods are needed. We aim to discover and validate immune biomarkers of MND prognosis in urine using Liquid Chromatography-Mass Spectrometry (LCMS), gene ontology (GO) analysis, and baseline neurological data.
From a cohort of 78 people with MND at baseline (first visit to neurologist) and 39 controls, two studies were completed. Neopterin was measured using multiple reaction monitoring (MRM) on a Thermo Altis QQQ mass spectrometer (MS). Neopterin quantified by LCMS was compared to an inhibition ELISA (Shepheard et al., 2022) in a subset of 44 MND and 19 controls. Differentially expressed urinary proteins were identified in a subset of 39 MND and 26 controls using a Thermo Orbitrap Exploris-480 MS in Data-Dependent Acquisition (DDA) mode for spectral library generation and Data-Independent Acquisition (DIA) mode for differential abundance analysis using Spectronaut. PANTHER was used for GO pathway analyses to identify candidates associated with neuroinflammation in MND. 10 candidate urinary proteins were verified using parallel reaction monitoring (PRM) and assessed for prognostication using clinical data (ALSFRS-R, ∆FRS, onset, age, gender, and time to death/ventilation) and SPSS.
Urinary neopterin quantified by LCMS/MRM correlated significantly with an inhibition ELISA in 44 people with MND (r=0.78, p<0.0001) (Shepheard et al., 2022). Neopterin levels were increased in 78 people with MND at baseline compared to 39 controls (170.7 ± 73.2 vs 135.2 ± 44.7 μmol neopterin/mol creatinine, p=0.0017) and was a weak, but independent predictor of prognosis (HR: 1.005, 95% CI:1.001-1.010, p=0.019). LCMS in DDA mode discovered 1,970 protein groups in the urinary proteome, with DIA mode detecting 1,350 protein groups common between MND and controls, including ≥257 differentially changed proteins found in MND (q<0.01, >1.5-fold difference). GO analysis highlighted key changes in immune alteration, cell death, and growth-factor receptor activity pathways. 10 protein candidates associated with neuroinflammation, neurodegeneration, and muscle degeneration were verified by PRM, identifying significant differences in 7 proteins at baseline. Cathepsin D (P07339, r=-0.34, p=0.035) and Fibulin-5 (Q9UBX5, r=-0.35, p=0.031) were significantly correlated to ALSFRS-R scores, however, when compared to prognostic factors neither could add value beyond the ∆FRS (HR: 2.60, 95% CI:1.49-4.55, p<0.001) or baseline urinary p75ECD quantified by ELISA (HR: 1.27, 95% CI:1.08-1.49, p=0.004). Ongoing validation is required in larger sample sizes and different MND phenotypes.
Urine is a valuable biofluid for discovering immune-related biomarkers and candidate proteins in MND. Ongoing validation studies using PRM mass spectrometry and stable isotope standards (SIS) will quantify predictive candidates of inflammation with absolute quantitative accuracy to assess their prognostic value. Panels of urine-based prognostic biomarkers will hopefully improve patient selection in clinical trials and reduce MND heterogeneity.
Keywords: Motor Neuron Disease (MND); amyotrophic lateral sclerosis (ALS); urine; biomarkers; urinary biomarkers; neuroinflammation; neopterin; proteomics; mass spectrometry (LCMS, DIA, PRM); prognosis; neurodegeneration; immune response; biomarker validation; clinical trials; heterogeneity reduction; p75ECD; gene ontology (GO) analysis
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Mary-Louise Rogers