Background. Radiotherapy induces systemic changes beyond the targeted tumour site, yet the biological mechanisms driving these effects remain poorly understood. This study aims to longitudinally profile acute systemic plasma proteomic responses to radiotherapy in patients with prostate, bladder, and head and neck cancers, providing insight into shared and tumour-specific effects and identifying biomarkers predictive of treatment-related toxicities.

Methods. We apply our previously developed Nano-proteomics workflow to comprehensively analyse longitudinal weekly plasma samples collected before and during radiotherapy. We perform mass spectrometry-based differential protein analysis to identify acute changes of the plasma proteome and enriched biological pathways involved. In the prostate cohort, we associate plasma proteomics with clinical toxicity outcomes.

Results. Our data indicate that the most significant systemic proteomic changes occur within the first two weeks of radiotherapy, highlighting a critical period for biomarker identification. Across all patient cohorts, we observe common biological responses: rapid activation of inflammatory and immune pathways, followed by structural reorganisation and immune resolution, regardless of tumour type or concurrent treatments. Despite this shared acute response, distinct protein mediators are found to be dysregulated in a tumour-specific manner. In the prostate cancer cohort, plasma profiling at baseline, one week after the initiation of radiotherapy, and at the end of radiotherapy we identify 28, 29, and 20 proteins, respectively, that are associated with subsequent bowel and urinary toxicities.

Conclusions. This study underscores the value of longitudinal proteomics in uncovering systemic effects of radiotherapy and supports the potential of plasma proteomic biomarkers to identify patients at increased risk of radiotherapy-induced toxicity, paving the way for personalised treatment strategies.