Figure 1. Study design and workflow.
This study enrolled 100 adult patients undergoing thoracic radiotherapy (RT) between 2019 and 2022, along with a control group of 24 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were collected at three time points: pre-RT (baseline), mid-RT (~6 weeks into treatment), and post-RT (1 month to 1 year after treatment). Follow-up visits concluded in September 2023, capturing acute and late RT-related outcomes through validated patient-reported questionnaires (EORTC QLQ-LC13, PRO-CTCAE) and medical records. Baseline demographics, cancer diagnostics, RT regimens, and medical history were collected from medical records and patient assessments. Genome integrity biomarkers, including telomere length (TL) and DNA repair capacity (DRC), were measured in PBMCs. TL was assessed using monochrome multiplex qPCR (MM-qPCR) and validated via flow-FISH and whole-genome sequencing (WGS). DRC was quantified using fluorescence multiplex host cell reactivation (FM-HCR) assays, covering all major DNA repair pathways. The study employed three analytical approaches: association analysis was used to identify factors influencing baseline TL and its response to RT; risk prediction was conducted to evaluate the predictive value of TL dynamics for RT-related health outcomes using cross-validation and receiver operating characteristic (ROC) analysis; mediation analysis was performed to assess whether DRC modulates the impact of RT on TL.