A Novel Cell-Free DNA Fragmentomics Assay and Its Application for Monitoring Disease Progression in Real-Time for Stage IV Cancer Patients

Background/Objectives: Conventional imaging assesses therapy response in stage IV solid-tumor patients in 8–12-week intervals, delaying detection of non-responders. We evaluated a real-time quantitative PCR (RT-qPCR) assay that interrogates size-distributed cell-free DNA (cfDNA) fragments to provide earlier insights into treatment efficacy. Methods: In this prospective study, 128 patients with metastatic lung, breast, or colorectal cancer provided plasma 12–21 days after the first dose of a new systemic regimen. RTqPCR targets multicopy retrotransposon elements in cfDNA fragments gt;80 bp, gt;105 bp, gt;265 bp, and internal control. A model integrates these quantities into a Progression Score (PS) ranging from 0–100; higher values indicate probable disease progression. Results: The PS model yielded an area under (AUC) the receiver-operating-characteristic (ROC) curve of 0.93 for predicting radiographic progression at first imaging. Scores were strongly bimodal: 92 % of patients with PS gt; 90 progressed, whereas 95 % with PS lt; 10 did not. Intermediate scores (10–90) comprised a mixed cohort. Assay performance was unaffected by tumor genomic profile. Conclusions: This cfDNA-based Progression Score (PS) assay enables tumor- and therapy-agnostic, non-invasive monitoring of treatment response as early as two weeks after initiation. By flagging ineffective regimens well before standard imaging, the test can accelerate clinical decision-making, reduce exposure to fu-tile therapy, and potentially improve outcomes in stage IV cancer. Early treatment plan changes may also avoid the high drug and administration costs of ineffective treatments, prevent downstream toxicity-related hospitalizations, and free up limited imaging and infusion-suite capacity—yielding savings for patients, payers, and healthcare systems.