Fragmented plasma cfDNA as a prognostic and surveillance biomarker for locoregional recurrence in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) frequently exhibits early locoregional recurrence despite adjuvant therapy. Circulating cell-free DNA (cfDNA) has emerged as a minimally invasive approach for evaluating tumor burden and disease dynamics. While most studies have focused on ctDNA mutations, the prognostic value of cfDNA fragmentomics-particularly mononucleosomal and dinucleosomal DNA-remains unclear in OSCC. Here, we investigated baseline cfDNA characteristics, mutational profiles, and fragmentation patterns, and evaluated their utility for recurrence prediction and longitudinal surveillance. Sixty-eight HPV-negative OSCC patients were prospectively enrolled, and plasma was collected at diagnosis and during follow-up. Baseline cfDNA was detected in 98.5% of patients (mean 6.65 μg/mL). Higher cfDNA levels correlated with aggressive clinical features, including advanced T stage, lymph node metastasis, and TNM III-IV disease, supporting its association with tumor burden. Targeted sequencing identified somatic variants in 30 genes, most commonly NOTCH1 (41%), FBXW7 (25%), and MGA (22%). Pathogenic variants were less frequent and did not correlate with TNM stage, and patients who recurred harbored fewer detectable mutations, indicating limited prognostic value of mutation-based profiling. Fragmentation analysis revealed distinct clinical implications for mono- and dinucleosomal cfDNA. Elevated baseline mononucleosomal cfDNA predicted recurrence, and a 3 ng/mL cutoff identified patients with significantly shorter recurrence-free survival. Dinucleosomal cfDNA, however, demonstrated superior value for longitudinal surveillance. Among 16 patients with locoregional recurrence, 13 (81%) showed detectable dinucleosomal cfDNA at or before radiologic confirmation, often during imaging-negative intervals. Persistent dinucleosomal cfDNA reflected ongoing local tumor activity, whereas metastatic recurrences rarely showed positivity. These results demonstrate that cfDNA fragmentomics provides prognostic information beyond mutation profiling in OSCC. Baseline mononucleosomal cfDNA stratifies patients at increased risk of recurrence, while the presence of dinucleosomal cfDNA offers a sensitive and clinically practical indicator of emerging locoregional disease. Together, these fragment-based biomarkers support the incorporation of cfDNA fragmentation analysis into postoperative surveillance strategies for earlier and more accurate detection of relapse.