Carbon and Nitrogen Isotopic Signatures as Metabolic Biomarkers of Nodal Metastasis and Recurrence in Oral Squamous Cell Carcinoma

Background/Objectives: Oral squamous cell carcinoma (OSCC) exhibits substantial biological heterogeneity, and current clinicopathological risk stratification incompletely reflects tumor metabolic behavior. Stable isotope ratio mass spectrometry enables the quantitative assessment of carbon and nitrogen isotopic composition, potentially capturing cumulative metabolic reprogramming associated with tumor aggressiveness. This study evaluated whether isotopic signatures of tumor tissue and surgical margins are associated with lymph node metastasis and survival outcomes in OSCC. Methods: In this prospective study, 54 consecutive patients undergoing primary surgical treatment for OSCC were enrolled. Paired samples derived from tumor tissue and surgical margins were analyzed using isotope ratio mass spectrometry to determine the relative abundance of nitrogen-15 and carbon-13 isotopes. The primary endpoint was pathological lymph node metastasis. Secondary endpoints included disease-free survival and overall survival. Paired comparisons were performed using Wilcoxon signed-rank tests with false discovery rate correction. Logistic regression models for nodal metastasis were constructed using Firth penalization with bootstrap internal validation, while survival outcomes were evaluated using Cox proportional hazards models with model complexity restricted according to the number of events. Results: Tumor tissues demonstrated significantly lower δ13C and δ15N values and higher nitrogen-to-carbon ratios compared with surgical margins (all adjusted p < 0.05). In multivariable analysis, tumor δ15N was independently associated with lymph node metastasis and modestly improved model discrimination. However, it was not independently associated with disease-free or overall survival. Exploratory analyses indicated that higher δ13C values in surgical margins were independently associated with shorter disease-free survival. Conclusions: These findings suggest that isotope ratio mass spectrometry-based isotopic profiling identifies reproducible metabolic differences between tumor and margin tissues in OSCC. Tumor δ15N is associated with lymph node metastasis, whereas margin δ13C may reflect recurrence risk and potentially capture metabolic field effects. These findings are hypothesis-generating and warrant validation in larger, independent cohorts.