Alterations of 13C and 15N Isotope Abundance as a Potential Biomarker for Tumor Biology and Risk Factor of Cervical Lymph Node Metastases in Oral Squamous Cell Carcinoma

Background: Cervical lymph node metastases are a major prognostic factor in patients with oral squamous cell carcinoma (OSCC). Despite advances in imaging, accurate preoperative prediction of nodal involvement remains a challenge. This study evaluated the utility of Isotope Ratio Mass Spectrometry (IRMS) in assessing the risk of lymph node metastases in patients with OSCC. We hypothesize that alterations in the abundance of 13C and 15N stable isotopes in OSCC tumor tissues reflect metabolic reprogramming associated with tumor progression and may correlate with cervical lymph node metastases. Methods: A prospective cohort of 61 patients with primary OSCC undergoing surgical treatment was analyzed. Tumor tissue samples were evaluated for the relative abundance of nitrogen-15 (¹⁵N) and carbon-13 (¹³C) isotopes using IRMS. Correlations between isotopic values and nodal metastases, as well as established clinicopathological risk factors, were assessed. Results: IRMS measurements of ¹³C and ¹⁵N abundance did not directly correlate with the presence of lymph node metastases but were associated with advanced tumor stages and negative prognostic features, such as angioinvasion/neuroinvasion. The median of the average nitrogen ¹⁵N content was higher in patients with more advanced clinical stages (11.89% in stage IV vs. 11.12% in stages I–III; p=0.04), and the median δ¹³C was lower in stage IV compared to stages I–III (-22.40‰ vs. -22.88‰; plt;0.05). Patients with angioinvasion/neuroinvasion also had a lower median δ¹³C (-22.26‰ vs. -22.75‰; plt;0.05). These findings suggest that IRMS reflects metabolic changes in tumor biology rather than specifically predicting nodal metastases. Multivariate logistic regression identified age, gender, and clinical tumor stage as independent predictors of nodal involvement. Conclusion: IRMS-based isotopic profiling may reflect key metabolic alterations associated with OSCC progression. Although IRMS parameters of carbon 13C and nitrogen 15N were not independently predictive of lymph node status, they were associated with key adverse prognostic features, indicating potential as an adjunctive biomarker and may complement traditional histopathological evaluation.