Amino Acids Signatures of Distance-Related Surgical Margins of Oral Squamous Cell Carcinoma

Abstract

Purpose: Histological assessment of the resected margins is the gold standard for determining the status of surgical margin of oral squamous cell carcinoma (OSCC). However, histological evaluation has significant deficiencies, such as the limitation of the thichness of slide, and other reasons, resulting the higher local recurrence (LR) although the margin status was negative. Many studies have demonstrated metabolic reprogramming in cancer or precancerous cells. Therefore, we aimed to identify a panel of metabolic molecular markers for evaluating the surgical margins of OSCC during the surgery. Experimental Design: A total of 28 cases (each case including Tumor, Margin-1, Margin-2and Margin-3 four sections) were enrolled in the study. 8 of the 28 cases were used for developing markers (Development group), and another 20 cases (Validation group) were used to validate the results of the Development group. Gas chromatography-mass spectrometry (GC-MS) untargeted analysis was used to evaluate the metabolic perturbation of the distance related surgical margins from development group. The acquired MS data of development group samples were further subjected to multivariate data analysis, and the significantly altered metabolites were identii¬ed. UHPLC-MS/MS targeted quantitative analysis was used to validate the results of the development group. Meanwhile, another 60 OSCC patients with dysplastic surgical margins were used to further validate the results of the development group by immunohistochemical exam key enzyme (asparagine synthetase, ASNS) expression, and correlate with clinicopathological parameters and clinical outcomes. Results: In development group, a panel of 10 amino acids was found to discriminate negative margin and another panel of 9 amino acids was found to discern the dysplastic margin. It was further determined 9 amino acids for negative margin biomarkers and 6 amino acids for dysplastic margin biomarkers from above markers (development group) by UHPLC-MS/MS targeted quantitative analysis in validation group. Individual amino acid biomarker for the ability to detect negative margin or dysplastic margin based on ROC curves, displayed good sensitivity and specificity. Moreover, the combination of the panel amino acids can significantly improve sensitivity and specificity of the diagnostic accuracy by multiple linear regressive analysis. Among them, aspartic acid and asparagine were found to be high in dysplastic margin and tumor tissues, so, we examined asparagine synthetase (aspartic acid metabolic key enzyme) expression in 60 OSCC samples with dysplastic margins by IHC analysis, which showed that ASNS positive expression in dysplastic surgical margins was correlated with tumor recurrence and local relapse-free survival (RFS). Conclusions: We developed a panel of amino acid markers to supplement the evaluation of negative and dysplastic margins, ASNS expression level was a predictor of tumor recurrence and poor prognosis in OSCC with dysplastic surgical margins. In conclusion, amino acids signatures of distance-related surgical margins of OSCC has positive clinical significance. Funding: This study was supported by Jiangsu Province’s Key Provincial Youth Talents Program (Grant No. QNRC2016841); Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2012); Center of Nanjing Clinical Medicine Tumor (Since 2014). Declaration of Interest: The authors have declared that no competing interests exist. Ethical Approval: This study was approved by the medical ethics committee of Stomatological Hospital, Medical School of Nanjing University, and carried out according to the recommendations of the Declaration of Helsinki.