Abstract Background Histological assessment of resected margins has some drawbacks. We therefore aimed to identify a panel of metabolic markers for evaluating the surgical margins of oral squamous cell carcinoma during surgery. Methods A total of 28 case of OSCC samples were enrolled in the study. Gas chromatography-mass spectrometry based untargeted metabolic analysis was employed to acquire the metabolic perturbation of the distance-related surgical margins in the development group. The acquired MS data were then subjected to univariate and multivariate analysis by MetaboAnalyst. Ultra–high performance liquid chromatography–tandem mass spectrometerbased targeted metabolomics for quantitative analysis of the validation group was performed to verify the results of the development group. Another 60 OSCC patients with dysplastic surgical margins were used to further validate the results of the development group by immunohistochemical examination of key enzyme expression, and correlate them with clinicopathological parameters and clinical outcomes. Findings We finally identified 4 amino acids as negative margin markers, and 6 amino acids as dysplastic margin markers. IHC analysis showed that asparagine synthetase positive expression in dysplastic surgical margins and its higher expression was correlated with tumor recurrence and local relapse-free survival. Interpretations We developed a panel of metabolic molecular markers to supplement the evaluation of negative and dysplastic margins. Fund This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2012); Center of Nanjing Clinical Medicine Tumor (Since 2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Keywords: Oral squamous cell carcinoma, Amino acids metabolomics, Surgical margins, Asparagine synthetase __________________________________________________________________ Research in context. Evidence before this study Obtaining negative surgical margins during surgery has always been the pursuit of tumor surgeons. In current clinical practice, 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 thickness 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. Added value of this study In this study, we first elucidated the characteristics of molecular changes in OSCC at different distances from surgical margins in terms of amino acid metabolism. We developed 4 amino acids as negative margin markers, and 6 amino acids for dysplastic margin markers. We further 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). These results indicate that the amino acid markers at the surgical margin have positive clinical significance. Implications of all the available evidence Evaluation of surgical margins at the molecular level is a promising new molecular diagnostic method, which can effectively supplement the traditional pathological evaluation. The report of molecular margin analysis in OSCC stems from 1953, the observation that histologically normal tissue harbors clonal populations of cells with premalignant change, namely field cancerization. However, most studies on molecular margin were restricted to single gene or protein, and lacked molecular combinations that effectively predicted the margin status. In this study, molecular markers of the surgical margin of oral squamous cell carcinoma were determined at the level of amino acid metabolism. It provides a theoretical basis for the development of amino acid diagnostic reagents and the improvement of margin evaluation. Alt-text: Unlabelled Box 1. Introduction Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers, with 6 million deaths worldwide every year [43][1]. Tobacco (smoked or chewed), alcohol consumption, and human papillomavirus infection are the most important risk factors for OSCC [44][2]. Although OSCC management has been greatly improved, only a minor improvement in OSCC survival has been obtained over the last 30 years, partly due to the difficulty in obtaining ‘clear’ resection margins. The pathological margin is an important prognostic factor for relapse-free survival (RFS) of oral cancer patients treated with primary surgery, because tumor cells or dysplastic epithelia that remain in the margins may lead to the local recurrence (LR) of OSCC [45][3], [46][4], [47][5], [48][6]. Our team previously reported that patients with mild dysplasia margins who did not undergo re-excision had a worse prognosis than those with negative margins [49][7]. Therefore, an overarching goal of oncologic surgery is to resect tumors with histological tumor-free margins, as adequate surgical resection is essential for good local control and improved prognosis. Paradoxically, excising dysplasia area adjacent to the cancer impairs the life quality of patients. Additionally, the LR rates still hover from 10% to 30% even if the histological surgical margins are ‘clear’ [50][8]. Therefore, determining the molecular markers of margins is especially crucial to supplement the pathological diagnosis. One of the most striking features of cancer cells is metabolic reprogramming, where cancer cells alter metabolic pathways to adapt to environmental stress and meet their growth need. For example, many cancers display an increasing demand for specific amino acids and become dependent on either an exogenous supply or upregulated de novo synthesis. Moreover, targeting the ‘addicted’ amino acids showed promising clinical applications [51][9,[52]10]. Therefore, a better understanding of the dysregulated amino acids and related pathways that control OSCC progression is essential for developing diagnostic and prognostic predictors. Recently, many tumor tissues and biological fluid samples have been studied in metabolic research [53][11,[54]12]. A list of metabolites has been identified to distinguish normal and tumor samples [55][13]. However, the metabolic study of OSCC margin has not been reported. Therefore, our purpose was to characterize the amino acids that clearly delineate OSCC margins and to supplement the clinicopathological diagnosis. 2. Materials and methods 2.1. Patients and tumor specimens Our study was approved by the medical ethics committee of the Nanjing stomatology hospital. A total of 28 cases (each case including Tumor, Margin-1, Margin-2 and Margin-3) were recruited 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. Two adjacent lumps of tissues (containing tumor and the longest margin, [56]Fig. 1A) were collected from every patient. One was used for pathological assessment, while the other was used for mass spectrometry (MS) analysis. All samples were divided into four groups: Tumor, Margin-1 (0–0.5 cm from tumor margin), Margin-2 (0.5–1 cm from tumor margin), Margin-3 (>1 cm from tumor margin) ([57]Fig. 1B). All tissues were frozen in liquid nitrogen and stored at −80 °C before sample preparation for gas chromatography–mass spectrometry (GC–MS) or ultra–high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS). Tumor, Margin-1, Margin-2 and Margin-3 samples were analyzed by GC–MS or UHPLC-MS/MS ([58]Fig. 1C). The clinical details of the 28 patients are shown in Supplemental Table 1 . The HE-stained samples were evaluated by professional pathologists. The Tumor group contained 80% tumor cells. The Margin-1 group contained dysplastic epithelium, while the Margin-2 and Margin-3 groups were histologically negative margins. And the Margin-3 group can be identified as normal tissues ([59]Fig. 1B). Table 1. Metabolic overall analysis among the four groups (Tumor, Margin-1, Margin-2 and Magin-3) by one way ANOVA and Tukey's honest significance difference (HSD) post Hoc test analysis. Image, table 1 [60]Open in a new tab Fig. 1. [61]Fig. 1 [62]Open in a new tab The overall design of this experiment. A Work flow. B HE staining of the whole tissue which were divided into four parts: Tumor (T), Margin-1 (Dysplasia), Margin-2 (Negative), Margin-3 (Normal). C MS analysis. D Data analysis. E Validation, including validating in 20 patients using targeted quantitative analysis and IHC stain of ASNS in 60 OSCC patients with dysplastic margins. 2.2. Gas chromatography-mass spectrometry untargeted analysis GC–MS experiment is under the guidance of professor Tong Xie (Nanjing University of Chinese Medicine). Materials and reagents for GC–MS analysis were prepared according to previous method. GC–MS analysis was performed by Trace 1310 Gas Chromatograph equipped with an AS 1310 auto sampler, which connected a TSQ 8000 triple quadrupole mass spectrometer (Thermo Scientific, Waltham, MA). See references for specific steps