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Saliva is a noninvasive biofluid that can contain metabolite signatures of oral squamous cell carcinoma (OSCC). Conductive polymer spray ionization mass spectrometry (CPSI-MS) is employed to record a wide range of metabolite species within a few seconds, making this technique appealing as a point-of-care method for the early detection of OSCC. Saliva samples from 373 volunteers, 124 who are healthy, 124 who have premalignant lesions, and 125 who are OSCC patients, were collected for discovering and validating dysregulated metabolites and determining altered metabolic pathways. Metabolite markers were reconfirmed at the primary tissue level by desorption electrospray ionization MS imaging (DESI-MSI), demonstrating the reliability of diagnoses based on saliva metabolomics. With the aid of machine learning (ML), OSCC and premalignant lesions can be distinguished from the normal physical condition in real time with an accuracy of 86.7%, on a person by person basis. These results suggest that the combination of CPSI-MS and ML is a feasible tool for accurate, automated diagnosis of OSCC in clinical practice.

The survival rate of oral squamous cell carcinoma (OSCC) can be greatly improved if intervention could be initiated as early as possible. This poses a technical demand for developing a sensitive screening and specific in vitro diagnosis method for OSCC. Herewith, a large cohort consisting of 241 healthy contrast (HC) and 578 OSCC patients were recruited for conducting the rapid metabolic profiling of trace volume serum using conductive polymer spray ionization mass spectrometry (CPSI‐MS). Statistical analysis picked out 65 metabolite ions as potential characteristic markers for differentiating OSCC from HC. With the aid of a supporting vector machine (SVM), OSCC can be distinguished from HC with an accuracy of 98.0% by cross‐validation in the discovery cohort and 89.6% accuracy in the validation cohort. Furthermore, orthogonal partial least square‐discriminant analysis (OPLS‐DA) also initially showed the potential for OSCC staging, especially between T1/T2 and T3/T4 stages with an accuracy of 90.1%. CPSI‐MS combined with SVM or OPLS‐DA can not only quickly distinguish OSCC from HC but also predict the OSCC progression from T1/2 to T3/4 stages in a few minutes, making it a promising tool for both screening and diagnosing high‐risk population. Key points Sixty‐five characteristic metabolite ions significantly changed in OSCC serum metabolic profile compared to that in the HC group. CPSI‐MS combined with SVM achieved 89.6% accuracy on the validation cohort for OSCC prediction. CPSI‐MS/OPLS‐DA can distinguish T1/T2 from T3/T4 stages with an accuracy of 90.1% by cross‐validation.

Purpose This was a retrospective analysis of the impact of the expression of p53 in the dys-plastic surgical margins of early oral squamous cell carcinoma (OSCC) (pT1-2, N0). Patients and methods Seventy-two patients with early oral squamous cell carcinoma (OSCC) were recruited. Margin characteristics were abstracted from the pathology report. Expression of p53 in dysplastic surgical margins was examined with the immunohistochemical method and was correlated with clinicopathological parameters and clinical outcomes. Results Patients with moderate/severe dysplasia had poor local relapse-free survival (RFS) compared to those with mild dysplasia. Thirty-two (44.4%) had at least one p53-positive margin, and there was a significant association between the expression of p53 and tumor recurrence (P

Background Although there is consensus that the optimal safe margin is ≥ 5mm, obtaining clear margins (≥5 mm) intraoperatively seems to be the major challenge. We applied a molecular diagnostic method at the lipidomic level to determine the safe surgical resection margin of OSCC by desorption electrospray ionisation mass spectrometry imaging (DESI-MSI). Methods By overlaying mass spectrometry images with hematoxylin-eosin staining (H&E) from 18 recruited OSCC participants, the mass spectra of all pixels across the diagnosed tumour and continuous mucosal margin regions were extracted to serve as the training and validation datasets. A Lasso regression model was used to evaluate the test performance. Findings By leave-one-out validation, the Lasso model achieved 88.6% accuracy in distinguishing between tumour and normal regions. To determine the safe surgical resection distance and margin status of OSCC, a set of 14 lipid ions that gradually decreased from tumour to normal tissue was assigned higher weight coefficients in the Lasso model. The safe surgical resection distance of OSCC was measured using the developed 14 lipid ion molecular diagnostic model for clinical reference. The overall accuracy of predicting tumours, positive margins, and negative margins was 92.6%. Interpretation The spatial segmentation results based on our diagnostic model not only clearly delineated the tumour and normal tissue, but also distinguished the different status of surgical margins. Meanwhile, the safe surgical resection margin of OSCC on frozen sections can also be accurately measured using the developed diagnostic model. Funding This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (since 2016) and the Centre of Nanjing Clinical Medicine Tumour (since 2014).

Purpose: The optimal safe margin distance of oral squamous cell carcinoma (OSCC) is still controversial. In this article, we apply a cost-effective molecular diagnosis method at the lipidomic level for determining the safe surgical-resection distance of OSCC by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). Methods: A total of 22 participants (each with a matching tumor and continuous mucosal margin tissues) were recruited to the study. All specimens were cut into 10-μm frozen cryosections and scanned by DESI-MSI in the negative ion mode. By overlaying the mass spectrometry image with the H&E staining image, we first precisely extracted the mass spectra of those pixels across the diagnosed tumor and normal regions which served as the training and validation datasets. A Lasso regression model was developed and externally validated for distinguishing the tumor and normal region. A set of feature ions which gradually decreased from tumor to normal tissue was highlighted by assigning them higher weight coefficients during the Lasso model development. The safe surgical-resection distance of OSCC on frozen sections was measured by using this developed molecular diagnostic model for clinical reference. Meanwhile, the genetic marker (p53) of the surgical-margin was used to verify the safe surgical-resection distance determined by DESI-MSI. Results: By leave-one-out validation, the Lasso model can achieve 88.6% accuracy. The Lasso model selected 179 features from an initially collected 22,955 ions across 7,243 pixels for distinguishing tumor and normal tissues. We further screened these 179 characteristic ions, which tend to decrease gradually along with the increase in distance from tumor to normal tissue. Finally, 14 decreasing characteristic ions were tentatively assigned as fatty acids and selected as potential markers for determining the safe surgical-resection distance. These 14 lipid ions gradually decrease from tumor to normal tissue and can be used to accurately distinguish tumors from normal tissues. The spatial segmentation results based on our diagnostic model can not only clearly delineate the tumor and normal tissue but also distinguish the different status of surgical margins. Meanwhile, the safe surgical-resection distance of OSCC on frozen sections can also be accurately measured by using the developed diagnostic model. Moreover, the genetic marker of the OSCC margin (p53) validated that the safe surgical-margin boundary determined by the DESI-MSI and p53 is highly consistent. Conclusions: A diagnostic model was successfully constructed by combining DESI-MSI with the Lasso regression, which could accurately measure the safe margin distance of OSCC on frozen sections. It provides a theoretical basis for a personalized evaluation of safe margin distance of OSCC. Funding Statement: This study was supported by Jiangsu Provincial Key Medical Discipline (since 2017), Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2016), National Natural Science Foundation of China (No.81772880, 81702680). Declaration of Interests: The authors have no competing interests. Ethics Approval Statement: This study was approved by the medical ethics committee of the Nanjing Stomatology Hospital, Medical School of Nanjing University. All participants signed the informed consent forms.

It is essential to explore potential molecular involved in oral squamous cell carcinoma (OSCC) malignant transformation and progression. The purpose of this study was to delineate the metabolic characteristics of OSCC patients. We firstly report GC-MS based metabolic profiling of OSCC tissues from 8 patients, including tumor tissues and its matched normal tissues, then another 81 OSCC patients with prognostic information were used to verify metabolomics results by exam key enzymes expression. A panel including ten metabolite biomarker were identified, which can distinguish OSCC tumor from normal controls. On the base of metabolites with significantly difference, we applied KEGG for further pathway analysis and found OSCC have highly active glutaminolysis metabolism. Therefore, glutaminolysis-related key enzymes GLS1 and GLUD1 were detected by immunohistochemical and results demonstrated that GLS1 and GLUD1 were highly expressed in OSCC. Moreover, Kaplan-Meier survival analysis also showed and GLS1 and GLUD1 expressions were correlated with poor prognosis and tumor progression in OSCC patients. In conclusion, our results indicated that OSCC have highly active glutaminolysis metabolism, and GLS1 and GLUD1 enzymes are potential diagnostic and prognostic factors for OSCC.

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.

The authors investigated the clinicopathological characteristics of keratocystoma of the parotid gland. Two cases of parotid gland keratocystoma in the files of Nanjing Stomatological Hospital were analysed. These slowly growing parotid gland tumours occurred in two women aged 29 and 49 years. The cut surface showed multilocular cystic lesions filled with keratin materials. Histologically, there were multi-cystic spaces and solid epithelium islands, containing keratinized lamellae. Without cytological atypia, the lining stratified squamous epithelium showed apparent keratinization through an orthokeratotic or parakeratotic pathway. No skin appendage formation was observed. Both cases immunoreactively stained positively for AE1/AE3 and CK5/6 but negatively for CK8/18, S-100 and Calponin. There was no evidence of recurrence 3 or 4 years after superficial parotidectomy. The data from these two cases and cases previously published suggest that keratocystoma of the parotid gland is a benign cystic neoplasm. Surgical resection is apparently sufficient for cure.

This study assessed the clinical and histological features and therapeutic efficacy of 25 cases of sublingual gland tumours from 1998 to 2008. There were 17 female patients and 8 male, the ratio of females to males was 2.1:1. The mean age was 48.6 years. 4 cases were benign tumours (16%). 21 cases were malignant sublingual gland tumours (84%) and of these, 18 were adenoid cystic carcinoma (86%). Adenoid cystic carcinoma was mainly of the histological type, and the other histological classifications included mucoepidermoid carcinoma, pleomorphic adenoma, myoepithelioma, oncocytoma and polymorphous low-grade adenocarcinoma. Sublingual gland tumours are rare and most are malignant. For malignant sublingual gland tumours, early diagnosis and aggressive surgical treatment, especially for tumours with nerve involvement, is the key to improving prognosis. Free radial forearm flap or pectoralis major myocutaneous flap are appropriate methods for mouth floor reconstruction. For benign sublingual gland tumours, the resection of tumour and sublingual gland is the preferred treatment.

The purpose of this study is to assess the diagnosis, treatment and prognosis of the dentinogenic ghost cell tumor (DGCT). This is a retrospective study of patients with DGCT who underwent one or multiple surgical interventions during the 10-year period from 1997 to 2007. Seven patients with DGCT were identified under the WHO 2005 classification guidelines. The lesions were located in the maxilla in 4 patients and in the mandible in 3 patients. Five patients experienced recurrences following conservative surgery. They were all treated with aggressive local resection. Two patients had no conservative surgery but aggressive local resection. After 7-105 months' follow-up, no recurrences were found in any of the patients. Intraosseous DGCT should be treated by resection with an adequate safety margin, of at least 0.5 cm, as recommended for ameloblastoma. Patients with a DGCT should remain in long-term follow-up.

Nodular fasciitis (NF) is a benign, reactive proliferation of fibroblasts in subcutaneous tissues which commonly occurs in the deep fascia. It can only be diagnosed by histopathological examination of a biopsy. A total of 23 orofacial NF patients was analysed, including those reported in the English language literature and six new patients from the files of this hospital. All patients were treated between 1994 and 2005. The reported lesions were located in cheek masseter muscle, parotid gland, upper neck, upper gingiva and body of mandibular. The clinical and histological features and differential diagnoses are discussed. All lesions were removed under general or local anaesthesia and no recurrence of the lesions was found.

Oral squamous papilloma and papillary squamous cell carcinoma are 2 clinicopathologically distinctive papillary epithelial tumors. The current study aims to compare the clinical and pathologic features of these oral papillary lesions in a patient population from eastern China. A retrospective review in a series of patients with clinical and pathologic diagnosis of oral squamous papilloma (n = 141) and papillary squamous cell carcinoma (n = 56) was conducted. The average age of oral squamous papilloma was 51.0 years (male-to-female ratio, 1.82), with the palate being the predominant site. The average age of oral papillary squamous cell carcinoma was 63.3 years (male-to-female ratio, 1.67), with the gingiva being the predominant site. Multivariate analysis revealed that the elderly patient with papillary lesion (≥60 years) was associated with 3.09-fold (95% confidence interval, 1.59-6.03) increased carcinoma risk compared with the nonelderly patient. The lesion located on the gingiva was associated with 4.98-fold (95% confidence interval, 1.96-12.63) increased carcinoma risk compared with other oral sites. Collectively, clinicopathologic features of oral squamous papilloma and papillary squamous cell carcinoma in eastern China were elucidated. Elderly patients with oral papillary lesions located on the gingiva correlate with higher carcinoma risk. It highlights the importance of using a histologic examination to confirm the clinical diagnosis for any suspicious papillary lesions.

The aim of this study was to demonstrate the extent of motor innervation of the trapezius muscle from the accessory nerve and branches of the cervical plexus using intraoperative electroneurography and histochemical staining. In 34 patients during radical neck dissection the accessory nerve and C2-C4 branches running to the trapezius were identified and stimulated. Potentials were registered under three conditions: intact accessory nerve, section of superior part of communication between the nerve and the cervical branches, and complete section of the nerve. Projections that did not elicit responses were analyzed for acetylcholinesterase activity. Before cutting the accessory nerve, its stimulation led to a recordable contraction in all parts of the trapezius muscle in all patients. C2 contributions were seen in 15, C3 in 21 and C4 in 20 patients. After sectioning of the upper half of the nerve, the results were similar. After the nerve was completely cut, C2 contributions were seen in only 2 patients, but C3 were seen in 20 patients and C4 in 19 patients. Histochemical staining revealed that the branches with no responses contained both motor and sensory axons. The accessory nerve provides the main motor input to the trapezius muscle, but preservation of the C2-C4 branches to the muscle during modified neck dissection should improve outcomes.