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Background: ANXA5, a notable tumor marker, displays irregular expression in diverse solid cancers, and links to local recurrence and metastasis rates. We aimed study the expression of ANXA5 in oral squamous cell carcinoma (OSCC) and its diagnostic and prognostic values. Methods: 520 head and neck squamous cell carcinoma (HNSCC) patients in TCGA database and 124 OSCC patients in Nanjing stomatology hospital were enrolled in our study. Immunohistochemical analyses were performed using ANXA5 antibodies. Chi‐square test was used to analyze the clinicopathological features. Survival rates were determined using the Kaplan–Meier method and log‐rank test. Results: Our results showed significantly elevated ANXA5 at the gene and protein levels in HNSCC and OSCC compared to non‐tumor tissues. Histopathologically, ANXA5 was broadly present in OSCC tumor cells and fibroblast‐like cells but absent in tumor‐infiltrating lymphocytes, particularly at the invasive tumor front. Patients exhibiting high ANXA5 expression in these cells demonstrated poor differentiation, aggressive invasion patterns, and heightened lymph node metastasis risk, contributing to poorer postoperative outcomes. Remarkably, ANXA5 in fibroblast‐like cells emerged as an independent risk factor impacting survival in OSCC patients. Gene set enrichment analysis (GSEA) highlighted ANXA5's involvement in key pathways like epithelial‐mesenchymal transformation (EMT), TGF‐beta signaling, and hypoxia, which correlated with adverse clinical outcomes in OSCC. Conclusion: ANXA5 emerges as a significant prognostic biomarker for OSCC, potentially influencing its metastasis via the EMT pathway. [ABSTRACT FROM AUTHOR]

Objective: The objective of the study was to assess the prognostic value of muscle invasion (MI), a key histopathological feature of tumor aggressiveness, and construct a superior prognostic prediction model combining the current TNM staging system. Materials and Methods: MI was analyzed in the whole‐slide images from a total of 301 patients with primary buccal mucosa squamous cell carcinoma (BMSCC). Survival times of patients with/without MI were evaluated by Kaplan–Meier analysis. MI was further combined with the TNM staging system to explore its predictive value for prognosis. Moreover, 204 cases of head and neck carcinoma from the TCGA database were included. Results: MI positive rate reached to 76% (229/301) in patients with BMSCC. MI was associated with poor overall survival (p = 0.012) and disease‐free survival (p = 0.022). The novel system (TNM staging combined with MI) revealed strong predictive performance, with the largest area under the curve (OS: p < 0.001, DFS: p < 0.004). MI and the established classification system were also had good predictive ability in the TCGA cohort. Conclusions: MI is an independent predictor of poor prognosis of BMSCC. The inclusion of MI in prediction system can augment the risk stratification of patients with oral squamous cell carcinoma and may assist in the clinical decision‐making process. [ABSTRACT FROM AUTHOR]

Objective: Disease metabolomes have been studied for identifying diagnostic and predictive biomarkers of pathology. Oral tongue squamous cell carcinoma (OTSCC) is one of the most prevalent subtypes of head and neck squamous cell carcinoma, yet the profile and diagnostic value of its tissue metabolite are unclear. Subjects and Methods: Tumor tissue samples and matched normal mucosal tissue samples were collected from 40 OTSCC patients. Untargeted metabolic analysis by liquid chromatography–mass spectrometry/mass spectrometry, in positive and negative ion modes, was used to identify dysregulated metabolites in OTSCC. Further, utilizing LASSO regression and receiver operating characteristic analyses, biomarker metabolites were selected and validated, and a diagnostic model was established. Results: One hundred and ninety metabolites were detected. The OTSCC had a total of 89 dysregulated metabolites, of which 73 were elevated. A diagnostic panel of nine metabolites was subsequently created that could accurately identify OTSCC with 100% sensitivity of 100%, 100% specificity and an AUC of 1.00. Conclusions: This study identified distinct metabolic characteristics of OTSCC and established a diagnostic model. Our research also contributes to the investigation of the pathogenesis of OTSCC. [ABSTRACT FROM AUTHOR]

Aims: Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological characteristics, and the individual histological characteristics of the tumors are poorly understood. Therefore, calculating the proportion of tumor cells in different regions that allow assessment of the prognostic outcomes for OSCC patients would be of great clinical significance. Methods and Results: We established an open‐source software‐based analytic pipeline that defines the inner tumor and invasive tumor front (ITF) in pancytokeratin‐stained whole slide images (WSIs) and quantifies the tumor‐stroma ratio (TSR) within the two regions. We applied this method to 114 patients with OSCC and predicted patient prognosis by the TSR. The proportion of tumor area in the inner tumor was generally higher than that in the ITF (p < 0.0001). TSR was an independent prognostic factor for overall survival (OS) (p = 0.016), disease‐free survival (DFS) (p = 0.026), and relapse‐free survival (RFS) (p = 0.037) in inner tumor, and TSR was an independent prognostic factor for OS (p = 0.00052), DFS (p = 0.035), and metastasis‐free survival (MFS) (p = 0.038) in the ITF. Tumor‐low status was associated with poorer prognosis. There was a significant correlation between the TSR and perineural invasion (PNI) in the inner tumor (p = 0.009). Conclusions: The histopathological characteristics of different regions of OSCC may be used to develop the potential prognostic markers. The TSR of the inner tumor is more targeted in predicting prognosis and accurately assesses the risk of PNI+. [ABSTRACT FROM AUTHOR]

Introduction: Metabolite stability is critical for tissue metabolomics. However, changes in metabolites in tissues over time from the operating room to the laboratory remain underexplored. Objectives: In this study, we evaluated the effect of postoperative freezing delay time on the stability of metabolites in normal and oral squamous cell carcinoma (OSCC) tissues. Methods: Tumor and paired normal tissues from five OSCC patients were collected after surgical resection, and samples was sequentially quenched in liquid nitrogen at 30, 40, 50, 60, 70, 80, 90 and 120 min (80 samples). Untargeted metabolic analysis by liquid chromatography–mass spectrometry/mass spectrometry in positive and negative ion modes was used to identify metabolic changes associated with delayed freezing time. The trends of metabolite changes at 30–120 and 30–60 min of delayed freezing were analyzed. Results: 190 metabolites in 36 chemical classes were detected. After delayed freezing for 120 min, approximately 20% of the metabolites changed significantly in normal and tumor tissues, and differences in the metabolites were found in normal and tumor tissues. After a delay of 60 min, 29 metabolites had changed significantly in normal tissues, and 84 metabolites had changed significantly in tumor tissues. In addition, we constructed three tissue freezing schemes based on the observed variation trends in the metabolites. Conclusion: Delayed freezing of tissue samples has a certain impact on the stability of metabolites. For metabolites with significant changes, we suggest that the freezing time of tissues be reasonably selected according to the freezing schemes and the actual clinical situation. [ABSTRACT FROM AUTHOR]

Background: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. Methods: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. Results: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial–mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. Conclusions: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy. [ABSTRACT FROM AUTHOR]

The Pattern Of Invasion (POI) of tumor cells into adjacent normal tissues clinically predicts postoperative tumor metastasis/recurrence of early oral squamous cell carcinoma (OSCC), but the mechanisms underlying the development of these subtypes remain unclear. Focusing on the highest score of POIs (Worst POI, WPOI) present within each tumor, we observe a disease progression-driven shift of WPOI towards the high-risk type 4/5, associated with a mesenchymal phenotype in advanced OSCC. WPOI 4-5-derived cancer-associated fibroblasts (CAFsWPOI4-5), characterized by high oxytocin receptor expression (OXTRHigh), contribute to local-regional metastasis. OXTRHigh CAFs induce a desmoplastic stroma and CCL26 is required for the invasive phenotype of CCR3+ tumors. Mechanistically, OXTR activates nuclear ERK5 transcription signaling via Gαq and CDC37 to maintain high levels of OXTR and CCL26. ERK5 ablation reprograms the pro-invasive phenotype of OXTRHigh CAFs. Therefore, targeting ERK5 signaling in OXTRHigh CAFs is a potential therapeutic strategy for OSCC patients with WPOI 4-5. Worst pattern of invasion (WPOI) is a parameter used to quantify tumor invasiveness of oral squamous cell carcinoma (OSCC). Here the authors show that a fibroblast subset characterized by the expression of the oxytocin receptor is enriched in highly invasive WPOI 4-5 OSCC tumors and can be targeted to reduce the desmoplastic stroma and tumor metastasis. [ABSTRACT FROM AUTHOR]

Background: PLIN2 (adipose differentiation-related protein) belongs to the perilipin family and is a marker of lipid droplets (LDs). Numerous types of tumor exhibit a high PLIN2 level, but its tumorigenic or tumor-suppressive role has been in debate. Recently, LDs serve as innate immune hubs and show antimicrobial capacity. We here aimed to investigate the heterogeneous functions of PLIN2 in the tumor microenvironment and immune regulation. Methods: This retrospective study included 96 oral squamous cell carcinoma (OSCC) samples and analyzed the spatial distribution of PLIN2 by immunohistochemistry (IHC) and LD level by oil red O staining. A total of 21 serial sections were obtained to analyze the relationship between PLIN2 and immune cells by IHC and immunofluorescence (IF). Single-cell sequencing was used to analyze the cell locations of PLIN2. The values of diagnosis and prognosis of PLIN2 were also evaluated. Tumor Immune Estimation Resource (TIMER), cBioPortal databases, and IHC analysis were used to investigate the relationship between PLIN2 and OSCC immune microenvironment. Results: PLIN2 was mainly expressed in tumor-infiltrating immunocytes (TIIs) of OSCC. Patients with high PLIN2 harbored more cytoplastic LDs. CD68+ tumor-associated macrophages (TAMs), instead of T cells and B cells, were found to be the main resource of PLIN2 in OSCC stroma and lung, pancreas, prostate, and testis. However, CD56+ NK cells also showed less extent of PLIN2 staining in OSCC. Moreover, patients with a high PLIN2 level in immune cells had a higher TNM stage and were susceptible to postoperative metastasis, but the escalated PLIN2 level in invasive tumor front independently predicted shorter metastasis-free survival. Furthermore, a high PLIN2 presentation in the microenvironment induced immune suppression which was featured as less infiltration of CD8+ T cells and more CD68+ TAMs and Foxp3+ Tregs, accompanied by more immune checkpoint molecules such as CSF1R , LGALS9 , IL-10 , CTLA-4 , and TIGIT. Conclusion: CD68+ TAM-derived PLIN2 might participate in regulating immune balance of OSCC patients, which provides new insight into immune checkpoint therapy. [ABSTRACT FROM AUTHOR]

As a key histopathological characteristic of tumor invasion, perineural invasion (PNI) assists tumor dissemination, whereas the current definition of PNI by dichotomy is not accurate and the prognostic value of PNI has not reached consensus. To define PNI status in each patient when mixed types of PNI occurred simultaneously, we here further subclassified the traditional PNI in 183 patients with oral squamous cell carcinoma (OSCC). The spatial localization of nerves in OSCC microenvironment was thoroughly evaluated and successfully concluded into four types of PNI: 0, tumor cells away from nerves; 1, tumor cells encircling nerves less than 33%; 2, tumor cells encircling nerves at least 33%; and 3, tumor cells infiltrating into nerve sheathes. Sequentially, patients were stratified by single and mixed types of PNI. Traditionally, types 0 and 1 were defined as PNI−, while types 2 and 3 were PNI+, which predicted shorter survival time. When multiple types of PNI existed within one tumor, patients with higher score of PNI types tended to have a relatively worse prognosis. Therefore, to define the status of PNI more precisely, the new variable worst pattern of PNI (WPNI) was proposed, which was taken as the highest score of PNI types present in each patient no matter how focal. Results showed that patients with WPNI 1 had longest survival time, and WPNI 2 correlated with better overall survival (p = 0.02), local-regional recurrence-free survival (p = 0.03), and distant metastasis-free survival (p = 0.046) than WPNI 3. Multivariate Cox analysis confirmed that only WPNI 3 could independently predict patients' prognosis, which could be explained by a more damaged immune response in WPNI 3 patients with less CD3+CD8+ T cells and CD19+ B cells. Conclusively, WPNI by trichotomy provide more meticulous and precise pathological information for tumor-nerve interactions in OSCC patients. [ABSTRACT FROM AUTHOR]

Background: Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a key enzyme that catalyzes the hydroxylation of lysine, plays a crucial role in the progression of several solid tumors. However, its spatial expression profile and prognostic significance in oral squamous cell carcinoma (OSCC) have not been revealed. Materials: Mass spectrometry was used to explore amino acid perturbations between OSCC tumor tissues and paired normal tissues of 28 patients. Then, PLOD2 mRNA and protein levels were assessed using several public databases and 18 pairs of OSCC patients' tissues. Additionally, PLOD2 spatial expression profiles were investigated in 100 OSCC patients by immunohistochemistry and its diagnostic and prognostic values were also evaluated. Lastly, gene set enrichment analysis (GSEA) was used to investigate the potential functions of PLOD2 in OSCC. Results: Lysine was significantly elevated in OSCC tissues and could effectively distinguish tumor from normal tissues (AUC = 0.859, p = 0.0035). PLOD2 mRNA and protein levels were highly increased in tumor tissues of head and neck squamous cell carcinoma (HNSCC) (p < 0.001) and OSCC compared with those in nontumor tissues (p < 0.001). Histopathologically, PLOD2 was ubiquitously expressed in tumor cells (TCs) and fibroblast-like cells (FLCs) of OSCC patients but absent in tumor-infiltrating lymphocytes (TILs). Patients with highly expressed PLOD2 in TCs (PLOD2TCs) and FLCs (PLOD2FLCs) showed poor differentiation, a worse pattern of invasion (WPOI) and more lymph node metastasis (LNM), contributing to higher postoperative metastasis risk and poor survival time. However, PLOD2FLCs rather than PLOD2TCs was an independent risk factor for survival outcomes in OSCC patients. Molecularly, GSEA demonstrated highly expressed PLOD2 was mainly enriched in epithelial–mesenchymal transformation (EMT), TGF-beta signaling and hypoxia pathway, which are associated with poor clinical outcomes of OSCC patients. Conclusions: PLOD2 was a poor prognostic biomarker for OSCC patients and may affect the metastasis of OSCC through EMT pathway. These findings might shed novel sights for future research in PLOD2 targeted OSCC therapy. [ABSTRACT FROM AUTHOR]