Patients with metastasis have an extremely poor prognosis in head and neck squamous cell carcinoma (HNSCC). Emerging studies have illuminated the impact of intratumor microbiota on cancer metastasis, though the specific role of Fusobacterium nucleatum (F. nucleatum) in HNSCC metastasis remains unresolved. This research found that intratumoral F. nucleatum abundance was elevated and correlated to diminished disease-free survival in HNSCC patients exhibiting lymph node metastasis. F. nucleatum invasion into primary and metastatic tumor tissues was observed using fluorescence in situ hybridization. F. nucleatum induced adhesion to endothelial cells and facilitated transendothelial migration via upregulating ESPN expression in HNSCC cells, which is an actin-binding protein. Mechanistically, F. nucleatum activated TLR4 signaling, inducing elevated expression of the transcription factor MYB, which subsequently stimulated ESPN transcription. Furthermore, metronidazole treatment significantly reduced metastatic incidence in vivo. These results indicate the significant potential of targeting F. nucleatum as a therapeutic approach in metastatic HNSCC.

OBJECTIVE:The objective is to evaluate the prognostic significance of tumor surface necrosis (TSN) in hypopharyngeal squamous cell carcinoma (HPSCC), a feature commonly observed during laryngoscopic examination. METHODS:We retrospectively analyzed 346 HPSCC patients who underwent laryngoscopy at our institution between January 2004 and December 2023. Following univariable and multivariable analyses and model diagnostics, a prognostic nomogram was constructed. The model's performance was assessed using discrimination and calibration measures. Comparative evaluation against the previous model was performed using integrated discrimination improvement (IDI) and net reclassification improvement (NRI). RESULTS:The presence of TSN was significantly associated with reduced overall survival (p = 0.0002), disease-free survival (p < 0.0001), local recurrence-free survival (p = 0.004), and regional recurrence-free survival (p < 0.0001). The final nomogram incorporated TSN (p < 0.001), tumor invasion depth (p = 0.001), lymphovascular invasion (p = 0.023), R_classification (p < 0.001), and pT_classification (p < 0.001). The area under the curve (AUC) values for 12-, 36-, and 60-month survival were 0.789, 0.791, and 0.759, respectively. Calibration curves demonstrated good concordance between predicted and observed outcomes. The new model showed significantly improved performance over the previous version (p < 0.001), with NRIs of 0.080, 0.123, and 0.129, and IDIs of 0.040, 0.049, and 0.051 at 12, 36, and 60 months, respectively. CONCLUSION:TSN identified via laryngoscopy was established as an independent risk factor for poor prognosis in HPSCC. The enhanced nomogram integrating TSN with key clinicopathological variables offers improved predictive accuracy, supporting better risk stratification and identification of high-risk patients. LEVEL OF EVIDENCE: 4:

Accurate preoperative diagnosis of thyroid nodules via fine-needle aspiration (FNA) biopsy remains challenging, particularly in cases with indeterminate cytology. This prospective, noninterventional, blinded, multicenter study establishes ThyroProt, a diagnostic classifier that integrates targeted mass-spectrometry-based quantification of a 3-protein signature with BRAFV600E mutation status, age, and gender. Developed and validated on 837 FNA samples, the classifier is evaluated in a prospective test set of 322 samples, achieving an area under the curve (AUC) of 0.94 with an overall accuracy of 90.7%. For the critical subgroup of Bethesda III/IV nodules, ThyroProt demonstrates an accuracy of 88.0%, with 82.4% sensitivity and 100% specificity. The classifier's robust performance is further evaluated in two independent multicenter cohorts, where it maintains an AUC of 0.87-0.91 and an accuracy of 84.3%-85.7%. This study supports the clinical utility of mass-spectrometry-based targeted proteomics for improving preoperative diagnosis of thyroid nodules, particularly those with indeterminate cytology.

Hypopharyngeal squamous cell carcinoma (HPSCC) patients with extranodal extension (ENE) are classified as pN3b and have the poorest prognosis. However, further risk stratification within this high-risk subgroup remains controversial. We retrospectively reviewed 141 HPSCC patients who underwent neck dissection and whose disease was confirmed as pN3b at our hospital between January 2012 and December 2021. The primary endpoint was disease-free survival (DFS). Survival analysis was performed using the Kaplan‒Meier method with the log-rank test for group comparisons. Multivariable analysis was conducted using Cox proportional hazards regression. Compared with patients without lymph node fusion (LNF, n = 30), those with LNF (n = 111) had significantly worse overall survival (OS, P = 0.0177), DFS (P = 0.0302), and distant metastasis-free survival (DMFS, P = 0.0404). Notably, patients with both LNF and soft tissue invasion (STI, n = 17) exhibited the poorest outcomes, with a median OS of only 19 months (95

OBJECTIVES:cN3 larynx and hypopharynx cancer is rare with a poor prognosis. This study evaluates treatment approaches, effectiveness, and prognosis for these patients in real-world settings. It aims to offer targeted and practical clinical guidance to enhance their treatment results and quality of life. METHODS:This retrospective cohort study (2014-2021) included 192 cN3 larynx and hypopharynx cancer patients from Eye & ENT Hospital of Fudan University. We collected detailed information on demographics, tumor characteristics, and treatment outcomes. The study focused on overall survival (OS), cancer-specific survival (CSS), and disease-free survival (DFS), using Kaplan-Meier and Cox regression analyses for independent prognostic analysis. RESULTS:This study encompassed 192 cN3 larynx and hypopharynx cancer patients, revealing a five-year overall survival rate of 40.60%. Treatment modalities analyzed included Surgery + CRT, ICT + Surgery + CRT, ICT + CRT, Surgery alone, and CRT alone. Patients receiving surgery followed by CRT demonstrated the most favorable prognosis (p < 0.0001) and the longest DFS (p < 0.0001), while those undergoing monotherapy had the poorest outcomes. Induction chemotherapy (ICT) achieved a 70% primary tumor response rate and a 48% neck lesion response rate, with patients showing a response to ICT exhibiting superior OS, CSS, and distant metastasis rate (DMR) (p < 0.05). Pathological type and primary tumor surgery were identified as independent prognostic factors for cN3 larynx and hypopharynx cancer. CONCLUSION:This study shows that combined treatments, especially surgery followed by chemoradiotherapy, improve survival and prognosis for cN3 larynx and hypopharynx cancer patients. It highlights the importance of tailored treatments and notes better outcomes for those responding to ICT. LEVEL OF EVIDENCE: 3:

MicroRNAs (miRNAs) are involved in carcinogenesis. However, the biological roles and underlying mechanism of miR-3690 in head and neck squamous cell carcinoma (HNSCC) progression are far from elucidated. In this study, we found that the expression level of miR-3690 in HNSCC tissues was significantly higher and correlated with poor clinical prognosis. HNSCC cells proliferation, migration, and invasion were promoted by miR-3690 overexpression, both in vitro and in vivo. Mechanistically, miRNA pulldown DNA-seq and luciferase reporter assays revealed that miR-3690 could directly activate CKS2 expression through targeting its promoter. Meanwhile, RNA pull down and mass spectrometry (MS) analysis suggested that upregulation of CKS2 by miR-3690 correlated with increased BPTF occupancy and H3K4me3 at CKS2 promoter. Additionally, luciferase reporter assays showed that miR-3690 facilitated Wnt/β-catenin signaling in HNSCC by repressing NKD1 expression through directly targeting its 3’-UTR. Finally, methylated RNA Immunoprecipitation (meRIP) and RNA pull down indicated that METTL3 and METTL14-mediated m6A modification accelerated pri-miR-3690 maturation through regulating the processing of pri-miR-3690 by DGCR8. In conclusion, miR-3690 may be a prognostic indicator and potential therapeutic target for HNSCC.

Smoking-associated laryngeal squamous cell carcinoma (LSCC) is characterized by high metastatic potential and poor prognosis. However, the underlying molecular mechanisms remain insufficiently understood. This study utilized single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to explore the heterogeneity of the tumor microenvironment in smoking-associated LSCC. Thirteen distinct cellular subpopulations within the tumor microenvironment are identified, with STC2 and ITGA5 emerging as smoking-associated prognostic markers. STC2 exhibited bifurcated differentiation within tumor epithelial cells, categorized as Tumor_C1 and Tumor_C2. The two subtypes are linked to vascular permeability and DNA replication pathways, respectively. Mechanistically, nicotine activated the JAK2/STAT3 signaling pathway through CHRNA5, resulting in direct STAT3 binding to the STC2 promoter and modulation of its transcription. STC2 subsequently upregulated TGFBI, which interacted with ITGA5 on endothelial cells, regulating vascular permeability and facilitating hematogenous dissemination of LSCC cells. Furthermore, STC2 knockdown altered F-actin cytoskeletal dynamics by modulating small GTPase signaling, impairing filopodia formation and epithelial polarity restoration. This study elucidates the tumor-endothelial interactions mediated by STC2 and ITGA5 in smoking-associated LSCC, emphasizing their roles in tumor progression and vascular permeability. These findings suggest potential prognostic biomarkers and therapeutic targets to improve the clinical management of smoking-associated LSCC.

Circular RNA (circRNA), a subtype of noncoding RNA, has emerged as a significant focus in RNA research due to its distinctive covalently closed loop structure. CircRNAs play pivotal roles in diverse physiological and pathological processes, functioning through mechanisms such as miRNAs or proteins sponging, regulation of splicing and gene expression, and serving as translation templates, particularly in the context of various cancers. The hallmarks of cancer comprise functional capabilities acquired during carcinogenesis and tumor progression, providing a conceptual framework that elucidates the nature of the malignant transformation. Although numerous studies have elucidated the role of circRNAs in the hallmarks of cancers, their functions in the development of chemoradiotherapy resistance remain unexplored and the clinical applications of circRNA-based translational therapeutics are still in their infancy. This review provides a comprehensive overview of circRNAs, covering their biogenesis, unique characteristics, functions, and turnover mechanisms. We also summarize the involvement of circRNAs in cancer hallmarks and their clinical relevance as biomarkers and therapeutic targets, especially in thyroid cancer (TC). Considering the potential of circRNAs as biomarkers and the fascination of circRNA-based therapeutics, the "Ying-Yang" dynamic regulations of circRNAs in TC warrant vastly dedicated investigations.

Objective. The objective of this study is to explore the utilization of next-generation sequencing (NGS) technology in evaluating the likelihood of identifying individuals with papillary thyroid microcarcinoma (PTMC ≤10 mm) who are at high or low risk. Design. NGS was used to analyze 393 formalin-fixed, paraffin-embedded tissues of PTC tumors, all of which were smaller than 15 mm. Results. The study found that bilateralism, multifocality, intrathyroidal spread, and extrathyroidal extension were present in 84 (21.4%), 153 (38.9%), 16 (4.1%), and 54 (13.7%) cases, respectively. Metastasis of cervical lymph nodes was identified in 226 (57.5%) cases and 96 (24.4%) cases with CLNM >5. Out of the total number of cases studied, 8 cases (2.3%) showed signs of tumor recurrence, all of which were localized and regional. Genetic alterations were detected in 342 cases (87.0%), with 336 cases revealing single mutations and 6 cases manifesting compound mutations. 332 cases (84.5%) had BRAFV600E mutation, 2 cases had KRASQ61K mutation, 2 cases had NRASQ61R mutation, 8 cases had RET/PTC1 rearrangement, 3 cases had RET/PTC3 rearrangement, and 1 case had TERT promoter mutation. Additionally, six individuals harbored concurrent mutations in two genes. These mutations were of various types and combinations: BRAFV600E and NRASQ61R (n = 2), BRAFV600E and RET/PTC3 (n = 2), BRAFV600E and RET/PTC1 (n = 1), and BRAFV600E and TERT promoter (n = 1). The subsequent analysis did not uncover a significant distinction in the incidence of gene mutation or fusion between the cN0 and cN1 patient cohorts. The presence of BRAFV600E mutation and CLNM incidence rates were found to be positively correlated with larger tumor size in PTMC. Our data showed that gene mutations did not appear to have much to do with high-risk papillary thyroid microcarcinoma (PTMC). However, when we looked at tumor size, we found that if the tumor was at least 5 millimeters in size, there was a higher chance of it being at high risk for PTM (P<0.001, odds ratio (OR) = 2.55, 95% confidence interval (CI): 1.57–4.14). Identification of BRAFV600E mutation was not demonstrated to be significantly correlated with advanced clinicopathological characteristics, although it was strongly associated with a bigger tumor diameter (OR = 4.92, 95% CI: 2.40–10.07, P<0.001). Conclusion. In clinical practice, BRAFV600E mutation does not consistently serve as an effective biomarker to distinguish high-risk PTMC or predict tumor progression. The size of the tumor has a significant correlation with its aggressive characteristics. PTMC with a diameter of ≤5 mm should be distinguished and targeted as a unique subset for specialized treatment.

[This corrects the article DOI: 10.3892/ol.2022.13554.].

Objective To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection. Methods We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People's Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics. Results The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.

The sensitivity of laryngeal squamous cell carcinoma (LSCC) to chemotherapy shows large heterogeneity. The role of miRNA in small extracellular vesicles (sEV) in chemotherapy resistance is under investigation. However, the regulation and sorting mechanism of sEV miRNAs remains unclear. In this study, small RNA sequencing was used to explore miRNA expression profiles in sEV of LSCC after cisplatin stimulation; RNA pull-down, mass spectrometry, and EMSA were used to clarify the binding of candidate RNA-binding protein (RBP) and candidate miRNA. Immunostaining and microRNA fluorescence in situ hybridization were performed to identify how candidate RBP affects miRNA stability and nuclear/cytoplasmic distribution. In vivo experiments were performed to verify the biological functions and response to cisplatin of candidate RBP. We found that cisplatin stimulation induced increased expression of miR-148a-3p and sEV sorting. ANXA11 binds to miR-148a-3p in a sequence-specific manner. ANXA11 inhibits tumor cell proliferation and drug resistance by binding to and retaining miR-148a-3p. Cisplatin stimulation reduced ANXA11 expression and promoted miR-148a-3p efflux through sEV pathways. ANXA11 overexpression reduced in vivo tumor proliferation and cisplatin-resistance. Taken together, ANXA11 mediates cisplatin resistance through sEV miRNA resorting. Mechanically, ANXA11 binds to miR-148a-3p in a sequence-specific manner to regulate its resorting and thus influences tumor proliferation and chemoresistance.

Nicotine, a crucial constituent of tobacco smoke, can bind to and activate nicotinic acetylcholine receptors (nAChRs), thereby regulating various biological functions. However, the specific mechanisms through which nicotine mediates nAChRs to regulate the metastasis of laryngeal squamous cell carcinoma (LSCC) remain elusive. In this study, smoking status was found to be closely associated with metastasis in patients with LSCC. In addition, nicotine exposure potentiated the hematogenous and lymphatic metastatic capacity of LSCC cells. Nicotine activates membrane-bound CHRNA5, promoting cell migration and invasion, EMT and cell-ECM adhesion in LSCC. Furthermore, this study demonstrated that the Ras superfamily protein RABL6 directly interacted with CHRNA5, which preferentially binds to the RABL6-39-279aa region, and this interaction was enhanced by nicotine. Nicotine-mediated activation of CHRNA5 enhanced its interaction with RABL6, triggering the JAK2/STAT3 signalling pathway and eventually augmenting the metastatic potential of LSCC cells. This study reveals a novel mechanism through which nicotine-mediated CHRNA5-RABL6 interaction promotes the metastasis of LSCC. The findings of this study may help to develop effective strategies for improving the outcome of patients with LSCC in clinical settings.

Glioma, originating from neuroglial progenitor cells, is a type of intrinsic brain tumor with poor prognosis. temozolomide (TMZ) is the first-line chemotherapeutic agent for glioma. Exploring the mechanisms of circTTLL13 underlying TMZ resistance in glioma is of great significance to improve glioma treatment. Bioinformatics was adopted to identify target genes. The circular structure of circTTLL13 and its high expression in glioma cells were disclosed by quantitative real time-PCR (qRT-PCR) and PCR-agarose gel electrophoresis. Functional experiments proved that oxidized LDL receptor 1 (OLR1) promotes TMZ resistance of glioma cells. CircTTLL13 enhances TMZ resistance of glioma cells via modulating OLR1. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pulldown, mRNA stability, N6-methyladenosine (m6A) dot blot and RNA total m6A quantification assays were implemented, indicating that circTTLL13 stabilizes OLR1 mRNA via recruiting YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and promotes m6A methylation of OLR1 pre-mRNA through recruiting methyltransferase-like 3 (METTL3). TOP/FOP-flash reporter assay and western blot verified that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. CircTTLL13 promotes TMZ resistance in glioma through regulating OLR1-mediated Wnt/β-catenin pathway activation. This study offers an insight into the efficacy improvement of TMZ for glioma treatment.

Despite the fact that metastasis is the leading cause of death in patients with head and neck squamous cell carcinoma, fundamental questions about the mechanisms that enable or inhibit metastasis remain unanswered. Tetraspanin CD63 has been linked to tumor progression and metastasis. However, few studies have examined the role of CD63 in HNSCC. In this study, we discovered that CD63 levels were abnormally altered in HNSCC tissue compared to adjacent tissue (n = 69 pairs), and that this was linked to prognosis. Through functional in vitro and in vivo experiments, the roles of CD63 in HNSCC were confirmed. Overexpression of CD63 inhibited the progression and metastasis of HNSCC cells. Using mass spectrometry and co-immunoprecipitation assays, we discovered that KRT1 could be a direct interacting partner of CD63. Furthermore, both CD63 and KRT1 expression was significantly decreased in metastatic tissue compared with primary tumor tissue (n = 13 pairs), suggesting that CD63 and KRT1 play a role in reducing the metastasis of HNSCC. In summary, we reveal a previously unrecognized role of CD63 in regulating KRT1-mediated cell cycle arrest in HNSCC cells, and our findings contribute to defining an important mechanism of HNSCC progression and metastasis.

BackgroundT2N0M0 glottic laryngeal squamous cell carcinoma (LSCC) is a common type of laryngeal cancer.ObjectivesThe objective of this research was to assess the predictive value of tumor size for the rates of overall survival (OS) and disease-free survival (DFS) as determined by postoperative pathological examination in patients with T2 LSCC.MethodsA retrospective study was conducted on 535 successive patients with T2 glottic LSCC who underwent operation from 2005 to 2010. The effect of tumor size on OS and DFS results was evaluated by the affected area.ResultsOf the cohort, 528 (98.7%) were male, and 7 (1.3%) were female, with an average age of 60.1 +/- 9.4 years. The 10-year DFS and OS rates were 72.1% and 76.3%, respectively. The tumor diameter and area cut-off values that best discriminated OS and DFS rates were 1.35 cm and 1 cm(2), respectively. Patients with glottis carcinoma with a longer tumor diameter and larger tumor area had inferior OS and DFS rates. Tumor diameter and tumor area were independent predictive factors for the rates of OS and DFS in patients with T2 glottic LSCC.Conclusion and significanceThis research showed that patients with T2 glottic LSCC with a carcinoma diameter >1.35 cm or a tumor area >1 cm(2) have worse survival outcomes. These factors independently predict survival outcomes in patients.

目的 探索病原体宏基因组学检测技术(mNGS)在神经外科中枢神经系统感染(CNSIs)中的诊断效能.方法 选取2021年1月至2022年7月收治44例疑似CNSIs患者作为研究对象,所有患者采集脑脊液后进行常规病原学检测和mNGS检测,依据脑脊液mNGS和脑脊液细菌培养结果分组:感染组(17例)和非感染组(27例).对比两种检测方法的灵敏度、特异度、曲线下面积(AUC)等差异.结果 44例患者中,确诊CNSIs 17例(38.6%).常规病原学检测灵敏度为52.9%,mNGS检测灵敏度为94.1%,差异有统计学意义(χ2=5.440,P=0.017).非感染组常规病原学检测特异度为85.2%,mNGS检测灵敏度为77.8%,差异无显著性(χ2=0.123,P=0.726).检测诊断符合率:传统检测为72.7%;mNGS检测为82.8%,差异有统计学意义(χ2=4.139,P=0.025).常规病原学检测诊断CNSIs的AUC为0.691,mNGS检测AUC为0.859,差异有统计学意义(P=0.048).结论 mNGS对比常规病原学检测诊断CNSIs,诊断效能更强,可为CNSIs诊断提供更为快速可靠的诊断依据.

Abstract Background As a human tumor disease, head and neck squamous cell carcinoma (HNSCC) is associated with a high mortality rate worldwide. Nicotinic acetylcholine receptors (nAChRs) are transmembrane receptor proteins and exert their biological effects following activation by nicotine. We aimed to construct a prognostic signature based on the expression of nAChRs among smokers with HNSCC. Methods The transcriptome profile of nAChRs was obtained from The Cancer Genome Atlas (TCGA). Following the integration of survival information, univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses were performed to screen the prognosis-related nAChRs and construct a prognostic signature. Kaplan–Meier (KM), receiver operating characteristic (ROC), principal component analysis (PCA), and independent prognostic analysis were utilized to verify the predictive power of the nAChR-associated prognostic signature. The expression of α5 nAChR in clinical samples was verified by quantitative reverse transcriptase PCR. Results Subunits α2, α5, α9, and β4 were related to the prognosis. The prognostic signature comprised the expression of subunits α5, α9, and β4. The nAChR-associated signature showed high sensitivity and specificity for prognostic prediction and was an independent factor for overall survival. Based on the clinical variables and expression of nAChRs, a nomogram was constructed for predicting the outcomes of HNSCC patients who were smokers in the clinical settings. In clinical specimens, α5 nAChR showed high expression in HNSCC tissues, especially among smokers. Conclusions The nAChR-associated signature constructed in this study may provide a better system for the classification of HNSCC patients and facilitate personalized treatment according to their smoking habits.

Objectives:The relationship between microbiota and HPSCC recurrence and metastasis remains uncertain. This study aimed to investigate the role of the tumour microbiota in the disease-free survival (DFS) of HPSCC patients. Materials and methods:Formalin-fixed paraffin-embedded (FFPE) tumour tissues were collected from 103 patients with HPSCC for 16S rRNA sequencing. We analysed the tumour microbiota in HPSCC patients with recurrence/metastasis and nonrecurrence/metastasis. The linear predictor score (LPS) was calculated based on the Cox regression model to assess the risk of recurrence and metastasis. Then, a time-dependent ROC curve was used to evaluate the prognostic power of the LPS. Results:The phyla Bacteroidota, Firmicutes and Proteobacteria were the most abundant bacterial taxa in the tumour tissues. Eubacterium_coprostanoligenes_group (hazard ratio [HR] = 0.289, 95% confidence interval [CI] 0.137-0.608, p= 0.001) and Prevotella (HR = 3.744, 95% CI 1.439-9.738, p= 0.007) were independent predictors of DFS. The predicting classifier for recurrence and metastasis risk yielded an area under the curve (AUC) of 0.838 at 3 years and 0.860 at 5 years. Conclusion:Our study demonstrated the relationship between tumour microbiota and recurrence and metastasis in patients with HPSCC.

Background: Head and neck squamous cell carcinoma (HNSCC) was the seventh most common cancer worldwide in 2018. Lymphatic metastasis (LM) is closely related to HNSCC prognosis and recurrence. However, the underlying mechanism of LM remains unclear. Therefore, this study aimed to identify the key genes in the LM of HNSCC. Methods: We used The Cancer Genome Atlas (TCGA) to identify differentially expressed genes (DEGs) between LM and non-LM cases. A random forest model, the Search Tool for the Retrieval of Interacting Genes, Cytoscape, and cytoHubba were used to identify hub genes among DEGs, including KRT20 (Cytokeratins 20). We analyzed the survival of KRT20 in TCGA, and we overexpressed KRT20 in HNSCC cell lines to investigate its effects on migration and invasion. We also correlated the expression of KRT20 in HNSCC tissue microarrays with survival and clinicopathological features. Results: We identified 243 DEGs-143 upregulated genes and 100 downregulated genes. Further analysis revealed that KRT20 is a potential key gene associated with LM and overall survival rates among patients with HNSCC. Overexpression of KRT20 increased the migration and invasion ability of HNSCC cell lines Tu686 and FD-LSC-1. Tissue microarray studies demonstrated an overexpression of KRT20 among N1+ patients (including N1-N3 patients). Survival analysis results and the clinicopathological features of HNSCC tissue microarrays were consistent with our analysis of TCGA. Thus, a high KRT20 expression level might suggest an adverse HNSCC prognosis. Our gene set enrichment analysis showed that KRT20 participates in many metabolic pathways, including those related to tumorigenesis and cancer development. Conclusions: We propose that KRT20 may be a key gene in HNSCC with LM.