Nicotine downregulates miR-375-3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells.

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375-3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375-3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375-3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375-3p specifically binds to 3' untranslated region (3'UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375-3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

Fusobacterium nucleatum facilitates proliferation and autophagy by activating miR-361-3p/NUDT1 axis through oxidative stress in hypopharyngeal squamous cell carcinoma.

BACKGROUND: To investigate how Fusobacterium nucleatum (Fn) promotes oxidative stress and mediates proliferation and autophagy in hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: The prognosis for 82 HPSCC cases was retrospectively analyzed. HPSCC cell line FaDu was co-cultured with Fn. Knockdown of NUDT1 (shNUDT1 group) was done after observing DNA damage response. CCK8 and tumorigenesis assays for proliferation observation, mitochondria ROS (MitoROS) measurement to examine intracellular oxidative stress, and ELISA to analyze concentration of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in cells. Dual-luciferase reporter assays clarified miR-361-3p connection with NUDT1. Autophagy flow was observed using electron microscopy and related proteins. RESULTS: Fn was highly associated with NUDT1. The shNUDT1 group experienced lower proliferation compared with normal FaDu (NC group) in vivo and in vitro. The shNUDT1 group showed 8-oxo-dG and γH2AX to be elevated. Intracellular ROS decreased in shNUDT1Fn group when compared to Fn group. Upregulating miR-361-3p could suppress NUDT1 expression and downstream proliferation and autophagy. Fn modulated miR-361-3p via OH-, which could be proven by H2O2 assay and N-acetylcysteine. CONCLUSIONS: Higher Fn in HPSCC patients suggests poorer prognosis. NUDT1 might affect cell proliferation and autophagy and modulate DNA damage response. The oxidative stress induced miR-361-3p/NUDT1 axis is first introduced in microbiome-carcinoma research.

Diagnostic and prognostic value of plasma cell-free DNA combined with VEGF-C in laryngeal squamous cell carcinoma.

BACKGROUND: Circulating cell-free DNA (cfDNA) and vascular endothelial growth factor-C (VEGF-C) can be utilized to detect cancer and predict its prognosis. However, their potential application in laryngeal squamous cell carcinoma (LSCC) is unclear. PURPOSE: This study aimed to identify the diagnostic and prognostic value of cfDNA and VEGF-C in LSCC patients. METHODS: The plasma cfDNA of 148 LSCC patients and 43 non-tumor patients were isolated. Quantitative real-time PCR (qRT-PCR) was performed to assess long and short DNA fragments in plasma by amplifying the ALU repeats. ALU-qPCR results (ALU247/ALU115) were used to calculate cfDNA integrity index. Vascular endothelial growth factor-C (VEGF-C) level was detected by ELISA assay. Correlation between cfDNA and clinical features was analyzed. For detecting the sensitivity and specificity of cfDNA and VEGF-C alone or in combination for diagnosing LSCC, receiver operator characteristic (ROC) was established. For evaluating the overall survival (OS) of LSCC, Kaplan-Meier curves were established. RESULTS: LSCC patients had significantly higher levels of plasma cfDNA (ALU115, ALU247, and cfDNA integrity index) and VEGF-C than those without cancer (p < 0.05), showing area under the curve (AUC) values of 0.79, 0.74, 0.62 and 0.80, when cutoff value was correspondingly defined at 2.14 ng/mL, 1.39 ng/mL, 0.73 and 412.90 pg/mL, respectively. The AUC for distinguishing LSCC patients from non-tumor patients by plasma cfDNA combined with VEGF-C was 0.89 (95% CI: 0.83-0.94). A significant correlation was found between plasma cfDNA levels and Ki-67, tumor size, pT stage, and smoking history (p < 0.05). Based on survival analysis, low VEGF-C concentration groups had longer OS than those with high VEGF-C concentration (p = 0.02). CONCLUSION: Indicators such as plasma cfDNA and VEGF-C may be used to diagnose and monitor LSCC for its noninvasiveness and rapid accessibility.

Fusobacterium nucleatum impairs DNA mismatch repair and stability in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Dysbiosis of the laryngeal microbiota has been demonstrated to the development of head and neck squamous cell carcinoma (HNSCC), but the association of Fusobacterium and Fusobacterium nucleatum (F. nucleatum) with DNA mismatch repair (MMR) and microsatellite instability (MSI) has not been investigated. METHODS: The abundance of Fusobacterium and F. nucleatum, the status of deficient MMR (dMMR) and MSI, and MMR-related gene expression were analyzed in 171 HNSCC tissues, 61 paired para-tumor tissues, and 60 vocal cord polyp tissues. The molecular mechanism of F. nucleatum and MMR-related gene expression were investigated in two human HNSCC cell lines (Tu 686 and FD-LSC-1). RESULTS: Our results demonstrated that a high Fusobacterium abundance was detected in the HNSCC tissues and was exaggerated in the recurrent patients. We further found that a high Fusobacterium abundance was detected in the HNSCC tissues with dMMR and MSI. The Fusobacterium abundance was negatively correlated with the expression of MLH1, MSH2, and MSH6 in the HNSCC tissues. The Fusobacterium abundance was closely associated with the F. nucleatum abundance in the HNSCC tissues. F. nucleatum increased miR-205-5p expression to suppress MLH1, MSH2, and MSH6 expression via the TLR4- and MYD88-dependent innate immune signaling pathway, resulting in dMMR, DNA damage, and cell proliferation in HNSCC. CONCLUSIONS: F. nucleatum impacts HNSCC epigenetic changes in tissues with dMMR to promote DNA damage and cell proliferation by suppressing MMR-related gene expression via the TLR4/MYD88/miR-205-5p signaling pathway, which is valuable in the development of efficient strategies for HNSCC prevention and treatment. LAY SUMMARY: This study clearly indicates that Fusobacterium induced head and neck squamous cell carcinoma (HNSCC) aggressiveness to affect poor prognosis in HNSCC patients by epigenetic alteration of DNA mismatch repair (MMR) and microsatellite instability. Moreover, the research has shown that Fusobacterium nucleatum ( F. nucleatum ) impacts HNSCC epigenetic changes in tissues with deficient MMR to promote DNA damage and cell proliferation by suppressing MMRrelated gene expression via the TLR4/MYD88/miR-205-5p signaling pathway.

A novel signature derived from metabolism-related genes GPT and SMS to predict prognosis of laryngeal squamous cell carcinoma.

BACKGROUND: A growing body of evidence has suggested the involvement of metabolism in the occurrence and development of tumors. But the link between metabolism and laryngeal squamous cell carcinoma (LSCC) has rarely been reported. This study seeks to understand and explain the role of metabolic biomarkers in predicting the prognosis of LSCC. METHODS: We identified the differentially expressed metabolism-related genes (MRGs) through RNA-seq data of The Cancer Genome Atlas (TCGA) and Gene set enrichment analysis (GSEA). After the screening of protein-protein interaction (PPI), hub MRGs were analyzed by least absolute shrinkage and selection operator (LASSO) and Cox regression analyses to construct a prognostic signature. Kaplan-Meier survival analysis and the receiver operating characteristic (ROC) was applied to verify the effectiveness of the prognostic signature in four cohorts (TCGA cohort, GSE27020 cohort, TCGA-sub1 cohort and TCGA-sub2 cohort). The expressions of the hub MRGs in LSCC cell lines and clinical samples were verified by quantitative reverse transcriptase PCR (qRT-PCR). The immunofluorescence staining of the tissue microarray (TMA) was carried out to further verify the reliability and validity of the prognostic signature. Cox regression analysis was then used to screen for independent prognostic factors of LSCC and a nomogram was constructed based on the results. RESULTS: Among the 180 differentially expressed MRGs, 14 prognostic MRGs were identified. A prognostic signature based on two MRGs (GPT and SMS) was then constructed and verified via internal and external validation cohorts. Compared to the adjacent normal tissues, SMS expression was higher while GPT expression was lower in LSCC tissues, indicating poorer outcomes. The prognostic signature was proven as an independent risk factor for LSCC in both internal and external validation cohorts. A nomogram based on these results was developed for clinical application. CONCLUSIONS: Differentially expressed MRGs were found and proven to be related to the prognosis of LSCC. We constructed a novel prognostic signature based on MRGs in LSCC for the first time and verified it via different cohorts from both databases and clinical samples. A nomogram based on this prognostic signature was developed.

Smoking-mediated nicotinic acetylcholine receptors (nAChRs) for predicting outcomes for head and neck squamous cell carcinomas.

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 ß4 were related to the prognosis. The prognostic signature comprised the expression of subunits α5, α9, and ß4. 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.

Preoperative tracheotomy in the treatment of upper airway obstruction of patients with advanced stage supraglottic carcinoma.

BACKGROUND: Preoperative tracheotomy is an effective option that secures upper airway patency in laryngeal carcinoma patients suffering from upper airway obstruction, but the influence of this treatment on oncologic outcomes of laryngeal carcinoma remains controversial. The purpose of this study was to determine the impact of preoperative tracheotomy on overall survival in supraglottic carcinoma patients with tumor obstruction of the upper airway, and explore the potential causes. MATERIALS AND METHODS: This retrospective study collected 243 consecutive patients with advanced stage supraglottic carcinoma from 2005 to 2010. Preoperative tracheotomy in the management of upper airway obstruction in patients with supraglottic carcinoma was analyzed. RESULTS: The mean age was 60.9 years at diagnosis, with men accounting for 98.4% of all patients. Thirty nine (16.0%) patients presenting with tumor obstruction of the upper airway required preoperative tracheotomy. T4 stage patients had higher rate of tracheotomy than those of patients with T3 stage (36.8% vs 12.2%). Patients with upper airway obstruction presented with greater tumor area compared with patients without (13.7 cm2 vs 9.0 cm2). The optimal cutoff value of tumor area for tracheotomy and OS rate were both at 10 cm2. Supraglottic patients with upper airway obstruction receiving preoperative tracheotomy had poorer OS rate compared with patients without. T stage and tumor area were correlated with upper airway obstruction, and these two variables were independent predictors of OS rate in supraglottic carcinoma patients. CONCLUSIONS: Advanced stage supraglottic carcinoma patients with upper airway obstruction undergoing preoperative tracheotomy experienced worse overall survival. Advanced T stage and greater tumor size were associated with upper airway obstruction, indicating that the negative influence of tumor obstruction on survival may be cause by these two preoperative variables. Therefore, preoperative tracheotomy acts only as an alternative procedure, and is not a prognostic agent.

Oncologic outcomes of transoral laser microsurgery versus open partial laryngectomies in the management of early stage glottic carcinoma.

BACKGROUND: The oncologic outcomes between transoral laser microsurgery (TLM) and open partial laryngectomy (OPL) using comprehensive analysis in one clinical center is rare. The purpose of this study was to evaluate the oncologic outcomes of TLM in patients with early stage glottic carcinoma, and to compare the results with OPL. SUBJECTS AND METHODS: Records of 425 glottic carcinoma patients with T1 - T2 stage treated with TLM, vertical partial laryngectomy (VPL), and cricohyoidoepiglottopexy (CHEP) from 2005 to 2010 were retrospectively analyzed. The overall survival (OS), disease-specific survival (DSS), and laryngeal function preservation (LFP) of these three treatments were assessed. RESULTS: One hundred and twenty-two patients were treated with TLM. Regarding OPL, 167 patients underwent VPL, and 136 patients underwent CHEP. The mean age was 59.7 years, with men accounting for 97.2 % of all cases. The OS, DSS, and LFP rates of patients with anterior commissure (AC) involvement undergoing TLM were worse than those of patients without AC involvement, but these differences were not statistically significant. The 5-year OS, DSS, and LFP of patients undergoing TLM were 88.4 %, 89.9 %, and 83.5 %, respectively, and the oncologic outcomes of patients undergoing TLM, VPL, and CHEP were not statistically different. CONCLUSION: Glottic carcinoma patients with early stage treated with TLM experience satisfactory oncologic outcomes. No compelling difference in oncologic outcomes among three treatments of TLM, VPL and CHEP, as well as VPL and CHEP can be alternatives to patients who are not suitable for receiving TLM.

Oropharynx microbiota transitions in hypopharyngeal carcinoma treatment of induced chemotherapy followed by surgery.

AIMS: To analyze changes in oropharynx microbiota composition after receiving induced chemotherapy followed by surgery for hypopharyngeal squamous cell carcinoma (HPSCC) patients. METHODS: Clinical data and swab samples of 38 HPSCC patients (HPSCC group) and 30 patients with benign disease (control group, CG) were enrolled in the study. HPSCC group was stratified into two groups: induced chemotherapy group (IC) of 10 patients and non-induced chemotherapy group (nIC) of 28 patients. The microbiota from oropharyngeal membrane was analyzed through 16S rRNA sequencing. RESULTS: Alpha-diversity (Shannon and Ace indexes) and weighted UniFrac based beta-diversity severely decreased in the HPSCC group when compared with CG. In pre-operative comparisons, PCoA and NMDS analyses showed microbial structures in the IC group were more similar to CG than nIC. Both IC group and nIC group yielded significantly diverse post-operative communities in contrast to their pre-operative counterparts, evident by the decrease in genera Veillonella and Fusobacterium and increase in genera Streptococcus and Gemella. Given that post-operative oropharynx microbiota showed no difference between IC and nIC groups, the IC group showed less accumulation in anaerobic communities. The abundance of genera Fusobacterium, Parvimonas, Actinomyces were enhanced in the advanced stages (III/IV). CONCLUSIONS: Oropharynx microbiota in the HPSCC group presents dysbiosis with low diversity and abundance. Induced chemotherapy is beneficial in adjusting the oropharynx microbial environment leading to fewer amounts of anaerobe accumulation after operation. Higher amounts of Fusobacterium in advanced stages (III/IV) may influence the progression of HPSCC.

miR-17-5p drives G2/M-phase accumulation by directly targeting CCNG2 and is related to recurrence of head and neck squamous cell carcinoma.

BACKGROUND: The human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC). METHODS: RT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression. RESULTS: In the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3'UTR. CONCLUSION: These findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.

Prognostic value of pathological tumor size in patients with supraglottic carcinoma.

BACKGROUND: The association between pathological tumor size and predictive value in patients with supraglottic carcinoma who receive surgery is poorly understood. To gain further insight into this association, the influence of postoperative tumor size assessed via pathological evaluation on survival outcomes in patients with supraglottic carcinoma was fully analyzed. MATERIALS AND METHODS: This study collected data from 375 consecutive supraglottic carcinoma patients who underwent primary surgical treatment between 2005 and 2010. The parameters of tumor size, including tumor diameter, tumor area, and tumor volume, were used to analyze the prognostic abilities of overall survival (OS) and disease-free survival (DFS) in supraglottic carcinoma. RESULTS: Twelve women and 363 men were included, with a mean age of 61.2 years. The 5-year OS and DFS rates were 65.4% and 56.2%, respectively. The mean tumor diameter, tumor area, and tumor volume of all patients was 3.1 cm, 8.6 cm2, and 9.1 cm3, respectively. Tumors with higher pT stages had a larger tumor diameter, tumor area, and tumor volume. These three factors were significantly correlated with pT stage, and tumor volume was the strongest factor contributing to pT stage. Patients with a larger tumor diameter, tumor area, and tumor volume had worse OS and DFS. Tumor area and tumor volume were independent prognostic factors of OS and DFS in supraglottic carcinoma patients. CONCLUSIONS: Patients with a larger tumor size have inferior survival outcomes, and tumor area and tumor volume are independent predictive parameters of survival in supraglottic carcinoma patients who are treated with primary surgery.

A nomogram for predicting occult lymph node metastasis in early hypopharyngeal cancer with cN0.

PURPOSE: To explore the risk factors of cervical occult lymph node metastasis (OLNM) in early cN0 hypopharyngeal squamous cell carcinoma (HPSCC), and construct a nomogram model to predict the risk of OLNM in patients with early cN0 HPSCC. METHODS: 78 cases of early (T1-T2) HPSCC patients who underwent hypopharyngectomy were retrospectively analyzed. Univariate and multivariate logistic regression analyses were used to determine independent risk factors and a nomogram was constructed according to the results of the multivariate logistic regression analysis. Model performance was assessed by constructing a receiver operating characteristic (ROC) curve, and discriminatory capacity assessed using the area under the curve (AUC). Calibration was completed using a plotted calibration curve accompanied by the Hosmer-Lemeshow test. RESULTS: Multivariate logistic regression analysis revealed that age (OR 0.928, 95% CI 0.863-0.997), history of drinking (OR 6.668, 95% CI 1.724-25.788), histological differentiation of tumor (OR 7.269, 95% CI 1.000-52.820), depth of invasion (OR 5.046, 95% CI 1.281-19.874) were independent risk factors of OLNM in early cN0 HPSCC. The ROC curve had an AUC of 0.811 (95% CI 0.713-0.909), which implies good discriminate capacity. The calibration curve and the Hosmer-Lemeshow test (P = 0.972) demonstrated good model fitted and high calibration. CONCLUSION: A nomogram model based on age, drinking history, histological differentiation of tumor, and depth of tumor invasion was successfully developed to predict occult cervical lymph node metastasis in patients with early cN0 hypopharyngeal cancer.

Preoperative tracheotomy as reflection of tumor size impacting oncologic outcomes of patients with advanced stage glottic carcinoma.

PURPOSE: The aim of this study was to evaluate the influence of preoperative tracheotomy on oncologic outcomes of advanced stage glottic carcinoma patients, and to explore the potential reason. METHODS: We retrospectively analyzed 413 consecutive advanced stage glottic carcinoma patients from January 2005 to December 2010. The correlation of preoperative tracheotomy and potential impacting factor of tumor size involving tumor diameter and tumor area with overall survival (OS) and disease-free survival (DFS) was fully assessed. RESULTS: Our cohort consisted of 302 (73.1%) patients with T3 and 111 (26.9%) patients with T4, and 98 (23.7%) patients received preoperative tracheotomy. The OS and DFS rates of patients receiving preoperative tracheotomy were worse than those without (5-year OS: 49.3% versus 69.8%; 5-year DFS: 45.3% versus 61.0%). The mean tumor diameter and tumor area of patients with preoperative tracheotomy were greater than those without (3.3 cm versus 2.4 cm, 8.9 cm2 versus 4.7 cm2). The optimal cutoff values of tumor diameter and tumor area for tracheotomy were 2.85 cm and 6.64 cm2. Tumor diameter and tumor area were correlated with tracheotomy intervention. Furthermore, when considering the potential effect of tumor area in multivariate model, we found that it was a significant factor in survival outcomes but variable of preoperative tracheotomy was not. CONCLUSION: This study indicates that tumor size is correlated with preoperative tracheotomy, and tracheotomy intervention may be reflection from effect of great tumor size that is a true adverse factor influencing oncologic outcomes of advanced stage glottic carcinoma patients.

Lack of miR-1246 in small extracellular vesicle blunts tumorigenesis of laryngeal carcinoma cells by regulating Cyclin G2.

Small extracellular vesicle (sEV) has precise impacts on tumor microenvironment and play vital functions in intercellular interaction. However, the functional role of sEV miRNA on laryngeal squamous cell carcinoma (LSCC) is largely unresolved. Here, the expression of miR-1246 in LSCC tissues and plasma sEV was examined. The internalization ability of sEV was determined by uptake assay. Then, the source and purity of sEV were checked through RNase and/or pharmacological inhibitors application. The invasion, migration, proliferation, and cell cycle assays were used to determine the altered abilities of miR-1246 in sEV in LSCC. Finally, target gene of miR-1246, Cyclin G2 (CCNG2), was stained immunohistochemically. In addition, the relationship between CCNG2 and clinicopathological features of patients was analyzed. We found that miR-1246 was higher in LSCC tissues and plasma sEV. MiR-1246 was enriched in sEV rather than soluble form. SEV could be internalized into adjacent cells. Lack of miR-1246 in sEV abrogated the tumorigenesis of LSCC. Furthermore, CCNG2 knockdown arrested the cell cycle and correlated to clinicopathological features and prognosis of LSCC patients. Taken together, we found that the function of sEV miR-1246 by regulating CCNG2 is responsible for LSCC advancement with emphasis on the main source of miR-1246 mainly root in sEV rather than in soluble form.

Throat Microbial Community Structure and Functional Changes in Postsurgery Laryngeal Carcinoma Patients.

The microbial community structure in the throat and its shift after laryngectomy in laryngeal squamous cell carcinoma (LSCC) patients were investigated. Thirty swab samples taken prior to laryngectomy (SLC), 18 samples 1 week after laryngectomy (SLCA1w), and 30 samples 24 weeks after laryngectomy (SLCA24w) from 30 LSCC patients were examined. Microbial diversity was profiled through sequencing the V3-V4 variable region of the 16S rRNA gene. Quantitative real-time PCR (qPCR) was used to validate the 16S rRNA sequence data for the V3-V4 region. The community structure and function of throat microbiota were assessed by PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) analysis. Both alpha and beta diversity results showed significant differences in the throat microbiota of LSCC patients before and after laryngectomy (P < 0.05). The drinking index of the SLC group was positively associated with the genus abundance of Prevotella (P < 0.05). The SLCA1w group had lower abundances of Fusobacterium, Leptotrichia, Lachnoanaerobaculum, and Veillonella than the SLC group (P < 0.05). The SLCA24w group had higher abundances of Streptococcus and Leptotrichia as well as lower abundances of Fusobacterium and Alloprevotella than the SLC group (P < 0.05). The throat microbiomes of the SLC group could be implicated in human cancer signaling pathways, as evidenced by PICRUSt analysis (P < 0.05). Our study clarifies alterations in throat microbial community structure and function in LSCC patients during the perioperative period and postoperative recovery period.IMPORTANCE Laryngeal squamous cell carcinoma greatly impacts patients' lives, and noninvasive means of prognostic assessment are valuable in determining the effectiveness of laryngectomy. We set out to study the microbial structure changes in the throat before and after laryngectomy and found the gene functions of several throat bacteria to be associated with human cancer signaling pathways. Our findings may offer insights into the disease management of patients with laryngeal squamous cell carcinoma. We hope to provide a means of using molecular mechanisms to improve the prognosis of laryngeal cancer treatment and to facilitate relevant research.

Prognostic values of preoperative platelet-to-lymphocyte ratio and platelet-related indices in advanced hypopharyngeal squamous cell carcinoma.

OBJECTIVES: To investigate the prognostic values of preoperative platelet-to-lymphocyte ratio (PLR) and platelet-related indices in advanced hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: The data of 247 eligible advanced HPSCC patients were reviewed retrospectively. Pretreatment haematological parameters were categorised into two groups based on the result of X-tile, and several variates were assessed using chi-square test, Kaplan-Meier method, Cox univariate and multivariate analysis. RESULTS: The optimal cut-off points of 171.4 for PLR, 260 × 109 /L for platelet, 10.4 fL for mean platelet volume (MPV) and 16.5% for platelet distribution width were defined. The haematological parameters PLR and MPV, postoperative metastasis and internal jugular vein invasion were statistically significant in OS and DFS analyses (P < .05). The high PLR (>171.4) or high MPV (>10.4 fL) was significantly associated with worse OS and DFS (P < .05). CONCLUSIONS: The preoperative levels of PLR and MPV could be considered as independent prognostic predictors in patients with advanced HPSCC.

Nicotine-induced activation of cholinergic receptor nicotinic alpha 5 subunit mediates the malignant behaviours of laryngeal squamous epithelial cells by interacting with RABL6.

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.

Integrative methylome and transcriptome analysis reveals epigenetic regulation of Fusobacterium nucleatum in laryngeal cancer.

The aetiological mechanisms of Fusobacterium nucleatum in laryngeal cancer remain unclear. This study aimed to reveal the epigenetic signature induced by F. nucleatum in laryngeal squamous cell carcinoma (LSCC). Combined analysis of methylome and transcriptome data was performed to address the functional role of F. nucleatum in laryngeal cancer. Twenty-nine differentially expressed methylation-driven genes were identified by mapping the methylation levels of significant differential methylation sites to the expression levels of related genes. The combined analysis revealed that F. nucleatum promoted Janus kinase 3 (JAK3) gene expression in LSCC. Further validation found decreased methylation and elevated expression of JAK3 in the F. nucleatum-treated LSCC cell group; F. nucleatum abundance and JAK3 gene expression had a positive correlation in tumour tissues. This analysis provides a novel understanding of the impact of F. nucleatum in the methylome and transcriptome of laryngeal cancer. Identification of these epigenetic regulatory mechanisms opens up new avenues for mechanistic studies to explore novel therapeutic strategies.

The Role of Parotid Gland Invasion in Adenoid Cystic Carcinoma of the External Auditory Canal.

OBJECTIVE: This study aimed to investigate the significance of parotid gland invasion in predicting distant metastasis of adenoid cystic carcinoma in the external auditory canal. STUDY DESIGN: Single-institution retrospective cohort study. METHODS: A retrospective review of patients with adenoid cystic carcinoma of the external auditory canal who underwent surgery was performed. Information on patient demographics, parotid gland invasion, tumor stage, perineural invasion, lymphovascular invasion, and follow-up data were collected and analyzed. RESULTS: One hundred twenty-nine patients were identified for review. Parotid gland invasion was noted in 45 patients (34.9%). Parotid gland invasion was significantly associated with tumor stage, perineural invasion, distant metastasis, and postoperative adjuvant therapy. Distant metastasis was noted in 30 patients (23.3%). Multivariate Cox proportional hazards analysis identified parotid gland invasion as an independent risk factor for predicting distant metastasis. The 5-year distant metastasis-free survival rate was 83.6% for patients without parotid gland invasion and 61.8% for patients with parotid gland invasion (p = 0.010). CONCLUSIONS: The parotid gland invasion rate is relatively high in adenoid cystic carcinoma of the external auditory canal and is significantly related to tumor stage. Parotid gland invasion is associated with worse distant metastasis-free survival. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:419-425, 2024.

Metagenomic shotgun sequencing reveals the enrichment of Salmonella and Mycobacterium in larynx due to prolonged ethanol exposure.

The exposure of ethanol increases the risk of head and neck inflammation and tumor progression. However, limited studies have investigated the composition and functionality of laryngeal microbiota under ethanol exposure. We established an ethanol-exposed mouse model to investigate the changes in composition and function of laryngeal microbiota using Metagenomic shotgun sequencing. In the middle and late stages of the experiment, the laryngeal microbiota of mice exposed to ethanol exhibited obvious distinguished from that of the control group on principal-coordinate analysis (PCoA) plots. Among the highly abundant species, Salmonella enterica and Mycobacterium marinum were likely to be most impacted. Our findings indicated that the exposure to ethanol significantly increased their abundance in larynxes in mice of the same age, which has been confirmed through FISH experiments. Among the species-related functions and genes, metabolism is most severely affected by ethanol. The difference was most obvious in the second month of the experiment, which may be alleviated later because the animal established tolerance. Notable enrichments concerning energy, amino acid, and carbohydrate metabolic pathways occurred during the second month under ethanol exposure. Finally, based on the correlation between species and functional variations, a network was established to investigate relationships among microbiota, functional pathways, and related genes affected by ethanol. Our data first demonstrated the continuous changes of abundance, function and their interrelationship of laryngeal microbiota under ethanol exposure by Metagenomic shotgun sequencing. Importance: Ethanol may participate in the inflammation and tumor progression by affecting the composition of the laryngeal microbiota. Here, we applied the metagenomic shotgun sequencing instead of 16 S rRNA sequencing method to identify the laryngeal microbiota under ethanol exposure. Salmonella enterica and Mycobacterium marinum are two dominant species that may play a role in the reconstruction of the laryngeal microenvironment, as their local abundance increases following exposure to ethanol. The metabolic function is most evidently impacted, and several potential metabolic pathways could be associated with alterations in microbiota composition. These findings could help us better understand the impact of prolonged ethanol exposure on the microbial composition and functionality in the larynx.