PFKL promotes cell viability and glycolysis and inhibits cisplatin chemosensitivity of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) with a high incidence and mortality rate, has a serious impact worldwide. Phosphofructokinase-1 liver type (PFKL) is a major enzyme in glycolysis progress, but its role in modulating tumorigenesis and cisplatin (DDP) chemosensitivity of LSCC was still unclear. The mRNA and protein levels of PFKL were detected by qRT-PCR and immunohistochemical assay. Cell Counting Kit-8 assay and flow cytometry were carried out to observe cell viability, as well as apoptosis and mitochondrial reactive oxygen species (mito-ROS). Extracellular acidification rate and lactate content were measured using extracellular flux analysis and lactate assay kit. Immunofluorescent staining was used to evaluate the expression of γ-H2AX foci. DNA damage was detected via single-cell gel electrophoresis. Western blotting was introduced to evaluate the protein level of PFKL, LDHA, γ-H2AX, cleaved PARP, H3K27ac, and H3K9ac. Mice xenograft model of LSCC was built for in vivo validation. The PFKL expression was significantly increased in LSCC and associated with poor survival of LSCC patients. Knockdown of PFKL in LSCC cells significantly inhibited cell viability, ECAR, lactate content, and LDHA expression, but promoted mito-ROS level. Furthermore, knockdown of PFKL enhanced response of LSCC cells to DDP by increasing DDP-induced apoptosis, promoting DDP-induced mito-ROS level, γ-H2AX foci, tail DNA, and the expression of γ-H2AX and cleaved PARP. However, the overexpression of PFKL in LSCC cells had opposite experimental results. Nude mice tumor formation experiment proved that downregulation of PFKL significantly enhanced response of cells to DDP, demonstrated by reduced tumor volume, weight and increased TUNEL-positive cells. Suppression of CBP/EP300-mediated PFKL transcription inhibited cell viability and glycolysis and promoted mito-ROS in LSCC. PFKL promotes cell viability and DNA damage repair in DDP-treated LSCC through regulation of glycolysis pathway.

The association of E2F1 and E2F2 single nucleotide polymorphisms with laryngeal squamous cell carcinoma pathomorphological features.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common types of cancer in the upper respiratory tract. It is well-known that it has a high mortality rate and poor prognosis in advanced stages. There are well-known risk factors for LSCC, though new specific and prognostic blood-based markers for LSCC development and prognosis are essential. The current study aimed to evaluate the impact of four different single nucleotide polymorphisms (SNPs), E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028), on LSCC development, morphological features, and patient 5-year survival rate. METHODS: A total of 200 LSCC patients and 200 controls were included in this study; both groups were matched by age and sex. In the present study, we analyzed four single nucleotide polymorphisms (SNPs) in the genes E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028) and evaluated their associations with the risk of LSCC development, its clinical and morphological manifestation, and patients 5-year survival rate. Genotyping was carried out using RT-PCR. RESULTS: None of the analyzed SNPs showed a direct association with LSCC development. E2F2 rs2075993 G allele carriers (OR = 4.589, 95% CI 1.050-20.051, p = 0.043) and rs3820028 A allele carriers (OR = 4.750, 95% CI 1.088-20.736, p = 0.038) had a statistically significantly higher risk for poor differentiated or undifferentiated LSCC than non-carriers. E2F1 rs3213180 GC heterozygotes were found to have a 3.7-fold increased risk for lymph node involvement (OR = 3.710, 95% CI 1.452-9.479, p = 0.006). There was no statistically significant association between investigated SNPs and patient 5-year survival rate. CONCLUSIONS: The present study indicates that E2F2 rs2075993 and rs3820028 impact LSCC differentiation, whereas E2F1 rs3213180 - on lymph node involvement.

LncRNA NEAT1 promotes proliferation, migration, and invasion of laryngeal squamous cell carcinoma cells through miR-411-3p/FZD3-mediated Wnt signaling pathway.

The lncRNA NEAT1 has been shown to promote the progression of several cancers, containing laryngeal squamous cell carcinoma (LSCC). However, the precise mechanism by which it promotes LSCC progression remains unclear. In this study, we verified the high expression of lncRNA NEAT1 in LSCC tissues and cells using RT-qPCR. Analysis of clinical data exhibited that high expression of lncRNA NEAT1 was associated with a history of smoking, worse T stage, lymph node metastasis, and later TNM stage in patients with LSCC. The promotion effect of lncRNA NEAT1 on LSCC cell proliferation, migration, invasion, and tumor growth in vivo was verified by CCK-8, plate clone formation, Transwell, and nude mouse tumorigenicity assays. Bioinformatics prediction and double luciferase reporter gene assay verified the binding of miR-411-3p to lncRNA NEAT1 and FZD3 mRNA, and inhibition of miR-411-3p reversed the inhibitory effect of lncRNA NEAT1 on FZD3 expression in LSCC cells. We also verified that lncRNA NEAT1-mediated FZD3 activation in the Wnt pathway affects LSCC development. In conclusion, we demonstrate that lncRNA NEAT1 promotes the progression of LSCC, and propose that the lncRNA NEAT1/miR-411-3p/FZD3 axis may be an effective target for LSCC therapy.

RUNX1-BMP2 promotes vasculogenic mimicry in laryngeal squamous cell carcinoma via activation of the PI3K-AKT signaling pathway.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck. Vasculogenic mimicry (VM) is crucial for tumor growth and metastasis and refers to the formation of fluid channels by invasive tumor cells rather than endothelial cells. However, the regulatory mechanisms underlying VM during the malignant progression of LSCC remain largely unknown. METHODS: Gene expression and clinical data for LSCC were obtained from the TCGA and Gene GEO (GSE27020) databases. A risk prediction model associated with VM was established using LASSO and Cox regression analyses. Based on their risk scores, patients with LSCC were categorized into high- and low-risk groups. The disparities in immune infiltration, tumor mutational burden (TMB), and functional enrichment between these two groups were examined. The core genes in LSCC were identified using the machine learning (SVM-RFE) and WGCNA algorithms. Subsequently, the involvement of bone morphogenetic protein 2 (BMP2) in VM and metastasis was investigated both in vitro and in vivo. To elucidate the downstream signaling pathways regulated by BMP2, western blotting was performed. Additionally, ChIP experiments were employed to identify the key transcription factors responsible for modulating the expression of BMP2. RESULTS: We established a new precise prognostic model for LSCC related to VM based on three genes: BMP2, EPO, and AGPS. The ROC curves from both TCGA and GSE27020 validation cohorts demonstrated precision survival prediction capabilities, with the nomogram showing some net clinical benefit. Multiple algorithm analyses indicated BMP2 as a potential core gene. Further experiments suggested that BMP2 promotes VM and metastasis in LSCC. The malignant progression of LSCC is promoted by BMP2 via the activation of the PI3K-AKT signaling pathway, with the high expression of BMP2 in LSCC resulting from its transcriptional activation by runt-related transcription factor 1 (RUNX1). CONCLUSION: BMP2 predicts poor prognosis in LSCC, promotes LSCC VM and metastasis through the PI3K-AKT signaling pathway, and is transcriptionally regulated by RUNX1. BMP2 may be a novel, precise, diagnostic, and therapeutic biomarker of LSCC.

Single-cell atlas profiling revealed cellular characteristics and dynamic changes after PD-1 blockade therapy of brain metastases from laryngeal squamous cell carcinoma.

Brain metastasis (BM) in laryngeal squamous cell carcinoma (LSCC) is uncommon but prognosis is poor. Anti-PD-1 immunotherapy benefits some advanced LSCC cases, yet its efficiency is limited by tumor complexity. We analyzed paired metastatic tumor samples from before and after immunotherapy using single-cell RNA sequencing (scRNA-seq), along with a primary LSCC dataset and bulk RNA sequencing. This identified changes post-immunotherapy and revealed differences in single-cell transcriptomes among LSCC, primBM, and neoBM. Our findings show that anti-PD-1 treatment suppresses metastasis-promoting pathways like VEGF and EMT in cancer cells, and alters immune cell functions. Notably, it upregulates T cell activation, leading to CD8 T cell exhaustion from excess heat shock proteins, notably HSPA8. However, CD8 T cell cytotoxic functions improve post-treatment. In myeloid cells, anti-PD-1 therapy enhances antigen presentation and promotes a proinflammatory shift post-metastasis. Additionally, NUPR1 is linked to BM in LSCC, and NEAT1 is a potential metastatic cancer cell cycle participant. Our study provides insights into cancer heterogeneity and the impact of PD-1 immunotherapy on metastasis, aiding precise diagnosis and prognosis.

The impact of margins in laryngeal cancer patients treated with transoral laser microsurgery: a systematic review and meta-analysis.

PURPOSE: In laryngeal squamous cell carcinoma (LSCC) treated with transoral laser microsurgery (TOLMS), the status of margins significantly affected local control. When a positive or close margin is present, there is no ubiquitous consensus regarding further treatments. The rationale of the present systematic review and meta-analysis is to investigate the survival impact of the status of the margins in patients affected by LSCC treated with TOLMS. DATA SOURCES: PubMed, EMBASE, and Cochrane Library. METHODS: We performed a systematic search, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Inclusion criteria were: patients affected by LSCC, staged according to the American Joint Committee on Cancer Staging System and treated by TOLMS without any previous treatment; margins status (close, positive, negative) and the adjusted hazard ratios (aHRs) with 95% confidence intervals (CIs) of overall survival, disease-specific survival, and disease-free survival has to be reported. RESULTS: Nine studies were deemed eligible for the qualitative analysis, and 3 for the quantitative analysis to investigate the association between margin status and OS. The cumulative number of patients was 3130. The sample size ranged from 96 to 747 patients. The follow-up period ranged from 0 to 201 months. The meta-analysis results show that positive margins have an aHR of 1.30 yet with CI range (0.56 to 2.97). CONCLUSIONS: Our current meta-analysis results are unable to definitively assess the real impact of resection margins on OS. Few authors provide accurate data regarding position and types of margins. Further prospective or high-quality studies are required.

Chemoradiotherapy vs radiotherapy for non-surgical locally advanced laryngeal squamous cell carcinoma patients: a propensity score-matched study and practical nomogram construction.

OBJECTIVE: To compare the cancer-specific survival (CSS) among patients with locally advanced laryngeal squamous cell carcinoma (LSCC) receiving chemoradiotherapy (CRT) and radiotherapy (RT) treatment, as well as to establish a prognostic nomogram for survival prediction in patients receiving CRT. METHOD: Using data from the Surveillance, Epidemiology, and End Results (SEER) database, patients with laryngeal cancer were identified between 2010 and 2015, with follow-up up to 2018. Propensity score matching (PSM) was performed to minimize disproportionate distributions of the potential confounding. Cox regression models were used to evaluate the CSS of two treatment groups. A prognostic nomogram for patients receiving CRT was then developed and evaluated. RESULTS: Totally 1085 non-surgical patients with locally advanced LSCC were included in this study (median [IQR] age, 62 [55-69] years; 829 [76.41%] males), of which 913 receiving CRT and 172 receiving RT. After PSM, significantly improved CSS was observed in locally advanced LSCC patients receiving CRT when compared to RT (HR: 0.62 [95% CI 0.42-0.92]; P = 0.014). Then, in the group of 639 locally advanced LSCC patients receiving CRT, a prognostic nomogram based on age, tumor size, N category, and marital status were developed and validated, of which the predictive performance was superior to that of TNM staging system (7th edition). CONCLUSION: CSS shows a statistically significant improvement in locally advanced LSCC patients who receipt of CRT when compared with RT. Furthermore, a prognostic nomogram for locally advanced LSCC patients receiving CRT was established, which shows a good calibration and identification accuracy.

Evaluation of Triglyceride-Glucose Index Elevation as a Biomarker and Risk Factor in Laryngeal Squamous Cell Carcinoma.

OBJECTIVES: Insulin resistance is associated with increased levels of IGF-1. IGF-1 has been shown to increase the risk of laryngeal squamous cell carcinoma. The Triglyceride-glucose index (TyG index) is a marker of insulin resistance. Our study aimed to investigate the relationship between the TyG index and laryngeal squamous cell carcinoma. DESIGN: Retrospective cohort study. SETTING: Two tertiary care academic hospitals. METHODS: The study included 53 patients with laryngeal squamous cell carcinoma (Group 1) and 48 healthy volunteers (Group 2). Laryngeal cancer patients were divided into two groups according to their stage. Stages I and II were named Group 1A, and Stages III and IV were called Group 1B. The TyG index was calculated as ln [fasting Triglycerides (mg/dL) × fasting plasma glucose (mg/dL)/2]. The effect of the TyG index on laryngeal cancer was investigated on the parameters of sex, age, body mass index, and stage of the disease. RESULTS: There were no significant differences in age, sex, and BMI between the groups. The TyG index of group 1 (4.75 ± 0.33) was significantly higher than that of group 2 (4.59 ± 0.15). The TyG index value of group 1B (4.84 ± 0.31) was significantly higher than both group 1A (4.61 ± 0.32) and group 2 (4.59 ± 0.15). There was no significant difference between the TyG index values of group 1A (4.61 ± 0.32) and group 2 (4.59 ± 0.15). CONCLUSION: The TyG index may be a promising laryngeal squamous cell carcinoma biomarker. People with a higher TyG index may have a higher incidence of laryngeal squamous cell carcinoma and a higher risk of progression.

MCTS1 enhances the proliferation of laryngeal squamous cell carcinoma via promoting OTUD6B-1 mediated LIN28B deubiquitination.

The aberrant upregulation of MCTS1 Re-Initiation and Release Factor (also known as Malignant T-cell-amplified sequence 1, MCTS1) can promote laryngeal squamous cell carcinoma (LSCC). It might act as a binding partner of multiple proteins. In this study, we further explored the expression of potential interaction between MCTS1 and OTU domain-containing protein 6B (OTUD6B) and its influence on the ubiquitination and degradation of OTUD6B's substrate in LSCC. LSCC cell lines AMC-HN-8 and TU177 were utilized for assessing protein-protein interaction, protein degradation and tumor growth in vitro and in vivo. The results showed that MCTS1 interacts with OUTD6B isoform 1 (OTUD6B-1) in the cell lines. Higher OTUD6B-1 expression is associated with significantly shorter progression-free interval in LSCC patients. OTUD6B positively modulated the expression of cyclin D1, cyclin E1 and c-Myc and LSCC cell proliferation in vitro and in vivo. MCTS1 negatively modulated the degradation of LIN28B in G1/S cells, via enhancing OTUD6B-mediated cleaving of K48-branched ubiquitin chains from LIN28B. OTUD6B or LIN28B shRNA weakened MCTS1 overexpression-induced cyclin D1 and c-Myc protein expression and LSCC cell proliferation. In summary, this study revealed that MCTS1 could enhance LSCC proliferation partially via the OTUD6B-LIN28B axis.

CD206+ tumor-associated macrophages interact with CD4+ tumor-infiltrating lymphocytes and predict adverse patient outcome in human laryngeal squamous cell carcinoma.

BACKGROUND: Tumor-associated macrophages (TAMs) are major component in the tumor microenvironment (TME) and play regulatory role in tumor progression. We aimed to investigate the infiltration and prognostic value of TAMs in laryngeal squamous cell carcinoma (LSCC) and to reveal the underlying mechanism of TAM subgroups in tumorigenesis. METHODS: Hematoxylin and eosin (HE) staining were performed to define the tumor nest and stroma of LSCC tissue microarrays. CD206 + /CD163 + and iNOS + TAM infiltrating profiles were obtained and analyzed through double-labeling immunofluorescence and immunohistochemical staining. The recurrence-free (RFS) and overall survival (OS) curves based on the infiltration of TAMs were plotted using the Kaplan-Meier method. Infiltration of macrophages, T lymphocytes and their corresponding subgroups were analyzed in fresh LSCC tissue samples by flow cytometry. RESULTS: We found that CD206+ rather than CD163+ M2-like TAMs were the most enriched population in the TME of human LSCC. CD206+ macrophages localized mostly in the tumor stroma (TS) rather than the tumor nest (TN) region. In contrast, relatively low infiltration of iNOS+ M1-like TAMs were found in the TS and almost none in the TN region. High level of TS CD206+ TAM infiltration correlated with poor prognosis. Interestingly, we identified a HLA-DRhigh CD206+ macrophage subgroup that was significantly associated with the tumor-infiltrating CD4+ T lymphocytes and showed different surface costimulatory molecule expression than that of the HLA-DRlow/-CD206+ subgroup. Taken together, our results indicate that HLA-DRhigh-CD206+ is a highly activated subgroup of CD206 + TAMs that may interact with CD4 + T cells through MHC-II axis and promote tumorigenesis.

The prognostic role of coagulation markers in the progression and metastasis of laryngeal squamous cell carcinoma.

BACKGROUND: The application of coagulation-related markers in laryngeal squamous cell carcinoma(LSCC) remains unclear. This study explored the prognostic role of coagulation markers in the progression and metastasis of LSCC. METHODS: Coagulation markers of patients with LSCC receiving surgery in the Second Affiliated Hospital of Fujian Medical University in China, from January 2013 to May 2022 were retrospectively analyzed and compared with those of contemporary patients with benign laryngeal diseases. The relationship between clinicopathological features of LSCC and coagulation markers was analyzed with the chi-square and rank sum tests. The ROC curve analysis was utilized to evaluate the diagnostic efficacy of seven coagulation markers for LSCC and its different clinicopathological features, and to find the optimal cutoff value of each coagulation marker. RESULTS: 303 patients with LSCC and 533 patients with benign laryngeal diseases were included in the present analysis. Compared to the control group, prothrombin time (PT) (p < 0.001), activated partial thromboplastin time (APTT) (p = 0.001), and Fib (p < 0.001) in patients with LSCC were significantly higher, while mean platelet volume (MPV) (p < 0.001) was significantly shorter. Significant increases were detected in PT (Z = 14.342, p = 0.002), Fib (Z = 25.985, p < 0.001), platelet count (PC) (Z = 12.768, p = 0.005), PCT (Z = 9.178, p = 0.027), MPV (F = 2.948, p = 0.033) in T4 stage. Fib had the highest prognostic value among the seven coagulation markers in different T stages (AUC = 0.676, p < 0.001), N stages (AUC = 0.717, p < 0.001), tumor stage (AUC = 0.665, p < 0.001), differentiation degree (AUC = 0.579, p = 0.022), and neurovascular invasion (AUC = 0.651, p = 0.007). Fib (Z = 25.832, p < 0.001), PC (Z = 23.842, p < 0.001), and PCT (Z = 20.15, p < 0.001) in N1 and N3 stages were significantly higher than in N0 stage. PT (Z = 12.174, p = 0.007), Fib (Z = 23.873, p < 0.001), PC (Z = 17.785, p < 0.001), and PCT (Z = 14.693, p = 0.002) were significantly higher in stage IV than in stage I and II. APTT (Z=-1.983, p = 0.047), Fib (Z=-2.68, p = 0.007), PC (Z=-2.723, p = 0.006), and PCT (Z=-2.592, p = 0.01) increased significantly when the tumor invaded neurovascular tissue. CONCLUSIONS: Coagulation markers have the potential to act as biomarkers for predicting pathological features of LSCC. The high level of Fib was helpful for the diagnosis of LSCC and the detection of advanced LSCC. TRIAL REGISTRATION: Not applicable.

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.

Identification of competing endogenous RNA network in laryngeal squamous cell carcinoma.

OBJECTIVE: This study was conducted to investigate key long noncoding RNAs (lncRNAs) involved in competitive endogenous RNA (ceRNA) network associated with laryngeal squamous cell carcinoma (LSCC). MATERIALS AND METHODS: Three mRNA datasets, two miRNA datasets, and one lncRNA dataset of LSCC were downloaded from GEO database. Following the identification of differentially expressed mRNAs (DEmRNAs), (microRNAs) miRNAs (DEmiRNAs), and lncRNAs (DElncRNAs) in LSCC compared with adjacent tissues, functional enrichment of DEmRNAs was performed. Then, construction of the ceRNA (DElncRNA-DEmiRNA-DEmRNA) regulatory network and functional analyses of all DEmRNAs in ceRNA regulatory network were conducted. Quantitative real-time polymerase chain reactions (qRT-PCR) were used to detect the expression levels of selected DEmRNAs, DEmiRNAs, and DElncRNAs. RESULTS: A total of 3449 DEmRNAs, 40 DEmiRNAs, and 100 DElncRNAs were identified in LSCC. The ceRNA networks, which contained 132 DElncRNA-DEmiRNA pairs and 287 DEmiRNA-DEmRNA pairs, involving 44 lncRNAs, 3 miRNAs, and 271 mRNAs, were obtained. DEmRNAs in ceRNA regulatory networks were significantly enriched in pathways in cancer, prostate cancer, and aldosterone-regulated sodium reabsorption. Except for HCG22 and hsa-miR-1246, expressions of the others in the qRT-PCR results played the same pattern with that in our integrated analysis, generally. CONCLUSIONS: We concluded that HCG22/EGOT-hsa-miR-1275-FAM107A and HCG22/EGOT-hsa-miR-1246-Glycerol-3-phosphate dehydrogenase 1 like interaction pairs may play a central role in LSCC.

Exosomal lncRNA LINC02191 Promotes Laryngeal Squamous cell Carcinoma Progression by Targeting miR-204-5p/RAB22A Axis and Regulating PI3K/Akt/mTOR Pathway.

Recent research has explored the potential use of serum-derived biomarkers in cancer screening, and mounting evidence has illustrated the pivotal roles of long noncoding RNAs (lncRNAs) in regulating laryngeal squamous cell carcinoma (LSCC) progression. LINC02191 is a newly identified lncRNA and no studies have investigated its role in malignant tumors. This study aims to explore the functions and mechanisms of lncRNA LINC02191 in LSCC. LINC02191 was knocked down in LSCC cells using shRNAs for loss-of-function experiments. RT-qPCR revealed that LINC02191 was upregulated in LSCC patients' serum exosomes, tissues and cells. Western blotting and RT-qPCR were implemented for detecting molecular protein and RNA levels. Colony formation, CCK-8, wound healing and Transwell assays were employed for examining LSCC cell malignant behaviors in vitro. A tumor-bearing mouse model (n = 4/group) was established for examining LINC02191 role in vivo. The results showed that LINC02191 silencing hindered LSCC cell proliferation, invasiveness, migration as well as EMT in vitro and impeded tumorigenesis in xenograft mouse model. Luciferase reporter assay was utilized for verifying the interaction between LINC02191, miR-204-5p and RAB22A. Pearson correlation analysis was employed to evaluate their expression correlation in LSCC tissue specimens (N = 30). Mechanistically, LINC02191 upregulated RAB22A by binding to miR-204-5p, and knocking down LINC02191 inhibited PI3K/Akt/mTOR signaling transduction in LSCC cells and tumor-bearing mice. Moreover, RAB22A overexpression reversed LINC02191 depletion-triggered suppression of LSCC cell aggressiveness and inactivation of PI3K/Akt/mTOR signaling. In conclusion, LINC02191 aggravates LSCC by targeting miR-204-5p/RAB22A/PI3K/Akt/mTOR signaling pathway, which indicates that LINC02191 may serve as a promising target for LSCC treatment.

A deep learning-based model predicts survival for patients with laryngeal squamous cell carcinoma: a large population-based study.

OBJECTIVES: To assess the performance of DeepSurv, a deep learning-based model in the survival prediction of laryngeal squamous cell carcinoma (LSCC) using the Surveillance, Epidemiology, and End Results (SEER) database. METHODS: In this large population-based study, we developed and validated a deep learning survival neural network using pathologically diagnosed patients with LSCC from the SEER database between January 2010 and December 2018. Totally 13 variables were included in this network, including patients baseline characteristics, stage, grade, site, tumor extension and treatment details. Based on the total risk score derived from this algorithm, a three-knot restricted cubic spline was plotted to exhibit the difference of survival benefits from two treatment modalities. RESULTS: Totally 6316 patients with LSCC were included in the study, of which 4237 cases diagnosed between 2010 and 2015 were selected as the development cohort, and the rest (2079 cases diagnosed from 2016 to 2018) were the validation cohort. A state-of-the-art deep learning-based model based on 23 features (i.e., 13 variables) was generated, which showed more superior performance in the prediction of overall survival (OS) than the tumor, node, and metastasis (TNM) staging system (C-index for DeepSurv vs TNM staging = 0.71; 95% CI 0.69-0.74 vs 0.61; 95% CI 0.60-0.63). Interestingly, a significantly nonlinear association between total risk score and treatment effectiveness was observed. When the total risk score ranges 0.1-1.5, surgical treatment brought more survival benefits than nonsurgical one for LSCC patients, especially in 70.5% of patients staged III-IV. CONCLUSIONS: The deep learning-based model shows more potential benefits in survival estimation for patients with LSCC, which may potentially serve as an auxiliary approach to provide reliable treatment recommendations.

The relation between hypoxia and proliferation biomarkers with radiosensitivity in locally advanced laryngeal cancer.

PURPOSE: Treatment decision-making in advanced-stage laryngeal squamous cell carcinoma (LSCC) is difficult due to the high recurrence rates and the desire to preserve laryngeal functions. New predictive markers for radiosensitivity are needed to facilitate treatment choices. In early stage glottic LSCC treated with primary radiotherapy, expression of hypoxia (HIF-1α and CA-IX) and proliferation (Ki-67) tumour markers showed prognostic value for local control. The objective of this study is to examine the prognostic value of tumour markers for hypoxia and proliferation on locoregional recurrent disease and disease-specific mortality in a well-defined cohort of patients with locally advanced LSCC treated with primary, curatively intended radiotherapy. METHODS: In pre-treatment biopsy tissues from a homogeneous cohort of 61 patients with advanced stage (T3-T4, M0) LSCC primarily treated with radiotherapy, expression of HIF-1α, CA-IX and Ki-67 was evaluated with immunohistochemistry. Demographic data (age and sex) and clinical data (T- and N-status) were retrospectively collected from the medical records. Cox regression analysis was performed to assess the relation between marker expression, demographic and clinical data, and locoregional recurrence and disease-specific mortality. RESULTS: Patients with high expression of HIF-1α developed significantly more often a locoregional recurrence (39%) compared to patients with a low expression (21%) (p = 0.002). The expression of CA-IX and Ki-67 showed no association with locoregional recurrent disease. HIF-1α, CA-IX and Ki-67 were not significantly related to disease-specific mortality. Clinical N-status was an independent predictor of recurrent disease (p < 0.001) and disease-specific mortality (p = 0.003). Age, sex and T-status were not related to locoregional recurrent disease or disease-specific mortality. CONCLUSION: HIF-1α overexpression and the presence of regional lymph node metastases at diagnosis were independent predictors of locoregional recurrent disease after primary treatment with curatively intended radiotherapy in patients with locally advanced LSCC.

A 5'-tiRNA fragment that inhibits proliferation and migration of laryngeal squamous cell carcinoma by targeting PIK3CD.

tRNA-derived small RNAs (tsRNAs) participate in several biological processes, including carcinogenesis. The correlations between tsRNAs and human cancers are attracting substantial attention. Nevertheless, the involvement of tsRNAs in laryngeal squamous cell carcinoma (LSCC) progression remains unclear. We constructed tsRNAs expression profiles in LSCC and adjacent normal tissues by next-generation sequencing. Interestingly, we identified a specific 5'-tiRNA fragment (tRF-33-Q1Q89P9L842205) that was significantly downregulated and was closely associated with lymph node metastasis and advanced stages of LSCC. Importantly, we found that tRF-33-Q1Q89P9L842205 suppressed cell growth, proliferation, migration, invasion and induced apoptosis in LSCC by directly silencing phosphoinositide 3-kinase catalytic subunit (PIK3CD). We speculated that tRF-33-Q1Q89P9L842205 is a potential diagnostic biomarker for LSCC and acts as a tumor suppressor by directly targeting PIK3CD.

Applying a nomogram based on preoperative CT to predict early recurrence of laryngeal squamous cell carcinoma after surgery.

PURPOSE: To identify the value of a computed tomography (CT)-based radiomics model to predict probability of early recurrence (ER) in patients diagnosed with laryngeal squamous cell carcinoma (LSCC) after surgery. MATERIALS AND METHOD: Pre-operative CT scans of 140 LSCC patients treated by surgery are reviewed and selected. These patients are randomly split into the training set (n = 97) and test set (n = 43). The regions of interest of each patient were delineated manually by two senior radiologists. Radiomics features are extracted from CT images acquired in non-enhanced, arterial, and venous phases. Variance threshold, one-way ANOVA, and least absolute shrinkage and selection operator algorithm are used for feature selection. Then, radiomics models are built with five algorithms namely, k-nearest neighbor (KNN), logistic regression (LR), linear support vector machine (LSVM), radial basis function SVM (RSVM), and polynomial SVM (PSVM). Clinical factors are selected using univariate and multivariate logistic regressions. Last, a radiomics nomogram incorporating the radiomics signature and clinical factors is built to predict ER and its efficiency is evaluated by receiver operating characteristic (ROC) curve and calibration curve. Decision curve analysis (DCA) is also used to evaluate clinical usefulness. RESULTS: Four features are remarkably associated with ER in patients with LSCC. Applying to test set, the area under the ROC curves (AUCs) of KNN, LR, LSVM, RSVM, and PSVM are 0.936, 0.855, 0.845, 0.829, and 0.794, respectively. The radiomics nomogram shows better discrimination (with AUC: 0.939, 95% CI: 0.867-0.989) than the best radiomics model and the clinical model. Predicted and actual ERs in the calibration curves are in good agreement. DCA shows that the radiomics nomogram is clinically useful. CONCLUSION: The radiomics nomogram, as a noninvasive prediction tool, exhibits favorable performance for ER prediction of LSCC patients after surgery.

Knockdown of Long Noncoding RNA CCAT2 Suppresses Malignant Phenotype in Human Laryngeal Squamous Cell Carcinoma.

This study aimed to explore the biological role and mechanism underlying the effects of colon cancer-associated transcript 2 (CCAT2), a long noncoding RNA (lncRNA) in human laryngeal squamous cell carcinoma (LSCC). CCAT2 expression levels in clinical LSCC samples and TU-212 cell line were evaluated by quantitative real-time PCR. The correlation of CCAT2 expression level with clinical-pathological characteristics of patients and their prognosis was analyzed. The functional role of CCAT2 in human LSCC was assessed by Cell Counting Kit-8, Transwell assay, flow cytometric analysis, and LSCC xenograft experiment in vivo. The expression of potential targeted proteins was detected by Western blotting and immunohistochemistry. We found that expression of CCAT2 was significantly elevated in LSCC tissues and TU-212 cells (p<0.05). Survival analysis showed that LSCC patients with high expression of CCAT2 had a shorter 5-year overall survival rate than those with low expression (p<0.05). In addition, CCAT2 silencing with short hairpin RNA significantly decreased the proliferative and invasive potential of TU-212 cells (p<0.05) and promoted their apoptosis. In Nude mice, CCAT2 knockdown suppressed the growth of tumor and decreased its volume and weight in comparison with the controls (p<0.05). In TU-212 cells, CCAT2 silencing with short hairpin RNA significantly down-regulated the expression of ß-catenin and CDK8 (p<0.05). Thus, knockdown of CCAT2 suppresses proliferation and invasion of the cells and inhibits Wnt/ß-catenin signaling pathway in LSCC, which indicates novel therapeutic targets and prognostic indicators in patients with LSCC.

Role of Human Papilloma Virus 16/18 In Laryngeal Carcinoma with Correlation to the Expression of Cyclin D1, p53, p16 and EGFR.

Objective: To investigate the role of Human Papilloma Virus (HPV) (16/18) in relation to the molecular genetic mechanisms of Cyclin D1, p53, p16, and Epidermal growth factor receptor (EGFR) in Laryngeal Squamous Cell Carcinoma (LSSC). Methods: A cross-sectional study of 88 (Formalin-fixed Paraffin Embedded) FFPE laryngeal biopsies were done at Basic Medical Sciences Institute, Jinnah Postgraduate Centre, Karachi from 2010 to 2019 with the application of Polymerase chain reaction (PCR) for HPV 16/18and Immuno-histochemical staining for molecular genetic expression of proteins, Cyclin D1, p53, p16, and EGFR. Results: Out of 88 cases of Laryngeal Squamous Cell Carcinoma (LSSC) there was female preponderance. Mean age of the participants was found as 50.7±12.8 years. High risk HPV 16/18 was positive in 28 cases (31.8%), largely related to Grade-II and Grade-III. Immunohistochemically, Cyclin D1 (87.5%) appeared as the most important driver mutation followed by p16 (86.4%), EGFR (65.9%), and, p53 was positive in (61.4%) of cases. Conclusion: The role of high-risk HPV 16/18 is concurred in the present study strongly in correlation to p16 as a surrogate marker. Moreover, the other driver mutations of Cyclin D1, p53, and EGFR are also implicated as cumulative molecular events in tumor progression as mostly seen in higher Grades.