Hongli Gong

The Composition of Microbiome in Larynx and the Throat Biodiversity between Laryngeal Squamous Cell Carcinoma Patients and Control Population

The throat is an ecological assemblage involved human cells and microbiota, and the colonizing bacteria are important factors in balancing this environment. However, this bacterial community profile has thus been poorly investigated. The purpose of this study was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. Barcoded pyrosequencing analysis of the 16S rRNA gene was used. We collected tissue samples from 29 patients with laryngeal carcinoma and 31 control patients with vocal cord polyps. The findings of high-quality sequence datasets revealed 218 genera from 13 phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx were Firmicutes (54%), Fusobacteria (17%), Bacteroidetes (15%), Proteobacteria (11%), and Actinobacteria (3%). The leading genera were Streptococcus (36%), Fusobacterium (15%), Prevotella (12%), Neisseria (6%), and Gemella (4%). The throat bacterial compositions were highly different between laryngeal carcinoma subjects and control population (p = 0.006). The abundance of the 26 genera was significantly different between the laryngeal cancer and control groups by metastats analysis (p<0.05). Fifteen genera may be associated with laryngeal carcinoma by partial least squares discriminant analysis (p<0.001). In summary, this study revealed the microbiota profiles in laryngeal mucosa from tissue specimens. The compositions of bacteria community in throat were different between laryngeal cancer patients and controls, and probably were related with this carcinoma. The disruption of this bio-ecological niche might be a risk factor for laryngeal carcinoma.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction.

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Microbiota in the Throat and Risk Factors for Laryngeal Carcinoma

ABSTRACT The compositions and abundances of the microbiota in the ecological niche of the human throat and the possible relationship between the microbiota and laryngeal cancer are poorly understood. To obtain insight into this, we enrolled 27 laryngeal carcinoma patients and 28 subjects with vocal cord polyps as controls. For each subject, we simultaneously collected swab samples from the upper throat near the epiglottis (site I) and tissue samples from the vestibulum laryngis to the subglottic region (site II). The microbiota of the throat were fully characterized by pyrosequencing of barcoded 16S rRNA genes. We found 14 phyla, 20 classes, 38 orders, 85 families, and 218 genera in the throats of enrolled subjects. The main phyla were Firmicutes (54.7%), Fusobacteria (14.8%), Bacteroidetes (12.7%), and Proteobacteria (10.6%). Streptococcus (37.3%), Fusobacterium (11.3%), and Prevotella (10.6%) were identified as the three most predominant genera in the throat. The relative abundances of 23 bacterial genera in site I were significantly different from those in site II (P < 0.05). The relative proportions of 12 genera largely varied between laryngeal cancer patients and control subjects (P < 0.05). Collectively, this study outlined the spatial structure of microbial communities in the human throat. The spatial structure of bacterial communities significantly varied in two anatomical sites of the throat. The bacterial profiles of the throat of laryngeal cancer patients were strongly different from those of control subjects, and several of these microorganisms may be related to laryngeal carcinoma.

The prognostic value of preoperative neutrophils, platelets, lymphocytes, monocytes and calculated ratios in patients with laryngeal squamous cell cancer

This study was aimed to examine the prognostic value of preoperative neutrophils, platelets, lymphocytes, monocytes and calculated ratios in patients with laryngeal squamous cell cancer (LSCC). From January 2007 to December 2011, 979 patients with LSCC were enrolled in our study. Preoperative neutrophils, platelets, lymphocytes, monocytes, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) were analyzed. Besides well-established clinicopathological prognostic factors, we evaluated the independent prognostic relevance of these hematological parameters by Cox regression models in disease-free survival (DFS) and cancer-specific survival (CSS). We found patients in the highest tertile of NLR (>2.40), PLR (>111.00) were at significantly higher risk of DFS and CSS (P<0.05) compared with those in the lowest tertile after multivariate analysis, whereas presenting significantly higher risk in the lowest tertile of lymphocytes (<1.60×109/L) and LMR (<3.50). Additionally, the tertile category of NLR as well as PLR increased and lymphocytes as well as LMR decreased in shorter DFS and CSS by the Kaplan-Meier method and the log-rank test. In conclusion, this study indicated that preoperative lymphocytes, NLR, PLR and LMR were significantly associated with LSCC progression, DFS and CSS, and these hematological parameters could be considered independent prognostic values for patients with LSCC.This study was aimed to examine the prognostic value of preoperative neutrophils, platelets, lymphocytes, monocytes and calculated ratios in patients with laryngeal squamous cell cancer (LSCC). From January 2007 to December 2011, 979 patients with LSCC were enrolled in our study. Preoperative neutrophils, platelets, lymphocytes, monocytes, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) were analyzed. Besides well-established clinicopathological prognostic factors, we evaluated the independent prognostic relevance of these hematological parameters by Cox regression models in disease-free survival (DFS) and cancer-specific survival (CSS). We found patients in the highest tertile of NLR (>2.40), PLR (>111.00) were at significantly higher risk of DFS and CSS (P<0.05) compared with those in the lowest tertile after multivariate analysis, whereas presenting significantly higher risk in the lowest tertile of lymphocytes (<1.60×109/L) and LMR (<3.50). Additionally, the tertile category of NLR as well as PLR increased and lymphocytes as well as LMR decreased in shorter DFS and CSS by the Kaplan-Meier method and the log-rank test. In conclusion, this study indicated that preoperative lymphocytes, NLR, PLR and LMR were significantly associated with LSCC progression, DFS and CSS, and these hematological parameters could be considered independent prognostic values for patients with LSCC.

Association between Helicobacter pylori Infection and Laryngeal Squamous Cell Carcinoma in a Chinese Male Population

Objectives: To investigate the presence of Helicobacter pylori in the larynx, and to identify the relationship between H. pylori infection and laryngeal squamous cell carcinoma in a male population. Methods: This study included 59 male patients with laryngeal squamous cell carcinoma and 41 control subjects. Nested polymerase chain reaction and target fragment sequencing were used to detect the presence of H. pylori in laryngeal mucosa. Logistic regression analysis was used to assess the association between H. pylori infection and laryngeal cancer. Results:H. pylori was present in a significantly greater number of patients with laryngeal carcinoma (76.3%) than in control subjects (31.7%) (p < 0.001). The correlation between H. pylori infection and laryngeal cancer was highly significant (OR = 9.82, 95% CI [3.35, 28.80], p < 0.001). Conclusions: The present study shows that H. pylori is present in the laryngeal mucosa of men, and supports a possible relationship between H. pylori infection and laryngeal squamous cell carcinoma in a male population.

Dickkopf-1 is a novel prognostic biomarker for laryngeal squamous cell carcinoma

Abstract Conclusions: Dickkopf-1 (DKK1) is a novel prognostic biomarker for laryngeal squamous cell carcinoma (LSCC). DKK1 may be a promising strategy for the future treatment of LSCC metastasis and recurrence. Objectives: DKK1 is reportedly involved in the metastasis and invasion of several tumor types. This study aimed to investigate the prognostic value of DKK1 in LSCC. Methods: DKK1 expression was measured in Hep-2 cell lines, as well as in tumor and peritumoral tissues, using quantitative real-time PCR and western blot analyses. The role of DKK1 in LSCC was investigated by depleting DKK1 using small interfering RNAs. Tissue microarrays of 102 LSCC patient samples were employed to immunohistochemically detect expression of DKK1, vascular endothelial growth factor C (VEGF-C), and β-catenin. Prognostic significance was assessed using Kaplan–Meier survival estimates. Results: DKK1 expression was elevated in the Hep-2 cell line and tumor samples. DKK1 depletion decreased cell proliferation, migration, and invasiveness. High DKK1 expression was significantly associated with T and clinical stage, lymph node metastasis, and tumor size (p < 0.05). Increased DKK1 levels in LSCC tissues correlated with elevated VEGF-C and β-catenin. Multivariate analyses revealed that DKK1 was an unfavorable predictor of overall survival.

Establishment and characterization of a novel HPV-negative laryngeal squamous cell carcinoma cell line, FD-LSC-1, with missense and nonsense mutations of TP53 in the DNA-binding domain

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in China; however, publically available LSCC cell lines are few and not established from Chinese populations. Hence, novel and well-characterized LSCC cell lines of Chinese origin are urgently needed to provide researchers with a comprehensive database for LSCC research. From 40 cases of LSCC, we established a novel cell line that was maintained for more than 100 passages in vitro and was found to have typical epithelial morphology and ultrastructure. In-depth characterization analysis revealed polyploidy in DNA content; a doubling time of some 24h; high tumorigenicity in immunodeficient mice; higher invasive potential and more sensitive to radiation and cisplatin compared with HeLa cell line; upregulated Ki67, Notch1, EGFR, and CK5 protein levels; negative infection of human papillomavirus (HPV) and mycoplasma; expression of head and neck squamous cell carcinoma (HNSCC) biomarkers; mutations of TP53 in exons 5 and 8; a near-triploid karyotype with complex structural aberrations; and dozens of dysregulated genes and miRNAs. Cell authentication testing by the American Type Culture Collection (ATCC) confirmed the human origin of this cell line. Our findings indicate that a novel and well-differentiated LSCC cell line recapitulating the primary tumors malignant characteristics is established and well characterized. It does not match any cell lines within the ATCC database and helps to elucidate the molecular pathogenesis of LSCC.

Alterations of microbiota structure in the larynx relevant to laryngeal carcinoma

The microbial communities that inhabit the laryngeal mucosa build stable microenvironments and have the potential to influence the health of the human throat. However, the associations between the microbiota structure and laryngeal carcinoma remain uncertain. Here, we explored this question by comparing the laryngeal microbiota structure in laryngeal cancer patients with that in control subjects with vocal cord polyps through high-throughput pyrosequencing. Overall, the genera Streptococcus, Fusobacterium, and Prevotella were prevalent bacterial populations in the laryngeal niche. Tumor tissue samples and normal tissues adjacent to the tumor sites (NATs) were collected from 31 laryngeal cancer patients, and the bacterial communities in laryngeal cancer patients were compared with control samples from 32 subjects. A comparison of the laryngeal communities in the tumor tissues and the NATs showed higher α-diversity in cancer patients than in control subjects, and the relative abundances of seven bacterial genera differed among the three groups of samples. Furthermore, the relative abundances of ten bacterial genera in laryngeal cancer patients differed substantially from those in control subjects. These findings indicate that the laryngeal microbiota profiles are altered in laryngeal cancer patients, suggesting that a disturbance of the microbiota structure might be relevant to laryngeal cancer.

Reduced expression of mutS homolog 2 and mutL homolog 1 affects overall survival in laryngeal squamous cell carcinoma patients: Investigation into a potential cause

The risk factors affecting the survival rates of laryngeal carcinoma are not well understood. In this study, we investigated the expression status of mutS homolog 2 (MSH2) and mutL homolog 1 (MLH1) and examined the relationship between these two molecules and overall survival rates in laryngeal cancer. We also explored the potential reason for the altered expression of these two genes. Using real-time polymerase chain reaction and western blotting, we detected MSH2 and MLH1 expression in laryngeal cancer tissue samples. We collected a retrospective cohort with 180 laryngeal cancer patients, and inspected MSH2 and MLH1 staining with tissue microarray immunohistochemistry. Prognostic value of clinicopathological characteristics was evaluated by statistical analysis. Laryngeal carcinoma cells were co-cultured with Helicobacter pylori (H. pylori) bacteria. MSH2 and MLH1 were expressed at lower levels compared to those of adjacent tissues in 21 laryngeal carcinoma patients. Patients with negative expression of MSH2 and MLH1 tended to have a higher risk of mortality compared to patients with positive expression (HR=4.38; HR=3.0, respectively). Cigarette smoking rate was higher in the MLH1 expression positive group. H. pylori infection reduced the MSH2 and MLH1 expression levels of laryngeal carcinoma cell lines within co-culture conditions. It is suggested that the altered expression levels of MSH2 and MLH1 probably affect the overall survival of laryngeal carcinoma patients. H. pylori infection may have an effect on the expression of MSH2 and MLH1 in laryngeal carcinoma patients.

Composition and abundance of microbiota in the pharynx in patients with laryngeal carcinoma and vocal cord polyps

The pharynx is an important site of microbiota colonization, but the bacterial populations at this site have been relatively unexplored by culture-independent approaches. The aim of this study was to characterize the microbiota structure of the pharynx. Pyrosequencing of 16S rRNA gene libraries was used to characterize the pharyngeal microbiota using swab samples from 68 subjects with laryngeal cancer and 28 subjects with vocal cord polyps. Overall, the major phylum was Firmicutes, with Streptococcus as the predominant genus in the pharyngeal communities. Nine core operational taxonomic units detected from Streptococcus, Fusobacterium, Prevotella, Granulicatella, and Veillonella accounted for 21.3% of the total sequences detected. However, there was no difference in bacterial communities in the pharynx from patients with laryngeal cancer and vocal cord polyps. The relative abundance of Firmicutes was inversely correlated with Fusobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes. The correlation was evident at the genus level, and the relative abundance of Streptococcus was inversely associated with Fusobacterium, Leptotrichia, Neisseria, Actinomyces, and Prevotella. This study presented a profile for the overall structure of the microbiota in pharyngeal swab samples. Inverse correlations were found between Streptococcus and other bacterial communities, suggesting that potential antagonism may exist among pharyngeal microbiota.