Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV). HPV-positive OPSCC is considered a distinct molecular entity with a better prognosis than HPV-negative cases of OPSCC. However, the exact pathogenic mechanisms underlying the differences in clinical and molecular behavior between HPV-positive and HPV-negative OPSCC remain poorly understood. Epigenetic events play an important role in the development of cancer. Hypermethylation of DNA in promoter regions and global hypomethylation are 2 epigenetic changes that have been frequently observed in human cancers. It is suggested that heterogeneous epigenetic changes play a role in the clinical and biological differences between HPV-positive and HPV-negative tumors. Unraveling the differences in methylation profiles of HPV-associated OPSCC may provide for promising clinical applications and may pave the road for personalized cancer treatment. This systematic review aims to assess the current state of knowledge regarding differences in promoter hypermethylation and global methylation between HPV-positive and HPV-negative OPSCC.     

Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma: A systematic review

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV). HPV-positive OPSCC is considered a distinct molecular entity with a better prognosis than HPV-negative cases of OPSCC. However, the exact pathogenic mechanisms underlying the differences in clinical and molecular behavior between HPV-positive and HPV-negative OPSCC remain poorly understood. Epigenetic events play an important role in the development of cancer. Hypermethylation of DNA in promoter regions and global hypomethylation are 2 epigenetic changes that have been frequently observed in human cancers. It is suggested that heterogeneous epigenetic changes play a role in the clinical and biological differences between HPV-positive and HPV-negative tumors. Unraveling the differences in methylation profiles of HPV-associated OPSCC may provide for promising clinical applications and may pave the road for personalized cancer treatment. This systematic review aims to assess the current state of knowledge regarding differences in promoter hypermethylation and global methylation between HPV-positive and HPV-negative OPSCC.     

Head and neck squamous cell carcinoma: role of the human papillomavirus in tumour progression

High risk human papilloma viruses (HPVs) have been shown to be independent risk factors for anogenital tract cancers, and have also been detected in head and neck squamous cell carcinomas (HNSCC). The aim of our study was to determine the prevalence of HPV DNA in a group of 47 squamous cell carcinomas of the oropharynx and the oral cavity, and to compare the clinical behaviour of HPV positive and negative tumours. We also assessed the proliferation index, as evaluated by Ki67 immunohistochemistry positivity, and the level of p53 reactivity. HPV DNA was found in 50% of carcinomas of the oropharynx and 36% in those of the oral cavity, the only genotype detected being HPV 16. Patients with HPV-positive carcinomas had a better overall survival than those with HPV-negative carcinomas. Our data suggest that HPV-positive oropharyngeal cancers comprise a distinct disease entity with an improved prognosis.     

Relationship between human oral lichen planus and oral squamous cell carcinoma at a genomic level: a datamining study

The leader gene approach is a data mining method based on the systematic search for genes involved in a specific process and their ranking according to the number of interconnections with the other genes identified. The genes with the strongest interconnections are termed leader genes, since they may be supposed to play an important role in the process. The potential of malignant progression of OLP to oral squamous cell carcinoma (OSCC) is still not completely clear. In this study, the leader gene approach is applied to investigate the association between OLP and OSCC at a molecular level. Results were integrated with those obtained in an experimental analysis (see paper 1 of this series). Genes involved in OLP and OSCC were identified by systematic queries to dedicated databases. Interconnections among identified genes were calculated and given a confidence value using STRING database. Leader genes were identified by clustering genes according to their interconnections. This theoretical analysis shows that OLP and OSCC share two leader genes: TP53 and CDKN1A, involved in the PI3K signalling events mediated by AKT pathway. This finding and those obtained in the experimental analysis suggest the possible involvement of some key genes/proteins LCK, PIK3CA, BIRC5, TP53 and CDKN1A in the malignant progression from OLP to OSCC. Moreover, these findings support the role of some molecular pathways, namely IL2 signalling events mediated by PI3K, PI3K signalling events mediated by AKT, and, possibly, Aurora A signalling in the association between OLP and OSCC.     

Serum exosome-derived biomarkers for the early detection of oral squamous cell carcinoma

Molecular and Cellular Biochemistry - Blood exosomes help regulate communication between tumour cells, moderating their behaviour. We sought to determine the protein content in serum exosomes...     

Cancer stem cell model in oral squamous cell carcinoma

The concept of "field cancerization" describes the presence of histological abnormal tissue surrounding oral squamous cell carcinoma (OSCC). Molecular model of multistep carcinogenesis indicates that an accumulation of genetic alterations forms the basis for the OSCC progression with genetic heterogeneity. Furthermore, we reviewed cancer stem cell (CSC) model, which suggests functional heterogeneity in the tumor mass and current supporting evidence in OSCC. According to CSC model, prevention from carcinogen exposure and eliminating the particular CSCs instead of targeting tumor mass could help obtain a more long-lasting therapeutic effect.     

Virology and molecular pathogenesis of HPV (human papillomavirus)-associated oropharyngeal squamous cell carcinoma

The current literature fully supports HPV (human papillomavirus)-associated OPSCC (oropharyngeal squamous cell carcinoma) as a unique clinical entity. It affects an unambiguous patient population with defined risk factors, has a genetic expression pattern more similar to cervical squamous cell carcinoma than non-HPV-associated HNSCC (head and neck squamous cell carcinoma), and may warrant divergent clinical management compared with HNSCC associated with traditional risk factors. However, a detailed understanding of the molecular mechanisms driving these differences and the ability to exploit this knowledge to improve clinical management of OPSCC has not yet come to fruition. The present review summarizes the aetiology of HPV-positive (HPV+) OPSCC and provides a detailed overview of HPV virology and molecular pathogenesis relevant to infection of oropharyngeal tissues. Methods of detection and differential gene expression analyses are also summarized. Future research into mechanisms that mediate tropism of HPV to oropharyngeal tissues, improved detection strategies and the pathophysiological significance of altered gene and microRNA expression profiles is warranted.     

Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.     

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.     

Salivary analytes in patients with oral squamous cell carcinoma

Literature data indicates that measurement of certain salivary constituents might serve as a useful diagnostic/prognostic tool in the patients with oral squamous cell carcinoma (OSCC). In 24 patients with OSCC (60 +/- 2.5 yrs) and in 24 controls (24 +/- 3.7 yrs) we have determined levels of salivary magnesium, calcium, copper, chloride, phosphate, potassium, sodium, total proteins and amylase. Sodium, potassium and chloride were determined by indirect potentiometry whereas copper, magnesium and phosphate were determined by atomic absorption spectrophotometry. Total proteins were determined by pyrogalol colorimetric method. Amylase levels were determined by continued colorimetric method. Statistical analysis was performed by use of chi2 test and Spearman's correlation test. The results of this study indicate that the concentrations of sodium and chloride were significantly elevated in patients with OSCC when compared to the controls. However, level of total protein was significantly decreased when compared to the healthy controls. Furthermore, there was a negative correlation between alcohol consumption and total protein concentration in patients with oral carcinoma. We might conclude that in patients with OSCC increased salivary sodium and chloride might reflect their overall dehydration status due to alcohol consumption rather than consequence of OSCC itself.     

Microbial diversity in saliva of oral squamous cell carcinoma

In the oral cavity, chronic inflammation has been observed at various stages of oral squamous cell carcinomas (OSCC). Such inflammation could result from persistent mucosal or epithelial cell colonization by microorganisms. There is increasing evidence of the involvement of oral bacteria in inflammation, warranting further studies on the association of bacteria with the progression of OSCC. The objective of this study was to evaluate the diversity and relative abundance of bacteria in the saliva of subjects with OSCC. Using 454 parallel DNA sequencing, ～58,000 PCR amplicons that span the V4-V5 hypervariable region of rRNAs from five subjects were sequenced. Members of eight phyla (divisions) of bacteria were detected. The majority of classified sequences belonged to the phyla Firmicutes (45%) and Bacteroidetes (25%). Further, 52 different genera containing approximately 860 (16.51%) known species were identified and 1077 (67%) sequences belonging to various uncultured bacteria or unclassified groups. The species diversity estimates obtained with abundance-based coverage estimators and Chao1 were greater than published analyses of other microbial profiles from the oral cavity. Fifteen unique phylotypes were present in all three OSCC subjects.     

Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA

The use of formalin-fixed paraffin-embedded (FFPE) tissues for HPV DNA detection by PCR from biopsy materials is not entirely clear in retrospective studies. The aim of our study was to evaluate the usefulness and efficiency of FFPE tissues from laryngeal cancer (LSCC) in HPV detection by immunohistochemistry reaction (IHC) and PCR-DNA enzyme immunoassay method (PCR/DEIA) and to compare with HPV detection from DFT. HPV-DNA was amplified from 54 FFPE tissues from LSCC specimens by the short PCR fragment (SPF10) primer set using PCR/DNA method and monoclonal anti Human Papillomavirus antibodies in IHC. In the same patients 54 specimens were collected and immediately deep-frozen and stored at (-70°C) to (-80°C). All the FFPE and deep-frozen tissue (DFT) specimens were positive for β-globin amplification. HPV was detected by two methods (SPF10 PCR/DEIA and IHC) in 14 (25.92%) out of 54 specimens from FFPE. Significant differences were found between the HPV detection using PCR/DEIA method and IHC method in FFPE tissues. The comparative analysis of the 54 samples after assuming PCR method in FFPE tissues showed accuracy of 92.6%, sensitivity of 90.5% and specificity of 93.9%. The FFPE tissues method has high sensitivity, specificity and accuracy when used to detect HPV DNA by PCR reaction and it is comparable to DFT results. DNA quality of FFPE samples is adequate and it can be used in HPV-DNA detection and in retrospective studies on LSCC.     

A mouse model for oral squamous cell carcinoma

Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell carcinomas. The carcinogenic 4-nitroquinoline-1-oxide (4NQO) model has proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has a suitable volume for scientific treatment. For this end we applied 4NQO 3 times a week during 16 weeks and measured the volume of tumor tissue each week until the end of the experiment at 40 weeks. Concurrent histopathology at different time points up to the end of the experiment revealed that all mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. The 4NQO model is a suitable alternative for preclinical gene therapy experiments with primary oral tumors. Future survey of therapeutic options in the carcinogenic 4NQO model should be conducted around 40 weeks after the start of the treatment.