TRH site-specific methylation in oral and oropharyngeal squamous cell carcinoma

Abstract

BackgroundThe incidence of oral squamous cell carcinoma (OSCC) continues to increase each year. Clinical examination and biopsy usually detect OSCC at an advanced stage that is difficult to treat, leading to poor prognosis. DNA methylation pattern is tissue specific and has emerged as a biomarker for the detection of cancers of tissue origin. Herein, we aimed to discover a novel site-specific methylation marker for OSCC.MethodsWe selected OSCC datasets analyzed using the IlluminaHumanMethylation27 BeadChip from the Gene Expression Omnibus repository of the National Center for Biotechnology Information using a bioinformatics approach. From 27,578 CG dinucleotide (CpG) sites, the CpG site with the highest difference in methylation level between healthy and cancerous cells was selected for further validation. A total of 18 mucosal tissue samples were collected from nine healthy controls and nine from OSCC subjects and subjected to microdissection for cell purification, followed by DNA extraction, bisulfite conversion, and pyrosequencing. Additionally, epithelial cells were collected from 2 cohorts including oral rinse from healthy controls, oral rinse and oral swab from OSCC subjects and oral rinse from oropharyngeal squamous cell carcinoma (SCC) were examined for their methylation status using real-time polymerase chain reaction (PCR).ResultsAmong the 27,578 differentially methylated CpG sites, cg01009664 of the thyrotropin-releasing hormone (TRH) gene showed the greatest difference in methylation level between healthy and cancerous cells. Validation of the TRH gene using pyrosequencing revealed a methylation percentage of 7% ± 3.43% in healthy cells in contrast to 63% ± 19.81% in cancerous cells. Screening of epithelial cells using real-time PCR showed that the DNA methylation level was significantly higher in oral swab and rinse samples collected from OSCC and oropharyngeal SCC subjects than those from healthy controls (p < 0.001). In addition, when using a cutoff at 3.31 ng/μL, the TRH methylation biomarker was able to distinguish OSCC and oropharyngeal SCC subjects from healthy controls with high level of area under the curve, sensitivity and specificity.ConclusionWe demonstrated cg01009664 of TRH as a potential biomarker for OSCC and oropharyngeal SCC screening using oral rinse and swab techniques.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4706-x) contains supplementary material, which is available to authorized users.

KRT84 is a potential tumor suppressor and good prognosis signature of oral squamous cell carcinoma

“…Oral squamous cell carcinoma (OSCC) is recognized as the most common phenotype of oral cancer, accounting for approximately 3% of the malignancies worldwide [1]. Annually, there are more than 300,000 patients diagnosed with OSCC worldwide, which is within the top 10 morbidity of all cancers and continues to increase [2][3][4]. Consumptions of tobacco and alcohol are responsible for almost 90% of the OSCC cases, while oral hygiene, human papillomavirus, and dental status are also considered as the risk factors [5].…”

Evaluation of NID2 promoter methylation for screening of Oral squamous cell carcinoma

“…We previously reported the Alu and TRH site-specific methylations for OSCC detection. However, the cutoff values of both markers have to be evaluated for cancer [14,21]. In this study, the calculation of cg22881914 NID2 methylation clearly indicated totally zero value in the normal oral epithelium, and eventually, the samples were used up to a concentration of 10 ng/μl.…”

“…The bioinformatic approach was previously reported [14]. In brief, we collected the methylation microarray data of GPL8490 (Illumina® HumanMethylation27 Bead-Chip Kit, Illumina Inc., San Diego, CA, USA) related to the following keywords: head and neck cancer, head and neck squamous cell carcinoma (HNSCC), OSCC, and normal oral epithelial cell.…”

“…One of these was cg01009664 of the TRH gene, which was a novel marker for screening. In this study, more interestingly and effectively, we have proposed another screening marker, nidogen 2 (NID2), and developed a method better than using cg01009664 of TRH for OSCC screening [14].…”

A Literature Review of the Potential Diagnostic Biomarkers of Head and Neck Neoplasms

“…In this manner, early diagnosis is potentially achieved and consequently leads to a better prognosis. Different papers focused on promoter hypermethylation and show that specific genes of patients with head and neck neoplasms have a higher prevalence of methylation in comparison to a healthy control group (3,6,11,12). Sushma et al suggest a promoter hypermethylation panel for oral squamous cell carcinoma (OSCC) consisting of the following genes: PTEN and p16 (12), which can be detected by a tissue biopsy.…”