Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers

Abstract

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with a typical 5 year survival rate of <40 %. DNA methylation in tumour-suppressor genes often occurs at an early stage of tumorigenesis, hence DNA methylation can be used as an early tumour biomarker. Saliva is an ideal diagnostic medium to detect early HNSCC tumour activities due to its proximity to tumour site, non-invasiveness and ease of sampling. We test the hypothesis that the surveillance of DNA methylation in five tumour-suppressor genes (RASSF1α, p16 INK4a, TIMP3, PCQAP/MED15) will allow us to diagnose HNSCC patients from a normal healthy control group as well as to discriminate between Human Papillomavirus (HPV)-positive and HPV-negative patients.MethodsMethylation-specific PCR (MSP) was used to determine the methylation levels of RASSF1α, p16 INK4a, TIMP3 and PCQAP/MED15 in DNA isolated from saliva. Statistical analysis was carried out using non-parametric Mann-Whitney’s U-test for individually methylated genes. A logistic regression analysis was carried out to determine the assay sensitivity when combing the five genes. Further, a five-fold cross-validation with a bootstrap procedure was carried out to determine how well the panel will perform in a real clinical scenario.ResultsSalivary DNA methylation levels were not affected by age. Salivary DNA methylation levels for RASSF1α, p16 INK4a, TIMP3 and PCQAP/MED15 were higher in HPV-negative HNSCC patients (n = 88) compared with a normal healthy control group (n = 122) (sensitivity of 71 % and specificity of 80 %). Conversely, DNA methylation levels for these genes were lower in HPV-positive HNSCC patients (n = 45) compared with a normal healthy control group (sensitivity of 80 % and specificity of 74 %), consistent with the proposed aetiology of HPV-positive HNSCCs.ConclusionsSalivary DNA tumour-suppressor methylation gene panel has the potential to detect early-stage tumours in HPV-negative HNSCC patients. HPV infection was found to deregulate the methylation levels in HPV-positive HNSCC patients. Large-scale double-blinded clinical trials are crucial before this panel can potentially be integrated into a clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2785-0) contains supplementary material, which is available to authorized users.

Methylation silencing of TGF-β receptor type II is involved in malignant transformation of esophageal squamous cell carcinoma

“…Methylation of specific sites may therefore be of biological and further clinical value in the early detection of ESCC, which is urgent for more favorable outcomes in the treatment of patients. Hotspots for DNA methylation are also valuable as biomarkers in the so-called liquid biopsy for cancer diagnosis and therapy since they are not only detected in resected tissues, but also in various body fluids, including peripheral blood [33][34][35][36], saliva [37][38][39], and urine [40][41][42]. In fact, methylated APC [43] and CDKN2A [44] have already been detected in the plasma of a subset of ESCC patients.…”

The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols

“…Volunteers were asked to refrain from eating and drinking for an hour prior to donating saliva samples as per our previous work 9 , 10 , 12 . The volunteers were asked to sit in a comfortable position and were asked to rinse their mouths with bottled water to remove food debris.…”

Salivary Protein Panel to Diagnose Systolic Heart Failure

“…Saliva as an alternative diagnostic medium is gaining attention due to its ease of collection, non-invasive nature, and the ability of multiple sample collections per patient at a single time point. Moreover, saliva collection reduces risk for contracting infections and contains about 30% of the biomolecules that are present in blood [12][13][14][15][16][17][18]. We were able to detect the SHF biomarkers N-terminal pro b-type natriuretic peptide (NT-proBNP) [15] and galectin-3 [18] in human saliva samples in previous studies.…”