Prognostic significance of TIMP3 hypermethylation in post-treatment salivary rinse from head and neck squamous cell carcinoma patients

Abstract

Hypermethylation in the promoter regions of genes is associated with suppression of gene expression and has been considered a potential molecular marker for several tumor types, including head and neck squamous cell carcinomas (HNSCC). Moreover, hypermethylation can be detected in body fluids such as saliva and can be useful for the diagnosis and prognosis of patients suffering from cancer. To evaluate the hypermethylation profile as a tool for early detection of tumor recurrences, this study determines the methylation status of 24 genes in salivary rinses collected from HNSCC patients at diagnosis, just after the last curative treatment and in the patients' follow-up visit at 6 months after treatment. In the analysis of salivary rinse samples taken at diagnosis of HNSCC patients, five genes (CCNA1, DAPK, DCC, MGMT and TIMP3) showed high specificity and sensitivity. Hypermethylation in any of these five genes was correlated with the presence of tumors in the oral cavity. Patients with TIMP3 methylation in samples collected 6 months after the last curative treatment had lower local recurrence-free survival (P = 0.008). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (P = 0.025). This study presents, for the first time, the detection of TIMP3 promoter hypermethylation in post-treatment salivary rinse as an independent prognostic maker for local recurrence-free survival in patients with HNSCC, justifying the use of DNA hypermethylation detection in saliva as a tool for identifying and monitoring HNSCC patients' subgroups with high risk of developing local recurrence.

Validation of methylation markers for diagnosis of oral cavity cancer

“…Since variations in the patterns of promoter hypermethylation in individual tumours may occur depending on alterations on specific molecular pathways, the use of multiple genes may provide greater applicability and coverage for different tumours when compared with a single gene for general detection, corroborating to results obtained by Carvalho et al [23] and Rettori et al [14] in different cohorts. From the initial screening of eight genes, four genes were selected as part of a panel to distinguish OSCC patients and healthy controls.…”

“…Carvalho et al [16] were able to confirm an elevated rate of promoter hypermethylation detected in HNSCC patient salivary rinses by using a panel of gene promoters previously described as methylated in HNSCC but not in control subjects, by the same group [23]. Rettori et al [14] in the analysis of salivary rinse samples taken at diagnosis of HNSCC patients, five genes (CCNA1, DAPK, DCC, MGMT and TIMP3) showed high specificity and sensitivity [24]. Thus, the detection of DNA methylation in body fluids opens the potential to develop biomarkers that can be useful for clinical use.…”

“…Carvalho et al (2008) showed an elevated frequency of promoter hypermethylation in HNSCC in a panel of gene promoters previously described as methylated in HNSCC as well as other solid tumours [23]. Moreover, Rettori et al (2013) found that CCNA1, DAPK, MGMT, SFRP1 and TIMP3 were able to distinguish HNSCC tumours from control samples with high specificity (>96%) and sensitivity (21-62%) in a different case/control study [14]. We, thus consider that the evaluation of epigenetic changes, such as DNA methylation, would be useful as a tool for diagnosis, surveillance and prognosis of OSCC.…”

“…Body fluids can potentially carry whole cells as well as protein, DNA, and RNA that allow for detection of cellular alterations related to cancer. Examples of relevant body fluids used for detection include analysis of sputum for lung cancer diagnosis [9,10], urine for urologic tumours [11,12], saliva for HNSCC [13][14][15][16], breast fluid [17], as well as serum or plasma for almost all types of cancer [18][19][20]. The detection of DNA methylation in body fluids such as saliva is a non-invasive technique that can easily obtain epithelial cells shed from the mucosal lining of the mouth and throat.…”

Novel DNA methylation targets in oral rinse samples predict survival of patients with oral squamous cell carcinoma

“…The broad collection of exfoliated epithelial cells across the oral cavity may be advantageous in the assessment of epigenetic alterations in the tumor cells and/or surrounding epithelium. While a number of studies have reported on the diagnostic potential of DNA methylation markers in oral rinse [9-19], few have evaluated the prognostic value [18, 20-22], and those that did were focused solely on promoter methylation of a limited subset of candidate tumor suppressor genes. Such a candidate approach has clear limitations, particularly given our incomplete understanding of genes involved in the biology of head and neck cancer and the crucial involvement of CpG methylation outside of the context of promoter regions in transcriptional regulation and carcinogenesis.…”