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.

BackgroundHypermethylation in the promoter regions is associated with the suppression of gene expression and has been considered a potential molecular marker for several tumor types, including head and neck squamous cell carcinomas (HNSCC).MethodsTo evaluate the gene hypermethylation profile as a prognostic marker, this retrospective study used a QMSP approach to determine the methylation status of 19 genes in 70 HNSCC patients.ResultsThe methylation profile analysis of primary HNSCC revealed that genes CCNA1, DAPK, MGMT, TIMP3 and SFRP1 were frequently hypermethylated, with high specificity and sensitivity. TIMP3 and CCNA1 hypermethylation was significantly associated with lower rates of second primary tumor-free survival (p = 0.007 and p = 0.001; log-rank test, respectively).ConclusionThis study, for the first time, presents CCNA1 and TIMP3 hypermethylation as a helpful tool to identify HNSCC subjects at risk of developing second primary carcinomas.

Background. Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. The purposes of this study were to determine the methylation profile of exfoliated tumors cells collected from patients with head and neck squamous cell carcinoma (HNSCC) and to evaluate its prognostic significance. Methods. The methylation profile and level of a 20-gene panel were evaluated by quantitative methylation-specific polymerase chain reaction (qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC. Results. CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in these samples. Patients with exfoliated tumors cells positive for DCC methylation showed a trend toward a lower local recurrence-free survival. Conclusion. These findings indicate that a low invasive method could be used to access the methylation profile of exfoliated cells from patients with HNSCC. Moreover, our data provide evidence that hypermethylation of DCC could be useful as prognostic indicator for this malignancy.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent types of cancer but the lack of early detection and high incidence of local-regional recurrence lead to high mortality. Therefore, it is important to develop new molecular markers to be used as diagnostic and prognostic indicators. An important mechanism for gene transcriptional inactivation is hypermethylation at the CpG islands within the promoter regions. The presence of hypermethylation might be useful as a molecular marker for tumor cell detection which could be useful for early detection of tumor recurrence. The aim of this study was to evaluate promoter hypermethylation of suppressor genes via quantitative methylation-specific PCR (Q-MSP) in tumor and body fluid samples (saliva) as a means to detect epigenetic alterations specific to HNSCC. Tumor samples and salivary rinse were collected before and after the last curative treatment from 180 consecutive HNSCC cases and every 6 months during the patient follow-up. The pattern of aberrant methylation of 24 genes was analyzed and allowed the selection of a panel of 5 genes that have a specificity of 95% and a sensitivity of 38% in the detection of hypermethylated cells in saliva samples from recently diagnosed HNSCC patients. The efficacy of this panel may be useful in early diagnosis of HNSCC recurrence is being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4910.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a high incidence and mortality disease. Despite significant improvements in therapeutic strategies, the overall survival rate in 5 years is around 50%. The presence of microscopic tumor in surgical margins is associated with treatment failure, locoregional recurrence and reduction of overall survival. For this reason, it is vital to ensure tumor-free surgical margins in these patients. Commonly, the extension of surgical resection is determined during the surgery through a low sensitivity histological exam of frozen tissue stained with Hematoxylin and Eosin. Since genetic alterations precede histological alterations, molecular markers may be useful to identify cells that have already started malignant transformation without having developed a histological phenotype that could be detected through conventional histopathological techniques. Also, novel molecular techniques make possible to obtain molecular data of surgical margins in a period of time compatible with the surgery, helping the surgeon in the resection of positive molecular margins in the same surgery and thereby reducing the risk of local recurrence. This study aimed to determine the gene expression pattern of specific markers in HNSCC surgical margins and verify the utility of these markers in detecting the presence of tumor cells in these tissues and predict tumor recurrence. Nine selected genes were evaluated and a panel of 5 genes showed a high specificity and good sensibility. Overexpression of at least one of these genes was found in 50% (23/46) of histologically free surgical margins analyzed and was associated with local recurrence. Patients with positive molecular margins were 7.3-fold more likely to have local recurrence. These preliminary results suggest that the approach used in this analysis is more sensible than traditional histopathological exams and could be useful for the intraoperative diagnosis of the presence of tumor cells in HNSCC surgical margins and as an informative tool to identify and monitor subsets of HNSCC patients with an increased risk of recurrence and consequently reduced survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2954.