[Show abstract][Hide abstract] ABSTRACT: Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP).
Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1.
We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8-80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2-6.5; P = 0.016).
We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.
Full-text · Article · May 2011 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: To evaluate aberrant promoter hypermethylation of candidate tumor suppressor genes as a means to detect epigenetic alterations specific to solid tumors, including head and neck squamous cell carcinoma (HNSCC).
Using promoter regions identified via a candidate gene and discovery approach, we evaluated the ability of an expanded panel of CpG-rich promoters known to be differentially hypermethylated in HNSCC in detection of promoter hypermethylation in serum and salivary rinses associated with HNSCC. We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21 genes in a limited cohort of patients with HNSCC and normal controls. Using sensitivity and specificity for individual markers as criteria, we selected panels of eight and six genes, respectively, for use in salivary rinse and serum detection and tested these in an expanded cohort including up to 211 patients with HNSCC and 527 normal controls.
Marker panels in salivary rinses showed improved detection when compared with single markers, including a panel with 35% sensitivity and 90% specificity and a panel with 85% sensitivity and 30% specificity. A similar pattern was noted in serum panels, including a panel with 84.5% specificity with 50.0% sensitivity and a panel with sensitivity of 81.0% with specificity of 43.5%. We also noted that serum and salivary rinse compartments showed a differential pattern of methylation in normal subjects that influenced the utility of individual markers.
Q-MSP detection of HNSCC in serum and salivary rinses using multiple targets offers improved performance when compared with single markers. Compartment-specific methylation in normal subjects affects the utility of Q-MSP detection strategies.
Full-text · Article · Feb 2008 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Promoter hypermethylation of tumor suppressor genes is common in head and neck cancer as well as other primary cancers resulting in epigenetic gene silencing. Tissue inhibitor of metalloproteinase-3 (TIMP-3) has been shown to have promoter hypermethylation in several solid tumors, but has not been identified in head and neck squamous cell carcinoma (HNSCC). Our objective was to determine if TIMP-3 promoter was hypermethylated in HNSCC, if there was any correlation with death associated protein kinase (DAPK), a tumor suppressor whose promoter has been hypermethylated at high levels in HNSCC, and if any clinical factors influence hypermethylation of either of these genes.
Tumor samples from 124 patients with HNSCC were evaluated for promoter hypermethylation for TIMP-3 and DAPK using quantitative methylation specific polymerase chain reaction (qMSP). We compared both TIMP-3 and DAPK hypermethylation in HNSCC with each other as well as with other clinical variables.
We found that TIMP-3 was hypermethylated in approximately 71.8% of the tumor samples and DAPK was hypermethylated in 74.2%. The presence of TIMP-3 and DAPK promoter hypermethylation was significantly higher than in control specimens. More importantly, TIMP-3 and DAPK hypermethylations in these samples were highly correlated with a concordance of 78% (P < .001). DAPK was also correlated with current alcohol consumption (P < .028), but neither TIMP-3 nor DAPK hypermethylation was significantly correlated with other clinical variables or with survival.
TIMP-3 promoter hypermethylation is elevated in HNSCC and is highly correlated with DAPK hypermethylation, implying a functional relationship between these genes.
[Show abstract][Hide abstract] ABSTRACT: Deleted in colorectal cancer (DCC) is a candidate tumor-suppressor gene located at chromosome 18q21. However, DCC gene was found to have few somatic mutations and the heterozygous mice (DCC(+/-)) showed a similar frequency of tumor formation compared with the wild-type mice (DCC(+/+)). Recently, DCC came back to the spotlight as a better understating of its function and relationship with its ligand (netrin-1) had shown that DCC may act as a conditional tumor-suppressor gene. We evaluated hypermethylation as a mechanism for DCC inactivation in head and neck squamous cell carcinoma (HNSCC). DCC promoter region hypermethylation was found in 75% of primary HNSCC. There was a significant correlation between DCC promoter region hypermethylation and DCC expression (assessed by immunohistochemistry; P = 0.021). DCC nonexpressing HNSCC cell lines JHU-O12 and JHU-O19 with baseline hypermethylation of the DCC promoter were treated with 5-aza-2'-deoxycytidine (a demethylating agent) and reexpression of DCC was noted. Transfection of DCC into DCC-negative HNSCC cell lines resulted in complete abrogation of growth in all cell lines, whereas additional cotransfection of netrin-1 resulted in rescue of DCC-mediated growth inhibition. These results suggest that DCC is a putative conditional tumor-suppressor gene that is epigenetically inactivated by promoter hypermethylation in a majority of HNSCC.