Purpose: Silencing of tumor suppressor genes (TSGs) plays an important role in head and neck carcinogenesis. Methylation of CpG islands in the promoter regions of genes acts as a significant mechanism of epigenetic gene silencing. In this study, we assessed the feasibility of epigenetic alterations in TSGs in post-treatment DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. A panel of four genes (KIF1A, EDNRB, p16, DCC) was evaluated in this study.

Experimental Design: Hypermethylation of the promoter regions was investigated by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) which provides more objective and rapid estimation of gene methylation status. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 83 patients with HNSCC. Post-treatment saliva samples were collected once in every 3 months. In addition, 46 normal saliva and 16 normal mucosal samples from healthy individuals were obtained. Then we evaluated the correlation of the results with the clinical parameters.

Results: KIF1A, EDNRB, p16 and DCC genes were methylated in 94%, 95%, 37% and 84% of primary HNSCC tissues, respectively and were only methylated in 2%, 6.8%, 5% and 10% of the salivary rinses from normal subjects. In addition, KIF1A, EDNRB, p16 and DCC were methylated in 35%, 69%, 8.4% and 38.6% of pre-treatment salivary rinses from HNSCC patients, respectively. We evaluated whether methylation levels of these genes in the post treatment saliva may predict the recurrence/prognosis or the chance of cure after treatment. Our data indicate that these genes may be potential biomarkers for HNSCC detection and follow up. Sixteen of 83 patients had recurrent tumors and KIF1A+EDNRB+p16+DCC methylation was observed in 37.5% of the patients in this group. The three gene panel (KIF1A, EDNRB and DCC) was also observed in 37.5% of the recurrent group and 46% of the overall patients, respectively.

Conclusion: Methylation of the KIF1A, EDNRB and DCC gene promoters is a frequent event in HNSCC and these genes are not methylated in normal salivary rinses, demonstrating potential that they may act as biomarkers in detection strategies. Patients with presence of methylation in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary methylated DNA may have promise for surveillance and early detection of recurrence.

Citation Format: Semra Demokan, Jatinder Kaur, Alice Y. Chuang, Wojciech K. Mydlarz, Kavita M. Pattani, Wayne M. Koch, David Sidransky, Nejat Dalay, Joseph A. Califano. Promoter methylation of tumor suppressor genes in convalescent saliva samples from patients with head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1384. doi:10.1158/1538-7445.AM2014-1384