Abstract 3554: Promoter hypermethylation of zinc finger proteins is the novel epigenetic biomarkers of head and neck squamous cell carcinomas.
Authors: Gaykalova Daria A. , Vatapalli Rajita , Wei Yingying , Wang Hao , Hennessey Patrick , Li Ryan , Zhang Chi , Tan Marietta , Khan Zubair , Westra William , Khan Tanbir , Zaboli David , Ochs Michael F. , Califano Joseph A. 
Published: August 2014

Abstract
Objective: Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer worldwide with five-year survival rate of 50% despite use of modern treatment modalities. We have employed a comprehensive genome-wide integrated analysis of epigenetic and transcriptional alteration in primary HNSCC tissues in order to discover novel biomarkers of this disease.
Experimental Design: A discovery cohort included 44 primary HNSCC and 25 normal mucosal samples was analyzed by Affymetrix GeneChip Human Exon 1.0 and Illumina Infinium Human Methylation 27 arrays. An independent validation cohort included 32 primary HNSCC and 15 normal mucosal samples to confirm differential methylation by bisulfate sequencing. A second validation cohort included salivary rinse and primary tissue from 59 HNSCC and 31 healthy patients. Detection of DNA methylation in bodily fluids and primary tissues from the later cohort was evaluated by quantitative methylation-specific PCR (qMSP).
Results: We have applied novel screening approach based on Cancer Outlier Profile Analysis (COPA) and Gene Set Enrichment Analysis (GSEA) to our array data for the discovery cohort. With use of this approach we determined 81 significant differentially expressed and 37 significant differentially methylated genes. Out of total 118 candidate genes we picked 36 tumor suppressing genes with strong correlation between hypermethylation and decrease of expression in tumor samples. 26 out of 36 genes (72%) demonstrated strong differential methylation in tumor samples that was confirmed on the independent validation cohort by bisulfate sequencing. Five of these 26 genes belong to Zinc Finger Proteins (ZNF) group. We analyzed the detection of the promoter methylation of ZNF14, ZNF160 and ZNF420 in salivary rinse of HNSCC and healthy patients. DNA methylation of at least one of ZNFs promoter was detected in HNSCC patients salivary rinses with sensitivity of 22% (95% CI: 12.3%-34.7%) and specificity 100% (95% CI: 89.9%-100%). Overall frequency and concordance of detection of DNA methylation from at least one ZNFs promoter in salivary rinse with signal found in primary tissues was 35.3% and 92.3%, respectively. ZNF methylation was strongly associated with oral cavity SCC (p = 0.0049).
Conclusions: Our new screening approach allowed the identification of at least 36 candidate tumor suppressor genes aberrantly methylated and transcriptionally suppressed in HNSCC. Promoter hypermethylation for three ZNF genes in salivary rinse was detected with 100% specificity to HNSCC and may be a valuable salivary rinse biomarker for HNSCC incidence and recurrence, especially among high-risk group population.
Citation Format: Daria A. Gaykalova, Rajita Vatapalli, Yingying Wei, Hao Wang, Patrick Hennessey, Ryan Li, Chi Zhang, Marietta Tan, Zubair Khan, William Westra, Tanbir Khan, David Zaboli, Michael F. Ochs, Joseph A. Califano. Promoter hypermethylation of zinc finger proteins is the novel epigenetic biomarkers of head and neck squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3554. doi:10.1158/1538-7445.AM2013-3554