Abstract 2202: Integrated screening for epigenetically controlled tumor suppressor genes and oncogenes in salivary adenoid cystic carcinoma
Authors: Shao Chunbo , Sun Wenyue , Cope Leslie , Bai Weiliang , Bhan Sheetal , Zhong Xiaoli , Sidransky David , Califano Joseph A. , Moskaluk Christopher , Ha Patrick K. 
Published: February 2011

Purpose: Epigenetic changes have been associated with cancer-specific expression differences in human malignancies, including salivary adenoid cystic carcinoma (ACC). It is widely accepted that promoter methylation can regulate the transcription of genes, including tumor suppressor genes (TSGs) and oncogenes. To date, a comprehensive, genome-wide study to identify TSGs and oncogenes regulated by promoter methylation in ACC has not been conducted. We used an integrative screening approach combining newer methylation array technology with expression arrays to identify candidate TSGs and oncogenes controlled by promoter methylation in ACC.

Experimental Design: The Illumina HumanMethylation27 methylation array (Illumina, San Diego, CA) was performed in 21 primary ACC tumors and 13 normal salivary gland tissues. Candidate genes that showed statistically significant differential methylation were identified. Next, expression microarray data (Affymetrix U133a array, (Affymetrix, Santa Clara, CA)) were obtained from a separate cohort of 18 primary ACC tumors and 8 normal salivary gland tissues. Significance Analysis of Microarrays (SAM) was performed to establish statistical significance for each transcript. Candidate genes showing differential expression were summarized. These two lists were combined by ranking the product of p values of the two analyses. The methylation status of the CpG islands of the target genes were then validated by bisulfite genomic sequencing in a separate cohort of normal salivary gland and ACC primary tumor samples.

Results: Using the methylation array, we analyzed 27,578 CpG sites located within the proximal promoter regions of transcription start sites of 14,475 consensus coding sequencing (CCDS) in the NCBI Database (Genome Build 36). With the U133a expression microarray, we analyzed the differential expressing levels of over 22,000 transcripts. With this high-throughput, integrated approach, we focused on14 candidate genes that showed either differential promoter methylation or differential expression or both. Among them, six were oncogene candidates and 8 were TSG candidates for ACC. After validating the methylation levels of the candidate genes in an independent cohort of ACC by bisulfite genomic sequencing, we found the Illumina methylation array results to be both sensitive and reproducible. Further validation of the expression levels of these candidates is underway.

Conclusions: The integration of data produced by methylation array and expression array offers a valid, focused approach to the potential discovery of novel TSGs or oncogenes that are epigenetically controlled in ACC.

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 2202.