S077 An Epigenetically Derived Monoclonal Origin for Recurrent Laryngeal Papillomas

Abstract

Background: Promoter methylation of tumor suppressor genes has been investigated by a variety of means, recently including pyrosequencing. Methods: Fresh tumor tissue and normal tissue from resection margin were obtained from 79 patients undergoing resection for squamous cell carcinoma. DNA was extracted and bisulphite treated. Polymerase chain reaction primers were designed to amplify 75– to 200– base pair regions of the CpG-rich gene promoters of p16, RAR-beta, E-Cad, CYGB, cyclin A1, MGMT, ATM, hMLH1, STAT1, and TIMP3. Methylation status of 5 to 22 individual CpG sites per gene was determined by pyrosequencing. Reverse transcriptase–PCR was used to correlate these data with mRNA expression. Results: Significant CpG methylation of gene promoters within tumor specimens was found in most genes studied; however, despite previous reports, there was no evidence in the mismatch repair genes ATM, STAT1, or hMLH1. Promoter methylation was in evidence in highly tumor-specific pattern for most genes; however, the quantitative nature of these data revealed that for RAR-beta and E-cadherin, the pattern was not tumor specific. Concordant methylation was demonstrated in this tumor series (P=.03), but it was difficult to identify a true methylator phenotype (CIMP). Cyclin A1 promoter methylation showed an inverse trend with histological grade. Promoter methylation of CYGB is common in head and neck SCC. Conclusions: Promoter methylation analysis using pyrosequencing reveals valuable quantitative data from several CpG sites. In contrast to qualitative data generated from methylation-specific PCR, our data clarify which genes are methylated in a tumor-specific pattern. Cytoglobin is a novel candidate tumor suppressor gene highly methylated in upper aerodigestive tract squamous cancer.