DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage Ian M. Smith 1 , Wojciech K. Mydlarz 1 , Suhail K. Mithani 2 and Joseph A. Califano 1 * 1 Department of Otolaryngology – Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD 2 Department of Surgery, Division of Plastic and Reconstructive Surgery, Johns Hopkins Medical Institutions, Baltimore, MD Epigenetic changes have been implicated in the pathogenesis of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Prior efforts have primarily examined regional pro- moter hypermethylation as a silencer of tumor suppressor gene expression. To analyze the global state of methylation in the HNSCC genome, we utilize pyrosequencing of repetitive elements (LINEs) to compare the state of global methylation in HNSCC to normal aerodigestive mucosa. 137 samples (119 HNSCC tumors and 18 normal mucosal tissues) were digested to extract DNA and subjected to bisulfite treatment. Treated DNA was amplified using PCR primers for the repetitive LINEs sequence and produced a heterogeneous sample of products, from many genomic loci. These products were pyrosequenced to quantitatively evaluate their global genomic methylation status. HNSCC specimens showed global hypomethylation, with a mean level of genomic methylation of 46.8% methylated with a standard deviation of 9.0. Conversely, the normal upper airway mucosa had a global methylation level of 54.0 and a standard deviation of 4.6 (Mann–Whitney p value < 0.001). The tumor specimens also showed an increasing degree of hypomethylation associated with advanced tumor stage (ANOVA p-value of 0.003). About 67% of HNSCC’s are globally hypome- thylated when evaluated against the minimum level of methylation in the normal mucosal specimens. Degree of global hypomethyla- tion was associated with smoking history, alcohol use and stage in univariate analysis (p-value 0.02), however, only HNSCC diagno- sis remained significant on multivariate analysis. Despite the pres- ence of regional promoter hypermethylation, HNSCC demon- strates global genomic hypomethylation. The effects of stage, alco- hol use and smoking on global hypomethylation were not independently significant. ' 2007 Wiley-Liss, Inc. Key words: DNA hypomethylation; head and neck squamous cell carcinoma; pyrosequencing; epigenetics; smoking exposure; tobacco Head and neck squamous cell carcinomas (HNSCCs) account for 3% of all cancers in the United States and 40,000 new cases each year. 1 Although significant progress has been made in the areas of early detection, diagnosis and treatment, the 5-year sur- vival rate for patients with HNSCC has shown only modest improvement in the past 40 years. 2 Comprehensive analysis of clinical and treatment factors has shown tumor-site specific improvements in 5-year survival for cancers of the nasopharynx, oropharynx and hypopharynx, and late-stage laryngeal cancer. 3 The molecular mechanisms of HNSCC carcinogenesis are undergoing intensive investigation. Despite significant genetic/ epigenetic alterations found in these cancers, few known altera- tions correlate with the clinical outcomes of the disease. Epige- netic changes have been characterized in the pathogenesis of HNSCC. By far, the most studied epigenetic mechanism has been promoter hypermethylation of tumor suppressor genes including: Cyclin A1, MGMT, DCC and p16. 4 Methylation of cytosine- guanine dinucleotides by the enzyme class of DNA methyltrans- ferases transfers a methyl group from S-adenosyl-methionine and is associated with gene silencing. Promoter hypermethylation has shown marked promise for identification of biomarkers and func- tional tumor suppressor genes implicated in the carcinogenesis of HNSCC. Reports have noted a paradox between regional DNA hyper- methylation in the promoters of tumor suppressor genes and a state of global genomic hypomethylation in solid tumors over the last few years. 5–7 In fact, there are several reasons to believe that hypomethylation may be a significant factor driving oncogenesis. Mice with disruption of DNA methyltransferase 1 (DNMT1) func- tion demonstrate significant genomic hypomethylation in all tissues and develop aggressive T-cell lymphomas with chromo- somal instability. 8 Murine embryonic cells with homozygous deletion of DNMT1 exhibit significantly elevated rates of genetic deletions. 9 DNMT mutation can lead to chromosomal instability in humans as well. Mutation of DNMT3b leads to numerous chro- mosome aberrations. 10 Meta-analysis of DNA global hypomethy- lation across various cancer types suggest a correlation between global hypomethylation and tumor stage. 7 Additionally, extensive demethylation of centromeric sequences is common in human tumors and may play a role in aneuploidy. 11 To date, the global methylation status of head and neck squa- mous cell cancer has not been adequately studied in patients. In a limited cohort of only 6 tumor samples, Chalitchagorn et al. 12 showed a promising trend toward hypomethylation in HNSCC versus normal mucosal tissues using combined bisulfite restriction analysis. In contrast, Piyathilake et al. found a higher percentage of cells positive for 5-methyl-cytosine immunostaining in 39 HNSCC samples versus normal oral epithelial cells. This evalua- tion was looking at the percentage of cells positive for staining and staining score, and does not necessarily correlate to the degree of DNA methylation per cell. To quantitate this finding globally, our group utilized the technique of pyrosequencing of LINE sequences to examine the degree of global genomic hypomethyla- tion in head and neck cancer tumor specimens compared to normal control mucosal tissue in an effort to determine if global DNA hypomethylation exists in head and neck cancer. Material and methods Histopathology All samples were analyzed by the Pathology Department at Johns Hopkins Hospital. Tissues were obtained via Johns Hopkins Institutional Review Board approved protocols. Normal samples were microdissected and DNA prepared from the mucosa. Normal upper aerodigestive mucosa was obtained during upper airway procedures for benign disease, primarily Uvulopalatopharyngo- plasty. Samples were confirmed to be upper aerodigestive mucosa by pathology and originated primarily from the soft palate. Tumor and normal samples were chosen randomly from available tissue banks comprised of patients treated at the Johns Hopkins Hospital from 1998 to 2006. Tumor samples were confirmed to be head and neck squamous and subsequently microdissected to separate tumor from stromal elements to yield at least 80% tumor cells. Micro- Grant sponsor: Damon Runyon Cancer Research Foundation; Grant number: CI-#9; Grant sponsor: Flight Attendant Medical Research Insti- tute; Grant sponsor: National Institute of Dental and Craniofacial Research; Grant number: 1R01DE015939-01; Grant sponsor: National Cancer Institute SPORE; Grant number: 5P50CA096784-05. *Correspondence to: Department of Otolaryngology – Head and Neck Surgery, Johns Hopkins Medical Institutions, 601 North Caroline Street, 6th Floor, 21287-0910 Baltimore, MD, USA. E-mail: jcalifa@jhmi.edu Received 1 February 2007; Accepted after revision 13 April 2007 DOI 10.1002/ijc.22889 Published online 20 June 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 121, 1724–1728 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer