title: Pyrosequencing assay to measure LINE-1 methylation level in esophageal squamous cell carcinoma.
abstract: BACKGROUND
  Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element 1 L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Nonetheless, no study has examined LINE-1 methylation in esophageal squamous cell carcinoma (ESCC). The aim of this study is to assess the precision of sodium bisulfite conversion and polymerase chain reaction (PCR) pyrosequencing assay for evaluating LINE-1 methylation in ESCC.
  
  
  METHODS
  To measure assay precision, we performed bisulfite conversion on 5 different DNA specimen aliquots (bisulfite-to-bisulfite) and repeated PCR pyrosequencing five times (run to run). Second, to assess heterogeneity of LINE-1 methylation levels within tumor, we made 5 different tissue sections from one tumor and examined LINE-1 methylation level of each section (section to section). Third, to evaluate LINE-1 methylation status in ESCC, we applied this assay to 30 ESCCs and 30 matched normal esophageal mucosa.
  
  
  RESULTS
  Bisulfite-to-bisulfite standard deviation (SD) ranged from 1.44 to 2.90 (median 2.32) in ESCCs; and 0.57 to 4.02 (median 1.23) in normal esophagus. Run-to-run SD ranged from 0.63 to 3.25 (median 1.54) in ESCCs. Section-to-section SD ranged from 1.37 to 3.31 (median 1.94). ESCC tissues showed significantly lower levels of LINE-1 methylation than matched normal mucosa (P < .0001; n = 30). There was no significant relationship between LINE-1 methylation level and tumor stage (P = 0.14).
  
  
  CONCLUSIONS
  Bisulfite conversion and PCR pyrosequencing assay can measure LINE-1 methylation in ESCC, and may be useful in clinical and research settings.
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Iwagami
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Baba
authors.forename: Masayuki
authors.initials: M
authors.lastname: Watanabe
authors.forename: Hironobu
authors.initials: H
authors.lastname: Shigaki
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyake
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ida
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nagai
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Ishimoto
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwatsuki
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Yuji
authors.initials: Y
authors.lastname: Miyamoto
authors.forename: Hideo
authors.initials: H
authors.lastname: Baba
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics* / Q000628:therapy; D016022:Case-Control Studies; D004247:DNA / Q000032:analysis / Q000235:genetics; D019175:DNA Methylation; D004938:Esophageal Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000628:therapy; D004947:Esophagus / Q000378:metabolism*; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D009367:Neoplasm Staging; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D011379:Prognosis; D013447:Sulfites / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D013447:Sulfites; D004247:DNA; C009279:sodium bisulfite