title: Reprimo methylation is a potential biomarker of Barrett's-Associated esophageal neoplastic progression.
abstract: OBJECTIVE
  Reprimo, a candidate tumor-suppressor gene, regulates p53-mediated cell cycle arrest at G2 phase, and tumor-suppressor gene methylation is involved in the pathogenesis and progression of esophageal cancer. Our aim was to determine whether and at what phase of neoplastic progression Reprimo methylation occurs in Barrett's adenocarcinogenesis, as well as its columnar or squamous cell-type specificity. We also sought to determine whether Reprimo expression could be restored in vitro by the demethylating agent 5-aza-deoxycytidine (5AzaC).
  
  
  METHODS
  Quantitative methylation-specific PCR for Reprimo was done using an ABI7700 (Taqman) apparatus on 175 endoscopic biopsy specimens. In addition, reverse transcription-PCR and quantitative methylation-specific PCR were done on esophageal carcinoma cells before and after treatment with 5AzaC.
  
  
  RESULTS
  In Barrett's esophagus (BE; P=0.001), high-grade dysplasia (HGD; P=0.001), and esophageal adenocarcinoma (EAC; P=0.00003), the level and frequency of Reprimo methylation were significantly higher than in normal esophagus (NE). There was no statistically significant difference between BE and EAC, HGD and EAC, or NE and esophageal squamous cell carcinoma (ESCC). Reprimo methylation occurred in 0 of 19 NE samples, 6 (13%) of 45 ESCC, 9 (36%) of 25 BE, 7 (64%) of 11 HGD, and 47 (63%) of 75 EAC. Analysis of Reprimo methylation in EAC versus NE revealed an area under the receiver-operator characteristic curve of 0.812 (P<0.00001; 95% confidence interval, 0.73-0.90). In vitro 5AzaC treatment of OE33 EAC cells reduced Reprimo methylation and increased Reprimo expression.
  
  
  CONCLUSIONS
  Reprimo methylation occurs significantly more frequently in BE, HGD, and EAC than in NE or ESCC, suggesting that this epigenetic alteration is a specialized columnar, cell-specific early event with potential as a biomarker for the early detection of esophageal neoplasia.
authors.affiliation: Department of Medicine, Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Hamilton
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Sato
authors.forename: Zhe
authors.initials: Z
authors.lastname: Jin
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Greenwald
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Ito
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Mori
authors.forename: Bogdan C
authors.initials: BC
authors.lastname: Paun
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kan
authors.forename: Yulan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Suna
authors.initials: S
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.forename: John M
authors.initials: JM
authors.lastname: Abraham
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Meltzer
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D000368:Aged; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001471:Barrett Esophagus / Q000235:genetics* / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor / Q000187:drug effects; D019175:DNA Methylation; D000077209:Decitabine; D018450:Disease Progression; D004935:Esophageal Diseases / Q000473:pathology; D004938:Esophageal Neoplasms / Q000473:pathology; D005260:Female; D005819:Genetic Markers; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011230:Precancerous Conditions / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D005819:Genetic Markers; D006023:Glycoproteins; C412566:RPRM protein, human; D000077209:Decitabine; D001374:Azacitidine