title: Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk.
abstract: Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC). Clinical neoplastic progression risk factors, such as age and the length of the esophageal BE segment, have been identified. However, improved molecular biomarkers predicting increased progression risk are needed for improved risk assessment and stratification. Using real-time quantitative methylation-specific PCR, we screened 10 genes (HPP1, RUNX3, RIZ1, CRBP1, 3-OST-2, APC, TIMP3, p16, MGMT, p14) for promoter hypermethylation in 77 EAC, 93 BE, and 64 normal esophagus (NE) specimens. A subset of genes manifesting significant differences in methylation frequencies between BE and EAC was then analysed in 20 dysplastic specimens. All 10 genes except p14 were frequently methylated in EACs, with RUNX3, HPP1, CRBP1, RIZ1, and OST-2 representing novel methylation targets in EAC and/or BE. p16, RUNX3, and HPP1 displayed increasing methylation frequencies in BE vs EAC. Furthermore, these increases in methylation occurred early, at the interface between BE and low-grade dysplasia (LGD). To demonstrate the silencing effect of hypermethylation, we selected the EAC cells BIC1, in which the HPP1 promoter is natively methylated, and subjected them to 5-aza-2'-deoxycytidine (Aza-C) treatment. Real-time RT-PCR indicated increased HPP1 mRNA levels after 3 days of Aza-C treatment, as well as decreased levels of methylated HPP1 DNA. Hypermethylation of a subset of six genes (APC, TIMP3, CRBP1, p16, RUNX3, and HPP1) was then tested in a retrospective longitudinal study of 99 BE and nine LGD specimens obtained from 53 BE patients undergoing surveillance endoscopy. Only high-grade dysplasia (HGD) or EAC were defined as progression end points. Two patient groups were compared: eight progressors (P) and 45 nonprogressors (NP), using Cox proportional hazards models to determine the relative progression risks of age, BE segment length, and methylation events. Multivariate analyses revealed that only hypermethylation of p16 (odds ratio (OR) 1.74, 95% confidence interval (CI) 1.33-2.20), RUNX3 (OR 1.80, 95% CI 1.08-2.81), and HPP1 (OR 1.77, 95% CI 1.06-2.81) were independently associated with an increased risk of progression, whereas age, BE segment length, and hypermethylation of TIMP3, APC, or CRBP1 were not independent risk factors. In combined analyses, risk was detectable up to, but not earlier than, 2 years preceding neoplastic progression. Hypermethylation of p16, RUNX3, and HPP1 in BE or LGD may represent independent risk factors for the progression of BE to HGD or EAC. These findings have implications regarding risk stratification, early EAC detection, and the appropriate endoscopic surveillance interval for patients with BE.
authors.affiliation: Division of Gastroenterology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
authors.forename: Karsten
authors.initials: K
authors.lastname: Schulmann
authors.forename: Anca
authors.initials: A
authors.lastname: Sterian
authors.forename: Agnes
authors.initials: A
authors.lastname: Berki
authors.forename: Jing
authors.initials: J
authors.lastname: Yin
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Sato
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Andreea
authors.initials: A
authors.lastname: Olaru
authors.forename: Suna
authors.initials: S
authors.lastname: Wang
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Mori
authors.forename: Elena
authors.initials: E
authors.lastname: Deacu
authors.forename: James
authors.initials: J
authors.lastname: Hamilton
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kan
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Krasna
authors.forename: David G
authors.initials: DG
authors.lastname: Beer
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Pepe
authors.forename: John M
authors.initials: JM
authors.lastname: Abraham
authors.forename: Ziding
authors.initials: Z
authors.lastname: Feng
authors.forename: Wolff
authors.initials: W
authors.lastname: Schmiegel
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Greenwald
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Meltzer
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D001471:Barrett Esophagus / Q000235:genetics*; D045744:Cell Line, Tumor; D050677:Core Binding Factor Alpha 3 Subunit; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics*; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012334:RNA, Neoplasm; C493729:Runx3 protein, human; C423567:TMEFF2 protein, human; D014157:Transcription Factors