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Reprimo Methylation Is a Potential Biomarker of Barrett's-Associated Esophageal Neoplastic Progression

Authors' Affiliations: ¹ Medicine (Gastroenterology), Johns Hopkins University School of Medicine; and ² Medicine (Gastroenterology) and ³ Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland

Requests for reprints: Stephen J. Meltzer, Division of Gastroenterology, Department of Medicine, Johns Hopkins School of Medicine, 1503 East Jefferson Street, Room 112, Baltimore, MD 21231. Phone: 410-502-6057; Fax: 410-502-1329; E-mail: smeltzer{at}jhmi.edu.

Purpose: 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).

Experimental Design: 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.