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 realtime 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 0 -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 highgrade 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.

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type O gene, PTPRO, was frequently methylated in rightsided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.

The activin type II receptor (ACVR2) gene is a putative tumor suppressor gene that is frequently mutated in microsatellite-unstable colon cancers (MSI-H colon cancers). ACVR2 is a member of the transforming growth factor (TGF)-␤ type II receptor (TGFBR2) family and controls cell growth and differentiation. SMAD proteins are major intracellular effectors shared by ACVR2 and TGFBR2 signaling; however, additional shared effector mechanisms remain to be explored. To discover novel mechanisms transmitting the ACVR2 signal, we restored ACVR2 function by transfecting wild-type ACVR2 (wt-ACVR2) into a MSI-H colon cancer cell line carrying an ACVR2 frameshift mutation. The effect of ACVR2 restoration on cell growth, SMAD phosphorylation, and global molecular phenotype was then evaluated. Decreased cell growth was observed in wt-ACVR2 transfectants relative to ACVR2-deficient vector-transfected controls. Western blotting revealed higher expression of phosphorylated SMAD2 in wt-ACVR2 transfectants versus controls, suggesting cells deficient in ACVR2 had impaired SMAD signaling. Microarray-based differential expression analysis revealed substantial ACVR2-induced overexpression of genes implicated in the control of cell growth and tumorigenesis, including the activator protein (AP)-1 complex genes JUND, JUN, and FOSB, as well as the small GTPase signal transduction family members, RHOB, ARHE, and ARHGDIA. Overexpression of these genes is shared with TGFBR2 activation. This observed similarity between the activin and TGF-␤ signaling systems suggests that activin may serve as an alternative activator of TGF-␤ effectors, including SMADs, and that frameshift mutation of ACVR2 may contribute to MSI-H colon tumorigenesis via disruption of alternate TGF-␤ effector pathways.

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