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 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.

Risk stratification of Barrett's oesophagus using a non-endoscopic sampling method coupled with a biomarker panel: a cohort study

“…22 Five genes shown to be methylated with increasing grade of dysplasia were tested: p16, ESR1, MYOD1, HPP1, and RUNX3. 21 After reviewing pilot data, only MYOD1 and RUNX3 were taken forward, as these were the most promising (See Appendix, p3).…”

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa

“…HPP1 methylation has also been detected in the dysplastic mucosa of ulcerative colitis patients (Sato et al, 2002). More recently, HPP1 methylation was shown to occur early in Barrett's-associated neoplastic progression to oesophageal adenocarcinoma, and was predictive of progression risk (Schulmann et al, 2005).…”

HPP1‐mediated tumor suppression requires activation of STAT1 pathways

“…[7][8][9][10][11] However, evidence supporting its tumor suppressive activity has been lacking. In this current study, we have demonstrated that ectopic expression of HPP1 in the colon cancer cell line, HCT116 results in a distinct alteration in cellular appearance consistent with reversion of transformation morphology.…”

“…Hypermethylation of HPP1 has been described in Barrett's-associated esophageal adenocarcinoma, gallbladder cancer, malignant mesothelioma and breast and lung cancer cell lines. [8][9][10][11] From a translational standpoint, there is growing interest in the study of HPP1 as a biomarker in gastrointestinal malignancies. HPP1 was among a panel of genes for which methylated DNA could be detected in the stool of patients with colorectal cancer.…”