DNA promoter hypermethylation of p16 and APC predicts neoplastic progression in Barrett's esophagus.

OBJECTIVES: Prediction of progression to cancer in patients with Barrett's esophagus (BE) is difficult using current techniques. We determined whether DNA promoter hypermethylation of genes frequently methylated in esophageal adenocarcinoma (p16 and APC) could be used as predictors of progression in BE. METHODS: We first performed a cross-sectional study to evaluate the prevalence of gene hypermethylation in biopsies from patients with normal esophagus (n=17), BE (n=102), and adenocarcinoma (n=42). We then performed a nested case-control study comparing gene hypermethylation in BE patients who progressed from baseline pathology to high-grade dysplasia or cancer (n=7) vs. patients who did not progress (n=50). RESULTS: None of the patients with normal esophagus had p16 or APC hypermethylation. Hypermethylation was prevalent in BE without dysplasia or low-grade dysplasia (p16=31% and APC=50%; P<0.01) and high-grade dysplasia or adenocarcinoma (p16=54% and APC=68%; P<0.001) compared with normal esophagus (not detected). Patients who progressed from baseline pathology to high-grade dysplasia or cancer had higher prevalence of hypermethylation in their initial esophagus biopsies compared with those who did not progress for both p16 (100 vs. 33%; P=0.008) and APC (86 vs. 40%; P=0.02). Hypermethylation of both p16 and APC was a strong predictor of subsequent progression to high-grade dysplasia or cancer during a mean follow-up time of 4.1 years (odds ratio (95% confidence interval)=14.97 (1.73,inf), P=0.01). Among patients who were negative for both p16 and APC hypermethylation, none progressed from baseline pathology to high-grade dysplasia or cancer. CONCLUSIONS: Hypermethylation of both p16 and APC strongly predicts progression to high-grade dysplasia or cancer in patients with BE. Absence of p16 and APC hypermethylation is associated with a benign course.

Promoter hypermethylation in sentinel lymph nodes as a marker for breast cancer recurrence.

PURPOSE: Promoter methylation of tumor suppressor genes in histologically negative sentinel lymph nodes (HNSN) of early stage breast cancer patients has not been extensively studied. This study evaluates the methylation frequency and pattern in HNSN to determine if detection of hypermethylation of one or more genes is associated with an increased recurrence risk in node negative breast cancer. EXPERIMENTAL DESIGN: In 1998, a prospective study of patients with early stage breast cancer and HNSN was initiated in order to correlate sentinel node analysis with clinical outcome. Nodal tissue was selected from 120 HNSN patients for methylation analysis in at least one and up to six sentinel nodes using a panel of nine genes. Corresponding primary breast tumors from 79 patients were also evaluated for hypermethylation. Methylation analysis was performed using nested Methylation Sensitive PCR (n-MSP). Logistical regression was used to evaluate the relationship between clinical recurrence and methylation status. RESULTS: Over a median follow-up of 79 months, 13 of the 120 patients had clinical recurrence. Hypermethylation of genes was frequently observed in HNSN, but there was no correlation of methylation pattern and clinical recurrence. However, increased frequency of gene methylation of the primary tumor correlated with clinical recurrence. CONCLUSIONS: Although hypermethylation of multiple genes occurs frequently in HNSN of breast cancer patients, it is not associated with breast cancer recurrence in the first 7 years of clinical follow-up.

Restraint stress and ethanol consumption in two mouse strains.

BACKGROUND: This study examined the interaction between restraint stress and ethanol drinking in mice that consume low and high amounts of ethanol. METHODS: Two strains of mice (129SVEV and C57BL/6J) underwent 1 hour of restraint stress twice per day for 4 days in the presence of a CRF-1 receptor antagonist, a glucocorticoid receptor antagonist or vehicle. Ethanol preference and consumption were assessed using a two bottle choice design. In another study, mice were implanted with pellets containing corticosterone; ethanol preference and consumption were assessed using a two bottle choice design. RESULTS: Restraint stress significantly increased ethanol preference and consumption in 129SVEV mice but not in C57BL/6J mice. Then 129SVEV mice underwent the identical stress procedure; however, mice received either the CRF-1 receptor antagonist, R121919 (15 or 20 mg/kg, ip) or vehicle 30 minutes prior to stress. R121919 did not block the stress-induced change in ethanol preference despite causing a significant blunting in the HPA axis. Negative results were also obtained using the CRF-1 receptor antagonist, Antalarmin (20 mg/kg, ip). In another study, 129SVEV mice were administered either the glucocorticoid receptor antagonist Mifepristone (25, 50 or 100 mug/kg, ip) or vehicle under the same procedure. Mifepristone did not alter ethanol preference. Moreover, the three receptor antagonist did not alter nonstress ethanol consumption either. In the last study, both mouse strains underwent active or sham adrenalectomy, then pellets containing corticosterone or placebo were implanted and preference for ethanol versus water was tested. Corticosterone administration decreased ethanol consumption in a strain-dependent manner. CONCLUSION: These data show the restraint model for stress can modestly increase ethanol consumption in 129SVEV mice but not in C57BL/6J mice. Pharmacologic manipulation of CRF and corticosterone did not blunt baseline or stress-induced change in ethanol preference nor did administration of corticosterone mimic the effects of restraint stress on ethanol consumption. These findings suggest the mechanism responsible for increasing ethanol consumption in this model is independent of the HPA axis and extra-hypothalamic CRF.

Evaluation of GATA-4 and GATA-5 methylation profiles in human pancreatic cancers indicate promoter methylation patterns distinct from other human tumor types.

The GATA-4 and GATA-5 transcription factors are increasingly recognized as playing a role in carcinogenesis of human tumors derived of endodermal and mesodermal origin. The pancreas is derived from endodermal tissues suggesting GATA-4 and GATA-5 gene methylation may play a critical role in the biology of human pancreatic cancer as well. We investigated GATA-4 and -5 by methylation-specific PCR (MSP) in normal and neoplastic pancreatic tissues, including isogenic xenografts or cultured cell lines derived from the coexistent primary cancer and/or metastases in patients with pancreatic carcinoma. The relationship of promoter methylation was correlated with mRNA expression for each gene, and methylation patterns were correlated with known clinicopathologic features of patients. GATA-4 demonstrated a significantly lower methylation frequency than GATA-5 in low passage pancreatic cancer xenografts or cell lines (1/34 versus 21/34, p < 0.001). GATA-4 and -5 were also evaluated in microdissected samples of normal duct epithelium and cancer from pancreas cancer tissues which confirmed infrequent GATA-4 methylation in pancreatic cancers as well as in normal duct epithelium. GATA-4 was frequently overexpressed at the mRNA level with 27 of 30 (90%) pancreatic cancers showing >5.0-fold overexpression compared to normal duct epithelial cells. By contrast, high frequency methylation of GATA-5 was confirmed in pancreatic cancers tissues, but was rarely methylated in normal duct epithelium, indicating hypermethylation of this gene during pancreatic cancer development. GATA-5 mRNA expression did not correlate with its promoter hypermethylation, and treatment with the demethylating agent 5-aza-2'-deoxycytidine only partially restored mRNA expression suggesting additional regulatory mechanisms of GATA-5 expression. The presence of GATA-5 methylation showed a trend towards worse long-term survival (14.0 +/- 9.2 months versus 19.5 +/- 3.9 months, p = 0.06). While hypermethylation of GATA-5 seems to be a universal feature among human tumors, infrequent methylation of GATA-4, and its corresponding overexpression, appears unique to pancreatic cancer from other tumor types reported thus far.