James P. Hamilton

MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3†

Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR‐21 was overexpressed in CCAs. miR‐21 differential expression in these 10 specimens was verified by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR). To validate these findings, qRT‐PCR for miR‐21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR‐21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the receiver operating characteristic curve of 0.995. Inhibitors of miR‐21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions: MiR‐21 is overexpressed in human CCAs. Furthermore, miR‐21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree. (HEPATOLOGY 2009.)

A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett’s esophagus

Esophageal adenocarcinoma risk in Barretts esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of eight BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors and 50 progressors using real-time quantitative methylation-specific PCR. Progressors were significantly older than nonprogressors (70.6 versus 62.5 years; P < 0.001). We evaluated a linear combination of the eight markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUC) were high in the 2-year, 4-year, and combined data models (0.843, 0.829, and 0.840; P < 0.001, <0.001, and <0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age versus age alone were substantial (Delta-AUC = 0.152, 0.114, and 0.118, respectively) in all 3 models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.

Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer.

Purpose: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation. Experimental Design:TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution. Results:TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barretts metaplasia (BE), dysplastic Barretts esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barretts metaplasia alone, 57.5% in dysplastic Barretts esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression. Conclusions:TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.

Silencing of Claudin-11 Is Associated with Increased Invasiveness of Gastric Cancer Cells

Background Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11 (CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region. Methodology/Principal Findings Levels of CLDN11 methylation and mRNA expression were measured in primary gastric cancer tissues, noncancerous gastric mucosae, and cell lines of gastric origin using quantitative methylation-specific PCR (qMSP) and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2′-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness. Conclusions/Significance These data suggest that hypermethylation of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.

Three-tiered risk stratification model to predict progression in Barrett's esophagus using epigenetic and clinical features

Background Barretts esophagus predisposes to esophageal adenocarcinoma. However, the value of endoscopic surveillance in Barretts esophagus has been debated because of the low incidence of esophageal adenocarcinoma in Barretts esophagus. Moreover, high inter-observer and sampling-dependent variation in the histologic staging of dysplasia make clinical risk assessment problematic. In this study, we developed a 3-tiered risk stratification strategy, based on systematically selected epigenetic and clinical parameters, to improve Barretts esophagus surveillance efficiency. Methods and Findings We defined high-grade dysplasia as endpoint of progression, and Barretts esophagus progressor patients as Barretts esophagus patients with either no dysplasia or low-grade dysplasia who later developed high-grade dysplasia or esophageal adenocarcinoma. We analyzed 4 epigenetic and 3 clinical parameters in 118 Barretts esophagus tissues obtained from 35 progressor and 27 non-progressor Barretts esophagus patients from Baltimore Veterans Affairs Maryland Health Care Systems and Mayo Clinic. Based on 2-year and 4-year prediction models using linear discriminant analysis (area under the receiver-operator characteristic (ROC) curve: 0.8386 and 0.7910, respectively), Barretts esophagus specimens were stratified into high-risk (HR), intermediate-risk (IR), or low-risk (LR) groups. This 3-tiered stratification method retained both the high specificity of the 2-year model and the high sensitivity of the 4-year model. Progression-free survivals differed significantly among the 3 risk groups, with p = 0.0022 (HR vs. IR) and p<0.0001 (HR or IR vs. LR). Incremental value analyses demonstrated that the number of methylated genes contributed most influentially to prediction accuracy. Conclusions This 3-tiered risk stratification strategy has the potential to exert a profound impact on Barretts esophagus surveillance accuracy and efficiency.

Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma

The nel-like1 (NELL1) gene maps to chromosome 11p15, which frequently undergoes loss of heterozygosity in esophageal adenocarcinoma (EAC). NELL1 promoter hypermethylation was examined by real-time methylation-specific polymerase chain reaction in 259 human esophageal tissues. Hypermethylation of this promoter showed highly discriminative receiver–operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and EAC from normal esophagus (NE) (P<0.001). NELL1 normalized methylation values were significantly higher in Barretts metaplasia (BE), dysplastic Barretts (D) and EAC than in NE (P<0.0000001). NELL1 hypermethylation frequency was zero in NE but increased early during neoplastic progression, to 41.7% in BE from patients with Barretts alone, 52.5% in D and 47.8% in EAC. There was a significant correlation between NELL1 hypermethylation and BE segment length. Three (11.5%) of 26 ESCCs exhibited NELL1 hypermethylation. Survival correlated inversely with NELL1 hypermethylation in patients with stages I–II (P=0.0264) but not in stages III–IV (P=0.68) EAC. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced NELL1 methylation and increased NELL1 mRNA expression. NELL1 mRNA levels in EACs with an unmethylated NELL1 promoter were significantly higher than those in EACs with a methylated promoter (P=0.02). Promoter hypermethylation of NELL1 is a common, tissue-specific event in human EAC, occurs early during Barretts-associated esophageal neoplastic progression, and is a potential biomarker of poor prognosis in early-stage EAC.

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

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 Barretts 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 Barretts 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.

Promoter hypermethylation of CDH13 is a common, early event in human esophageal adenocarcinogenesis and correlates with clinical risk factors

Although the CDH13 gene has been shown to undergo epigenetic silencing by promoter methylation in many types of tumors, hypermethylation of this gene in Barretts‐associated esophageal adenocarcinogenesis has not been studied. Two hundred fifty‐nine human esophageal tissues were therefore examined for CDH13 promoter hypermethylation by real‐time methylation‐specific PCR. CDH13 hypermethylation showed discriminative receiver‐operator characteristic curve profiles, sharply demarcating esophageal adenocarcinoma (EAC) from esophageal squamous cell carcinoma (ESCC) and normal esophagus (NE) (p < 0.0001). CDH13 normalized methylation values (NMV) were significantly higher in Barretts esophagus (BE), dysplastic BE (D) and EAC than in NE (p < 0.0000001). CDH13 hypermethylation frequency was 0% in NE but increased early during neoplastic progression, rising to 70% in BE, 77.5% in D and 76.1% in EAC. Both CDH13 hypermethylation frequency and its mean NMV were significantly higher in BE with than without accompanying EAC. In contrast, only 5 (19.2%) of 26 ESCCs exhibited CDH13 hypermethylation. Furthermore, both CDH13 hypermethylation frequency and its mean NMV were significantly higher in EAC than in ESCC, as well as in BE or D vs. ESCC. Interestingly, mean CDH13 NMV was significantly lower in short‐segment than in long‐segment BE, a known clinical risk factor for neoplastic progression. Similarly, BE segment length was significantly lower in specimens with unmethylated than with methylated CDH13 promoters. 5‐aza‐2′‐deoxycytidine treatment of OE33 EAC and KYSE220 ESCC cells reduced CDH13 methylation and increased CDH13 mRNA expression. These findings suggest that hypermethylation of CDH13 is a common, tissue‐specific event in human EAC, occurs early during BE‐associated neoplastic progression, and correlates with known clinical neoplastic progression risk factors.

Epigenetics: Principles and Practice

Epigenetics is defined as heritable changes in gene expression that are, unlike mutations, not attributable to alterations in the sequence of DNA. The predominant epigenetic mechanisms are DNA methylation, modifications to chromatin, loss of imprinting and non-coding RNA. Epigenetic regulation of gene expression appears to have long-term effects and wide-ranging effects on health. Diet and environmental exposures may potentially alter the level and scope of epigenetic regulation, thus interesting developments in the study of epigenetics might explain correlations that researchers have found between lifestyle and risk of disease. Aberrant epigenetic patterns have been linked to a number of digestive diseases including Barrett’s esophagus, cirrhosis, inflammatory bowel disease, and numerous gastrointestinal malignancies. In fact, many exciting discoveries about epigenetics in general have been made by studying diseases of the gastrointestinal tract and hepatobiliary tree. Epigenetic modifications of DNA in cancer and precancerous lesions offer hope and the promise of novel biomarkers for early cancer detection, prediction, prognosis, and response to treatment. Furthermore, reversal of epigenetic changes represents a potential target of novel therapeutic strategies and medication design. In the future, it is anticipated that innovative diagnostic tests, treatment regimens, and even lifestyle modifications will be based on epigenetic mechanisms and be incorporated into the practice of medicine.

Hypermethylation of the somatostatin promoter is a common, early event in human esophageal carcinogenesis.

The promoter of somatostatin (SST), a primary inhibitor of gastrin‐stimulated gastric acid secretion, is hypermethylated in 80% of human colon cancers. The aim of the current study was to investigate whether and at what stage promoter hypermethylation of SST is involved in human esophageal carcinogenesis.