Takeichi Yoshida

DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients : Its possible involvement in the formation of epigenetic field defect

Accumulation of aberrant DNA methylation in normal‐appearing gastric mucosae, mostly induced by H. pylori infection, is now known to be deeply involved in predisposition to gastric cancers (epigenetic field defect), and silencing of protein‐coding genes has been analyzed so far. In this study, we aimed to clarify the involvement of microRNA (miRNA) gene silencing in the field defect. First, we selected three miRNA genes as methylation‐silenced after analysis of six candidate “methylation‐silenced” tumor‐suppressor miRNA genes. Methylation levels of the three genes (miR‐124a‐1, miR‐124a‐2 and miR‐124a‐3) were quantified in 56 normal gastric mucosae of healthy volunteers (28 volunteers with H. pylori and 28 without), 45 noncancerous gastric mucosae of gastric cancer patients (29 patients with H. pylori and 16 without), and 28 gastric cancer tissues (13 intestinal and 15 diffuse types). Among the healthy volunteers, individuals with H. pylori had 7.8–13.1‐fold higher methylation levels than those without (p < 0.001). Among individuals without H. pylori, noncancerous gastric mucosae of gastric cancer patients had 7.2–15.5‐fold higher methylation levels than gastric mucosae of healthy volunteers (p < 0.005). Different from protein‐coding genes, individuals with past H. pylori infection retained similar methylation levels to those with current infection. In cancer tissues, methylation levels were highly variable, and no difference was observed between intestinal and diffuse histological types. This strongly indicated that methylation‐silencing of miRNA genes, in addition to that of protein‐coding genes, contributed to the formation of a field defect for gastric cancers.

Cancer development based on chronic active gastritis and resulting gastric atrophy as assessed by serum levels of pepsinogen and Helicobacter pylori antibody titer

Our study investigated the relationship between gastric cancer development and activity of Helicobacter pylori‐associated chronic gastritis or the resulting chronic atrophic gastritis (CAG). A cohort of 4,655 healthy asymptomatic subjects, in whom serum pepsinogen (PG) and H. pylori antibody titer had been measured to assess the activity and stage of H. pylori‐associated chronic gastritis, was followed for up to 16 years, and cancer development was investigated. In subjects with a serologically diagnosed healthy stomach (H. pylori‐negative/CAG‐negative), cancer incidence rate was low, at 16/100,000 person‐years. With the establishment of H. pylori infection and progression of chronic gastritis, significant stepwise cancer risk elevations were seen from CAG‐free subjects (H. pylori‐positive/CAG‐negative) [hazard ratio (HR) = 8.9, 95% confidence interval (CI) = 2.7–54.7] to subjects with CAG (H. pylori‐positive/CAG‐positive) (HR = 17.7, 95% CI = 5.4–108.6) and finally to subjects with metaplastic gastritis (H. pylori‐negative/CAG‐positive) (HR = 69.7, 95% CI = 13.6–502.9). In H. pylori‐infected CAG‐free subjects, significantly elevated cancer risk was observed in the subgroup with active inflammation‐based high PG II level or potent immune response‐based high H. pylori antibody titer; the former was associated with a particularly high risk of diffuse‐type cancer, and both subgroups showed high cancer incidence rates of around 250/100,000 person‐years, comparable to that in subjects with CAG. No such risk elevation was observed in H. pylori‐infected subjects with CAG. These results clearly indicate that gastric cancer develops mainly from the gastritis‐atrophy‐metaplasia‐cancer sequence and partly from active inflammation‐based direct carcinogenesis, and that serum levels of PG and H. pylori antibody titer provide indices of cancer development in H. pylori‐infected subjects.

Demonstration of the usefulness of epigenetic cancer risk prediction by a multicentre prospective cohort study

Background Epigenetic alterations accumulate in normal-appearing tissues of patients with cancer, producing an epigenetic field defect. Cross-sectional studies show that the degree of the defect may be associated with risk in some types of cancer, especially cancers associated with chronic inflammation. Objective To demonstrate, by a multicentre prospective cohort study, that the risk of metachronous gastric cancer after endoscopic resection (ER) can be predicted by assessment of the epigenetic field defect using methylation levels. Design Patients with early gastric cancer, aged 40–80 years, who planned to have, or had undergone, ER, were enrolled at least 6 months after Helicobacter pylori infection discontinued. Methylation levels of three preselected genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy, and the primary endpoint was defined as detection of a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrolment. Results Among 826 patients enrolled, 782 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1–2, 2–3 and ≥3 years after the enrolment, respectively. The highest quartile of the miR-124a-3 methylation level had a significant univariate HR (95% CI) (2.17 (1.07 to 4.41); p=0.032) and a multivariate-adjusted HR (2.30 (1.03 to 5.10); p=0.042) of developing authentic metachronous gastric cancers. Similar trends were seen for EMX1 and NKX6-1. Conclusions Assessment of the degree of an epigenetic field defect is a promising cancer risk marker that takes account of life history.

Alu and Satα hypomethylation in Helicobacter pylori-infected gastric mucosae.

Global hypomethylation and regional hypermethylation are supposed to be hallmarks of cancer cells. During gastric carcinogenesis, in which Helicobacter pylori infection is causally involved, aberrant hypermethylation is already present in H. pylori‐infected gastric mucosae. In contrast, little is known about global hypomethylation, which can be caused by hypomethylation of individual repetitive elements and other sequences. We, therefore, investigated hypomethylation of individual repetitive elements and the global 5‐methylcytosine content in four groups of gastric mucosal samples that represented the time course of H. pylori infection and gastric carcinogenesis [gastric mucosae of H. pylori‐negative healthy volunteers (G1, n = 34), H. pylori‐positive healthy volunteers (G2, n = 42), H. pylori‐positive gastric cancer patients (G3, n = 34) and H. pylori‐negative gastric cancer patients (G4, n = 20)] and 52 primary gastric cancers. Major variants of Alu, LINE1 and Satα were identified, and their methylation levels were quantified by bisulfite pyrosequencing. Compared with G1, the Alu methylation level was decreased in G2, G3, G4 and cancers (89.2–97.1% of that in G1, p < 0.05). The Satα methylation level was decreased in G2 (91.6%, p < 0.05) and G3 (94.3%, p = 0.08) but not in G4 and cancers. The LINE1 methylation level was decreased only in cancers. The 5‐methylcytosine content was at similar levels in G2, G3 and G4 and highly variable in cancers. These results showed that Alu and Satα hypomethylation is induced in gastric mucosae by H. pylori infection during gastric carcinogenesis, possibly in different target cells, and that global hypomethylation is not always present in human gastric cancers.

Development of gastric cancer in nonatrophic stomach with highly active inflammation identified by serum levels of pepsinogen and Helicobacter pylori antibody together with endoscopic rugal hyperplastic gastritis

This study aimed to elucidate groups at high risk of developing cancer among patients with serologically identified Helicobacter pylori infection and nonatrophic stomach. Annual endoscopy was performed for a mean of 5.4 years in 496 asymptomatic middle‐aged men who were H. pylori antibody‐positive and pepsinogen (PG) test‐negative. Subjects were stratified according to the activity of H. pylori‐associated gastritis measured by serum levels of PG and H. pylori antibody, and/or by endoscopic findings of rugal hyperplastic gastritis (RHG), and cancer development was investigated. During the study period, seven cases of cancer developed in the cohort (incidence rate, 261/100,000 person‐years), with 85.7% developing in the group showing a PGI/II ratio ≤3.0, reflecting active inflammation‐based high PGII levels. Cancer incidence was significantly higher in this group (750/100,000 person‐years) than in groups with less active gastritis. Furthermore, cancer incidence for this group was significantly higher in the subgroup with high H. pylori antibody titers than in the low‐titer subgroup. Meanwhile, endoscopic findings revealed that 11.7% of subjects showed RHG reflecting localized highly active inflammation, and cancer risk was significantly higher in patients with RHG than in patients without. Combining the two serum tests and endoscopic examination for RHG allowed identification of subjects with more active gastritis and higher cancer risk. No cancer development was observed in these high‐risk subjects after H. pylori eradication. Subjects with highly active gastritis identified by the two serological tests and endoscopic RHG constitute a group at high risk of cancer development with H. pylori‐infected nonatrophic stomach.

Usefulness of contrast-enhanced endoscopic sonography for discriminating mural nodules from mucous clots in intraductal papillary mucinous neoplasms: a single-center prospective study.

The aim of this study was to evaluate the ability of contrast‐enhanced endoscopic sonography for discrimination of mural nodules from mucous clots in intraductal papillary mucinous neoplasms of the pancreas.

Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis

BackgroundChronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer.MethodsMethylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II.ResultsMethylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation.ConclusionsAlteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.

Contrast-enhanced endoscopic ultrasonography can predict a higher malignant potential of gastrointestinal stromal tumors by visualizing large newly formed vessels

The aim of this study was to elucidate the histologic and clinical implications of detection of intratumoral vessels on contrast‐enhanced endoscopic ultrasonography (CE‐EUS) in gastrointestinal stromal tumors (GISTs).

Hypomethylation of Alu repetitive elements in esophageal mucosa, and its potential contribution to the epigenetic field for cancerization

BackgroundAberrant hypermethylation of specific genes is present in esophageal squamous cell carcinomas (ESCCs). Such hypermethylation is also present in normal-appearing esophageal mucosae of ESCC patients and is considered to contribute to the formation of a field for cancerization. On the other hand, the presence of global hypomethylation in ESCCs or in their background esophageal mucosae is unknown.MethodWe collected 184 samples of esophageal mucosae (95 normal mucosae from healthy subjects, and 89 non-cancerous background mucosae from ESCC patients) and 93 samples of ESCCs. Methylation levels of repetitive elements (Alu, LINE1) and cancer/testis antigen genes (NY-ESO-1, MAGE-C1) were measured by bisulfite pyrosequencing and quantitative methylation-specific PCR, respectively.ResultsMethylation levels of Alu, LINE1, NY-ESO-1, and MAGE-C1 were significantly lower in ESCCs than in their background and normal mucosae. Also, in the background mucosae, a significant decrease of the Alu methylation level compared with the normal mucosae was present. In ESCCs, methylation levels of the two repetitive elements and the two cancer/testis antigen genes were correlated with each other.ConclusionThis is the first study to show the presence of global hypomethylation in ESCCs, and even in their non-cancerous background mucosae. Alu hypomethylation might reflect the severity of an epigenetic field for cancerization.

Tumor vessel depiction with contrast-enhanced endoscopic ultrasonography predicts efficacy of chemotherapy in pancreatic cancer.

Objectives Contrast-enhanced endoscopic ultrasonography (CE-EUS) is a new imaging modality for pancreatic lesions. The aim of this study was to evaluate if CE-EUS is useful for predicting treatment efficacy before pancreatic cancer chemotherapy by assessing intratumoral vessel flow. Methods Thirty-nine patients with unresectable advanced pancreatic cancer underwent CE-EUS before chemotherapy. The patients were divided into 2 groups according to the intratumoral vessel flow observed with CE-EUS: vessel sign–positive and vessel sign–negative groups. Patient prognosis was investigated according to presence or absence of the vessel sign. Results Two patients were excluded due to poor visualization of CE-EUS images; therefore, 37 patients were analyzed. Contrast-enhanced EUS revealed positive vessel sign in 20 patients, whereas it revealed negative vessel sign in 17 patients. Both progression-free survival and overall survival were significantly longer in the positive- versus negative vessel sign groups (P = 0.037 and P = 0.027, respectively). Multivariate analysis demonstrated that the positive vessel sign was an independent factor associated with longer overall survival (hazard ratio, 0.22; 95% confidence interval, 0.08–0.53). Conclusions Evaluation of intratumoral vessel flow by CE-EUS could be useful for predicting efficacy of chemotherapy in patients with pancreatic cancer. Contrast-enhanced EUS could be used before chemotherapy for inoperable pancreatic cancer.