Identification of gastric cancer risk markers that are informative in individuals with past H. pylori infection.

BACKGROUND: Epigenomic damage induced by Helicobacter pylori infection is accumulated in gastric mucosae before the development of malignancy. In individuals without current H. pylori infection, DNA methylation levels of specific CpG islands (CGIs) are associated with gastric cancer risk. Because risk estimation in individuals with past infection is clinically important, we here aimed to identify the risk markers that reflect epigenomic damage induced by H. pylori infection, and that are informative in these individuals. METHODS: Gastric mucosae were obtained from 55 gastric cancer patients (GC-Pt) (21 with current infection and 34 with past infection) and 55 healthy volunteers (HV) (7 never-infected, 21 with current infection, and 27 with past infection). Hypermethylated CGIs were searched for by methylated DNA immunoprecipitation-CGI microarray, and methylation levels were analyzed by quantitative methylation-specific polymerase chain reaction (PCR). RESULTS: By microarray analysis of a pool of three samples from GC-Pt with past infection and another pool of samples from HV with past infection, 15 hypermethylated CGIs in the former pool were isolated. Seven of them had significantly higher methylation levels in GC-Pt with past infection (n = 10) than in HV with past infection (n = 10) (P < 0.001). In a validation cohort (21 GC-Pt with past infection and 14 HV with past infection), the seven new markers had large areas under the receiver-operating characteristic curves (0.78-0.84) and high odds ratios (12.7-36.0) compared with two currently available markers (0.60-0.65, 5.0-5.7). CONCLUSIONS: We identified seven novel gastric cancer risk markers that are highly informative in individuals with past infection.

Steakhouse syndrome causing large esophageal ulcer and stenosis.

A 66-year-old man developed dysphagia during dinner and was evaluated 2 d later in our hospital because of persistent symptoms. Upper gastrointestinal endoscopy showed no impacted food, but advanced esophageal cancer was suspected based on the presence in the upper esophagus of a large irregular ulcerative lesion with a thick white coating and stenosis. Further imaging studies were performed to evaluate for metastases, revealing circumferential esophageal wall thickening and findings suggestive of lung and mediastinal lymph node metastases. However, dysphagia symptoms and the esophageal ulcer improved after hospital admission, and histopathological examination of the esophageal mucosa revealed only nonspecific inflammation. At the time of symptom onset, the patient had been eating stewed beef tendon (Gyusuji nikomi in Japanese) without chewing well. Esophageal ulceration due to steakhouse syndrome was therefore diagnosed. The lung lesion was a primary lung cancer that was surgically resected. Although rare, steakhouse syndrome can cause large esophageal ulceration and stenosis, so care must be taken to distinguish this from esophageal cancer.

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.

Simultaneous measurement of pazufloxacin, ciprofloxacin, and levofloxacin in human serum by high-performance liquid chromatography with fluorescence detection.

In this study, three fluoroquinolones, pazufloxacin, ciprofloxacin and levofloxacin, were simultaneously determined in spiked human serum by high-performance liquid chromatography (HPLC) method with fluorescence detection. Chromatography was performed using a C8 column with an isocratic mobile phase consisting of 1% triethylamine (pH 3.0)/acetonitrile (86/14, v/v). Protein precipitation was conducted using perchloric acid and methanol. The calibration curves for the three fluoroquinolones were linear over concentrations ranging from 0.1 to 20.0 microg/mL. The within-day and between-day coefficients of variation obtained from three fluoroquinolones were less than 7%, and relative errors ranged from -1.6% to 9.3%. Mean recoveries of pazufloxacin, ciprofloxacin, and levofloxacin from spiked human serum were 97%, 88%, and 90%, respectively. The proposed method proved to be simple and reliable for the determination of three fluoroquinolones.

Persistence of a component of DNA methylation in gastric mucosae after Helicobacter pylori eradication.

BACKGROUND: Helicobacter pylori (HP) infection potently induces aberrant DNA methylation in gastric mucosae, and its accumulation is associated with gastric cancer risk. Cross-sectional analysis of methylation levels (fraction of methylated DNA molecules) and temporal analysis of methylation incidence suggested that methylation levels decrease after HP infection discontinues. We aimed to demonstrate the decrease in methylation levels. METHODS: Thirty-five patients with HP infection who had undergone curative endoscopic resection and 11 healthy volunteers were recruited. Methylation levels were quantified by real-time methylation-specific PCR. Histology was evaluated according to the updated Sydney System. RESULTS: In the 20 patients with successful eradication, the FLNc methylation level, along with infiltration of inflammatory cells, decreased from 0.6 to 0.4% at 6 weeks (P = 0.049) and remained low at 1 year. The THBD methylation level (30.1%) remained high at 6 weeks, but decreased to 19.0% at 1 year (P = 0.0032). Nine healthy volunteers with successful eradication tended to show a decrease of both FLNc and THBD at 6 weeks. However, the methylation levels after the decrease were still higher than those of healthy individuals without HP infection. In the 15 patients with persistent infection, the methylation levels remained the same. Before eradication, the THBD methylation level correlated with the degree of inflammatory cell infiltration (P < 0.05). CONCLUSIONS: Methylation levels in gastric mucosae decreased to certain levels after HP eradication in profiles unique to individual markers. Involvement of chronic inflammation in methylation induction was suggested.

Diagnosis of pancreatic tuberculosis by combined, contrast-enhanced sonography and endoscopic ultrasound-guided fine-needle aspiration.

A 79-year-old woman complaining of epigastric pain was examined by her local physician, who found an abdominal mass and referred the patient to our department. Abdominal plain computed tomography revealed a mass, 50 mm in size, with slight calcification on the ventral side of the head of the pancreas. On abdominal ultrasound, the mass lesion consisted of an aggregation of hypoechoic masses, with a heterogeneous hyperechoic region at its center. On contrast ultrasonography, only the hyperechoic region was stained. (18)F-Fluorodeoxyglucose-positron emission tomography (FDG-PET) revealed FDG accumulation in the same region. It was difficult to differentiate between a malignant pancreatic tumor and an inflammatory disease on imaging, but since QuantiFERON TB2G testing was positive, pancreatic tuberculosis was suspected, and endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) was performed to obtain a definitive diagnosis. Samples from the hypoechoic region consisted of necrotic tissue, while those from the hyperechoic region consisted of pancreatic tissue together with granulation tissue. BCG immunostaining was positive, and a diagnosis of pancreatic tuberculosis was made. If EUS-FNA is performed on stained areas seen on contrast ultrasonography, this will probably enable a more accurate diagnosis of pancreatic tuberculosis with low invasiveness.

The presence of a methylation fingerprint of Helicobacter pylori infection in human gastric mucosae.

Aberrant DNA methylation is deeply involved in human cancers, but its inducers and targets are still mostly unclear. Helicobacter pylori infection was recently shown to induce aberrant methylation in gastric mucosae, and produce a predisposed field for cancerization. Here, we analyzed the presence of target genes in methylation induction by H. pylori and the mechanism for the gene specificity. Noncancerous gastric mucosae were collected from 4 groups of individuals (with and without a gastric cancer, and with and without current H. pylori infection; N = 11 for each group), and methylation of promoter CpG islands of 48 genes that can be methylated in gastric cancer cell lines was analyzed by methylation-specific PCR. In total, 26 genes were consistently methylated in individuals with current or past infection by H. pylori, whereas 7 genes were not methylated at all. In addition, 14 genes were randomly or intermediately methylated in individuals with gastric cancers and the remaining 1 gene was methylated in all the cases. The methylation-susceptible genes had significantly lower mRNA expression levels than the methylation-resistant genes. H. pylori infection did not induce mRNA and protein expression of DNA methyltransferases; DNMT1, DNMT3A or DNMT3B. Gene specificity was present in the induction of aberrant DNA methylation by H. pylori infection, and low mRNA expression, which could precede methylation, was one of the mechanisms for the gene specificity. These findings open up the possibility that a methylation fingerprint can be used as a novel marker for past exposure to a specific carcinogenic factor.

Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae.

The presence of high levels of aberrant DNA methylation in gastric mucosae correlates with risk of gastric cancer. Some gastric cancers are known to have methylation of multiple CpG islands (CGI), which is referred to as the CGI methylator phenotype (CIMP). In the present study, we aimed to clarify the possible association between the CIMP in cancers and high methylation levels in their background mucosae by accurate quantitative methylation analysis of 14 carefully selected promoter CGI. Methylation levels were measured in 66 cancers and their background mucosae, along with 19 normal mucosae of healthy volunteers. Methylation in cancers was classified as absent (methylation level = 0%) or positive. The number of methylated CGI in a cancer showed a continuous distribution, and cancers were classified as CIMP high (21 cases), CIMP low (30 cases), or CIMP negative (15 cases). CIMP-high gastric cancer patients had significantly better survival rates than CIMP-negative patients. Of the Epstein-Barr virus-positive gastric cancers studied, eight out of nine presented as CIMP high. Methylation in background mucosae showed a unimodal distribution, and was assessed by their degree. The gastric mucosae of cancer patients showed higher levels than normal gastric mucosae of healthy volunteers. Finally, the CIMP-high, CIMP-low, and CIMP-negative statuses in cancers were not associated with methylation levels of individual genes and their means in the background mucosae. These showed that the CIMP statuses in gastric cancers had no association with methylation levels in the background gastric mucosae.

Silencing of the UCHL1 gene in human colorectal and ovarian cancers.

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2'-deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (> or =8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk.

INTRODUCTION: Risk prediction of gastric cancers is important to implement appropriate screening procedures. Although aberrant DNA methylation is deeply involved in gastric carcinogenesis, its induction by Helicobacter pylori, a strong gastric carcinogen, is unclear. Here, we analyzed the effect of H. pylori infection on the quantity of methylated DNA molecules in noncancerous gastric mucosae and examined its association with gastric cancer risk. EXPERIMENTAL DESIGN: Gastric mucosae were collected from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and 72 cases with differentiated-type gastric cancers (29 H. pylori negative and 43 H. pylori positive) by endoscopy. The numbers of DNA molecules methylated and unmethylated for eight regions of seven CpG islands (CGI) were quantified by quantitative PCR after bisulfite modification, and fractions of methylated molecules (methylation levels) were calculated. RESULTS: Among healthy volunteers, methylation levels of all the eight regions were 5.4- to 303-fold higher in H. pylori positives than in H. pylori negatives (P < 0.0001). Methylation levels of the LOX, HAND1, and THBD promoter CGIs and p41ARC exonic CGI were as high as 7.4% or more in H. pylori-positive individuals. Among H. pylori-negative individuals, methylation levels of all the eight regions were 2.2- to 32-fold higher in gastric cancer cases than in age-matched healthy volunteers (P < or = 0.01). Among H. pylori-positive individuals, methylation levels were highly variable, and that of only HAND1 was significantly increased in gastric cancer cases (1.4-fold, P = 0.02). CONCLUSIONS: It was indicated that H. pylori infection potently induces methylation of CGIs to various degrees. Methylation levels of specific CGIs seemed to reflect gastric cancer risk in H. pylori-negative individuals.