Kazuyuki Nakazawa

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 ≤ 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.

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.

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 (≥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.

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. (Cancer Sci 2007; 98: 1853–1861)

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.

Subclinical syphilitic hepatitis, which was markedly worsened by a Jarisch-Herxheimer reaction

Early syphilitic hepatitis is uncommon and tends to be overlooked. However, the diagnosis of this disease is important, because appropriate treatment results in rapid resolution of the hepatitis. We report a case of subclinical early syphilitic hepatitis exaggerated by a Jarisch-Herxheimer reaction. This reaction helped to realize the diagnosis in this case.

Contrast Harmonic Sonographically Guided Radio Frequency Ablation for Spontaneous Ruptured Hepatocellular Carcinoma

Received January 19, 2005, from the Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan (H.T., M.O., H.M., N.S., T.K., S.E., T.S., M.I., K.N., K.A., K.Y., Y.S., H.N., M.I.); and Department of Gastroenterology, University of Tokyo, Tokyo, Japan (M.F., N.Y., S.S.). Revision requested February 7, 2005. Revised manuscript accepted for publication March 1, 2005. Address correspondence to Hideyuki Tamai, MD, Second Department of Internal Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama City, Wakayama 640-0012, Japan. Abbreviations CT, computed tomography; HCC, hepatocellular carcinoma; PEIT, percutaneous ethanol injection therapy; RFA, radio frequency ablation; S, segment; TAE, transcatheter arterial embolization ntraperitoneal bleeding due to a ruptured tumor is a serious complication in patients with hepatocellular carcinoma (HCC). According to data compiled by the Liver Cancer Study Group of Japan,1 ruptured HCC accounts for around 10% of deaths in these patients. Clinical features include the sudden onset of abdominal pain and distension and, if bleeding is massive, the presence of shock. Other causes of an acute abdominal emergency must be ruled out. Diagnostic imaging generally includes sonography, contrast computed tomography (CT), and angiography. In patients with ruptured HCC, prompt diagnosis and treatment is essential to avoid hepatocyte necrosis and secondary hepatic failure associated with shock and decreased hepatic perfusion due to bleeding. The underlying liver disease varies in such patients with ruptured HCC. Chronic hepatitis, cirrhosis, or both may be present, and the severity of hepatic dysfunction as well as the size, number, and progression of the neoplastic lesions present varies from case to case. A common feature is the presence of a responsible lesion on or protruding from the surface of the liver. If hemostasis can be achieved early after HCC rupture, then overall prognosis depends on the patient’s liver function and degree of tumor progression. Although there is a risk of intraperitoneal seeding, long-term survival is possible if the tumor can be completely resected by hepatectomy. One study has already reported a good 5-year survival rate after resection of ruptured and nonruptured HCC.2 In another study, rather than performing emergency surgery, Marini et al3 used transcatheter arterial embolization (TAE) to control bleeding; in those patients who could then undergo surgery, elective hepatectomy was associated with long-term survival. Treatment of ruptured HCC involves more than just hemostasis. Subsequent therapy is important, and, whenever possible, complete resection should be performed after bleeding has been controlled.

Migration of gastrostomy site: rare complication of percutaneous endoscopic gastrostomy.

Although percutaneous endoscopic gastrostomy (PEG) is relatively safe and easy to carry out, complications can occur during and after the procedure. Here, we report a rare case with complications involving a gastrostomy tube. An 82-year-old woman with a history of cerebral hemorrhage underwent PEG using a Safety PEG kit (Boston Scientific Japan, Tokyo, Japan) with the pull technique at the anterior wall of the lower gastric body (Fig. 1A). Esophagogastroduodenoscopy (EGD) showed normal findings in the stomach and duodenum (Fig. 1B). Exchanges of the balloontype (Foley-type) gastrostomy tube had been carried out in an irregular manner with intervals of 1–6 months without using endoscopy. EGD 4 years after initial placement of the gastrostomy revealed a longitudinal ulcer scar extending from the anterior wall of the lower gastric body to the anterior wall of the pylorus ring, with shortening of the lesser curvature of the stomach (Fig. 1C). The internal balloon was placed in the bulbus (Fig. 1D). The ulcer scar of the stomach appeared to continue from the position where gastrostomy A B

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.

M1699 Inhibitory Effects of Japanese Apricot, (Prunus Mume Siebold ET Zucc.; Urne), on Helicobacter pylori-Related Chronic Gastritis

BACKGROUND: Recently, there has been a growing body of evidence suggesting a relationship between H. pylori gastritis and hypoferritinemia or IDA . AIM: To systematically review the role of H. pylori infection in hypoferritinemia and IDA, and to perform a meta-analysis of case-control studies. METHODS: Selection of studies: Case-control studies comparing a) the prevalence of H. pylori infection in patients with and without IDA/hypoferritinemia, and b) the prevalence of IDA/hypoferritinemia in patients with and without H. pylori infection. Search strategy: electronic and manual bibliographical searches. Data synthesis: Meta-analysis combining the Odds Ratios (OR). RESULTS: a) Eight studies compared the prevalence of H. pylori infection in patients with (637 patients) and without (2,305 patients) IDA, showing a higher prevalence of H. pylori infection in anaemic patients (34% vs. 22%; OR=1.6; 95%CI= 1.26-2.03). Only one study compared the prevalence of infection in patients with and without hypoferritinemia (29% vs. 19%; p<0.05). b) Seven studies compared the prevalence of IDA in patients with (3093 patients) and without (1926 patients) H. pylori infection, showing a slightly (and almost statistically significant) higher prevalence of IDA in H. pyloripositive patients (16% vs. 14%; OR=1.4; 95%CI=0.98-2.0). Finally, 5 studies compared the prevalence of hypoferritinemia in patients with (297 patients) and without (129 patients) H. pylori infection, showing a higher prevalence of hypoferritinemia in H. pylori-positive patients (33% vs. 13%; OR=1.7; 95%CI=1.31-2.32). Results were heterogeneous for all comparisons. CONCLUSION: Some epidemiologic studies suggest an association between H. pylori infection and lower iron stores or IDA. However, these data should be interpreted with caution due to marked heterogeneity among studies.