Epigenetic silencing of miR-137 is a frequent event in gastric carcinogenesis.

MicroRNAs (miRNA) are involved in posttranscriptional regulation of gene expression and are dysregulated during carcinogenesis. CpG island methylation of miR-137 is a common event in different cancers; however, the role of miR-137 in gastric cancer (GC) remains largely unexplored. In this study we aimed to characterize the epigenetic alterations of miR-137 in gastric carcinogenesis. We analyzed total 295 tissues including paired primary gastric cancer (T-GC) with corresponding adjacent gastric mucosa (N-GC), paired primary colorectal cancer (CRC) tissues with corresponding non-tumorous mucosa, gastric tissues from controls (N), and patients with chronic/atrophic gastritis (CG) with and without Helicobacter pylori infection. Bisulfite pyrosequencing and TaqMan RT-PCR were used to analyze miR-137 methylation and expression, respectively. Survival differences were evaluated using Kaplan-Meier analyses. miR-137 CpG island methylation was more frequent in tumorous compared to non-tumorous conditions and higher in CRC than in GC. In comparison to N-GC, miR 137 methylation level was lower in N and CG tissues, which correlates with Correas cascade. MiR-137 methylation inversely correlates with global LINE-1 methylation and miR-137 expression. miR-137 methylation was higher in intestinal type GC compared to diffuse one, and higher in antrum compared to cardia and corpus, however, miR-137 methylation was associated with worse prognosis in diffuse, but not in intestinal type of GC. The expression in colon was significantly higher compared to any gastric tissues suggesting functional difference. In summary, miR-137 methylation is a frequent event in gastrointestinal cancers which occurs early in stepwise manner during gastric carcinogenesis and inversely correlates with global methylation. © 2015 Wiley Periodicals, Inc.

Helicobacter Pylori Serology in Relation to Hepatitis C Virus Infection and IL28B Single Nucleotide Polymorphism.

The aim of the study was to evaluate the serological rate of Helicobacter pylori (H. pylori) infection in patients with chronic hepatitis C virus (HCV) infection and determine any correlations with liver damage and IL28B single-nucleotide polymorphism (SNP). One hundred eighty-nine patients with chronic HCV infection were included in the study, and H. pylori status was defined based on anti-H. pylori-IgG or anti-CagA-IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Liver damage was assessed using histology or transient elastography. IL28B C/T polymorphism (rs12979860) was evaluated in circulating blood cells using a PCR-based restriction fragment length polymorphism assay. Overall H. pylori serology was positive in 38.1% of our HCV-infected subjects. Among those, the anti-CagA-IgG positivity rate was 43.1% and was within the range of previously described populations of the same region. Highest prevalence of H. pylori was found in patients between 31 and 40 years compared to other age subgroups. The seropositivity rate was higher in the non-cirrhotic group than the cirrhotic one (45.4% vs. 20.0%, p < 0.05). No difference was found in IL28B genotype between H. pylori-positive and -negative cohorts. However, we observed a trend for the lower anti-CagA-IgG expression level in relation to the IL28B T-allele. Our results do not support an association between HCV and H. pylori infection. Whether IL28B SNP has a functional role in modulation of serological response to H. pylori CagA needs further investigation.

Influence of laboratory-related and endoscopy-related factors on the assessment of serum pepsinogens and gastrin-17.

BACKGROUND AND AIM: Serum pepsinogen I (PGI) and pepsinogen II (PGII) are noninvasive parameters in the detection of atrophic gastritis. The diagnostic add-on value of serum gastrin-17 (G-17) remains uncertain. The aim of this study was to assess the stability of these serum parameters over time and to evaluate the influence of clinical factors, such as upper gastrointestinal (GI) endoscopy and bowel cleansing, on serum PGI, PGII, and G-17 assessment. PATIENTS AND METHODS: A prospective study was carried out in healthy individuals and patients. For the stability analyses, the plasma and serum samples from 23 individuals were processed at different time points with and without the addition of a stabilizer. Ten patients were included to evaluate the influence of upper GI endoscopy and 18 patients to evaluate the effect of bowel cleansing before colonoscopy. RESULTS: PGI, PGII, and G-17 levels were not statistically different in the serum and plasma. PGI and PGII serum levels were stable over time. G-17 is associated with time-dependent degradation (P=0.0001). The addition of the G-17 stabilizer showed no improvement in stability. Upper GI endoscopy and bowel preparation before colonoscopy were associated with minimal variations in PGI and PGII, whereas G-17 showed patient-specific alterations. CONCLUSION: PGI and PGII serum levels are stable over time. However, G-17 stability is strongly dependent on the time of processing and storage; therefore, samples for G-17 analysis need to be processed no later than 6 h after blood collection. Upper GI endoscopy and colonoscopy preparation lead to minimal nonsignificant changes in basal PGI, PGII, and G-17 levels.

LINE-1 hypomethylation is not a common event in preneoplastic stages of gastric carcinogenesis.

LINE-1 hypomethylation is widely accepted as marker for global genomic DNA hypomethylation, which is a frequent event in cancer. The aim of the study was to evaluate LINE-1 methylation status at different stages of gastric carcinogenesis and evaluate its prognostic potential in clinical settings. LINE-1 methylation was analyzed in 267 tissue samples by bisulfite pyrosequencing including primary colorectal cancer tissues (T-CRC) with corresponding adjacent colon mucosa (N-CRC), gastric cancer tissues (T-GC) with corresponding gastric mucosa (N-GC), normal gastric tissues (N), chronic non-atrophic and atrophic gastritis (CG). LINE-1 methylation level was lower in both T-GC and T-CRC when compared to paired adjacent tissues. No difference was observed for LINE-1 methylation status between patients with normal gastric mucosa, CG and N-GC. LINE-1 methylation in T-GC but not N-GC tended to correlate with age. Subgroup stratification analysis did not reveal significant differences in LINE-1 methylation status according to tumor stage, anatomical location, histological subtype, differentiation grade. We observed similar overall survival data between patients with high or low LINE-1 levels. In summary, LINE-1 hypomethylation is a characteristic feature in GC but not very common in early preneoplastic stages of gastric carcinogenesis. Prognostic role of LINE-1 hypomethylation in GC patients could not be confirmed in this cohort.

Helicobacter pylori vacA genotype is a predominant determinant of immune response to Helicobacter pylori CagA.

AIM: To evaluate the frequency of Helicobacter pylori (H. pylori) CagA antibodies in H. pylori infected subjects and to identify potential histopathological and bacterial factors related to H. pylori CagA-immune response. METHODS: Systematic data to H. pylori isolates, blood samples, gastric biopsies for histological and molecular analyses were available from 99 prospectively recruited subjects. Serological profile (anti-H. pylori, anti-CagA) was correlated with H. pylori isolates (cagA, EPIYA, vacA s/m genotype), histology (Sydney classification) and mucosal interleukin-8 (IL-8) mRNA and protein expression. Selected H. pylori strains were assessed for H. pylori CagA protein expression and IL-8 induction in co-cultivation model with AGS cells. RESULTS: Thirty point three percent of microbiologically confirmed H. pylori infected patients were seropositive for CagA. Majority of H. pylori isolates were cagA gene positive (93.9%) with following vacA polymorphisms: 42.4% vacA s1m1, 23.2% s1m2 and 34.3% s2m2. Anti-CagA-IgG seropositivity was strongly associated with atrophic gastritis, increased mucosal inflammation according to the Sydney score, IL-8 and cagA mRNA expression. VacA s and m polymorphisms were the major determinants for positive (vacA s1m1) or negative (vacA s2m2) anti-CagA serological immune response, which also correlated with the in vitro inflammatory potential in AGS cells. In vitro co-cultivation of representative H. pylori strains with AGS cells confirmed functional CagA translocation, which showed only partial correlation with CagA seropositivity in patients, supporting vacA as major co-determinant of the immune response. CONCLUSION: Serological immune response to H. pylori cagA+ strain in H. pylori infected patients is strongly associated with vacA polymorphism, suggesting the crucial role of bacterial factors in immune and clinical phenotype of the infection.

Differential expression of microRNAs in preneoplastic gastric mucosa.

Gastric carcinogenesis is a multifactorial H.pylori-triggered dynamic process that goes through a cascade of preneoplastic conditions. The expression of miRNAs in the stomach with regard to preneoplastic precursor conditions and H.pylori infection has not been investigated systematically. In this prospective proof-of-principle study, we evaluated the miRNA expression in gastric antrum and corpus mucosa from patients with chronic non-atrophic gastritis (CNAG), atrophic gastritis (AG), and GC compared to controls. Gastric normal mucosa shows a unique expression pattern for miR-21, miR-155 and miR-223, which is specific for different regions. In correlation with progression of Correa's cascade and H.pylori infection, we observed a gradual increase in miR-155 and miR-223 both in corpus and antrum and miR-21 only in the antrum mucosa. Using miRNA expression we calculated a score that allowed us to discriminate patients with AG from subjects with normal mucosa with high diagnostic accuracy in testing and validation cohorts reproducibly. In summary, the expression pattern of miRNAs in the gastric mucosa is gradually increased with progression of Correa's cascade and H.pylori infection, suggesting miRNAs as potential biomarkers for preneoplastic precursor conditions. However, differences of miRNA expression between the gastric antrum and the corpus need to be considered in future studies.

MMP2 and MMP7 at the invasive front of gastric cancer are not associated with mTOR expression.

BACKGROUND: Regulation of MMP expression by activation of mTOR signalling has been demonstrated for several tumor types, but has thus far not been confirmed in gastric cancer. FINDINGS: The study compromised 128 patients who underwent gastric resection for cancer (66.4 % male; 86 intestinal, 42 diffuse type). Immunohistochemical staining of MMPs was performed to analyse the topographical pattern of MMP expression at the tumor center and the invasive front, respectively. MMP2 showed higher expression at the invasive front compared to the tumor center, whereas MMP7 staining scores were higher in the tumor center, and there was no difference for MMP9. The expression of p-mTOR was higher in the tumor center than at the invasive front, with a similar trend for mTOR. For intestinal type gastric cancer there was a weak correlation of MMP9 with expression of mTOR in the tumor center. Otherwise, there was no correlation of the MMPs with mTOR. By treatment of MKN45 gastric cancer cells with rapamycin, a reduction of p-mTOR in the Western blot was achieved; however, expression of MMPs remained unaffected. CONCLUSIONS: Expression of MMP2 and MMP7 in gastric cancer is not associated with mTOR, MMP9 expression might be related to mTOR signalling in a subset of tumors.

Different antibiotic susceptibility between antrum and corpus of the stomach, a possible reason for treatment failure of Helicobacter pylori infection.

AIM: To assess whether antibiotic resistance varies between the antrum and corpus of the stomach of patients that are either Helicobacter pylori (H. pylori) therapy-naive or pre-treated. METHODS: H. pylori strains were isolated from antrum and corpus biopsies from 66 patients that received a diagnostic gastroduodenoscopy for variant clinical indications. Antimicrobial susceptibility to amoxicillin, clarithromycin, tetracycline, metronidazole, levofloxacin and rifabutin was tested with the E-test method on Iso-Sensitest agar with 10 vol% defibrinated horse blood. In patients with a different antibiotic susceptibility pattern between the isolates from the antrum and corpus, DNA fingerprinting via random amplified polymorphic DNA analysis was performed to detect differences among DNA patterns of H. pylori isolates. RESULTS: Primary, secondary and tertiary resistance to clarithromycin was 6.9%, 53.8% and 83.3%, retrospectively. Metronidazole and levofloxacin resistance also increased according to the number of previous treatments (17.2%, 69.2%, 83.3%; 13.8%, 23.1%, 33.3%). Tertiary resistance to rifabutin was detected in 12.5% of patients. In none of the 66 patients a resistance against amoxicillin or tetracycline was detectable. Discordant antibiotic susceptibility between antrum and corpus isolates for different antibiotics was seen in 15.2% (10/66) of the patients. Two out of those ten patients were naive to any H. pylori antibiotic treatment. The remaining eight patients previously received at least one eradication therapy. DNA fingerprinting analysis revealed no substantial differences among DNA patterns between antrum and corpus isolates in the majority of patients suggesting an infection with a single H. pylori strain. CONCLUSION: Different antibiotic susceptibility between antrum and corpus biopsies is a common phenomenon and a possible explanation for treatment failure. Resistant H. pylori strains may be missed if just one biopsy from one anatomic site of the stomach is taken for H. pylori susceptibility testing.

Antibiotic susceptibility of Helicobacter pylori in central Germany and its relationship with the number of eradication therapies.

OBJECTIVES: Helicobacter pylori eradication rates show a constant decline over the last few years. The main reason for H. pylori treatment failure is the increasing antibiotic resistance.We assessed antibiotic susceptibility of H. pylori in a region of mid-Germany and analyzed the relationship of antibiotic resistance with the number of eradication therapies over a period of 7 years (2005-2012). METHODS: H. pylori strains were isolated from 436 patients who underwent gastroscopy for different clinical indications. Susceptibility to amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and rifabutin was determined using the E-test. RESULTS: Primary, secondary, and tertiary resistances against clarithromycin were 7.5, 63.2, and 75.4%, respectively. Primary, secondary, and tertiary resistances to levofloxacin were 11.7, 17.6, and 36.4% and to metronidazole were 32.7, 63.2, and 80.1%, respectively. The resistance rates against tetracycline and rifabutin were comparatively low (<5%), even in patients with previous exposure to these antibiotics. Resistance to rifabutin increased to 6.2% in patients who received more than two previous eradication therapies. Amoxicillin resistance was not detectable in all patients. CONCLUSION: In our region, we observed a stable, but constantly increasing, resistance rate to antibiotics commonly used for the treatment of H. pylori infection. Knowledge of the local antibiotic resistance rates is essential for developing successful treatment strategies for H. pylori eradication.