Promoter methylation correlates with reduced NDRG2 expression in advanced colon tumour.

BACKGROUND: Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current study to validate our previous microarray data and to identify novel hypermethylated gene promoters. METHODS: The validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour tissues from 30 patients with CRC. RESULTS: Data from our previous genome search have received confirmation in the new set of 8 patients with CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated. CONCLUSION: The findings highlight the usefulness of combining gene expression patterns and epigenetic data to identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being associated with a more advanced stage.

Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication.

BACKGROUND: Gene promoter methylation is an epigenetic event leading to gene silencing. This mechanism is particularly relevant in cancer since it can interfere with the activity of specific "suppressor" genes. AIM: To evaluate promoter methylation of CDH1, p16, APC, MLH1, and COX2 in patients with H. pylori (Hp) infection before and after eradication. METHODS: Fifty-seven dyspeptic outpatients who had never performed previous endoscopy or Hp testing and treatment underwent clinical interview, endoscopy with three paired gastric biopsy specimens from the antrum, angulus, and corpus, and (13)C-urea breath test (UBT). Biopsies were scored for the presence of Hp and intestinal metaplasia (IM). DNA methylation of five tumor-related genes (CDH1, p16, MLH1, APC, and COX2) was evaluated by methylation-specific PCR in each biopsy. Infected patients were given a standard eradicating treatment and, after 1 yr, underwent endoscopy with biopsies and UBT. RESULTS: Hp infection was found in 45 patients. IM was detected in 17 out of 45 (38%) infected patients. Mean number of methylated genes was 0, 1.1 +/- 0.9, and 1.6 +/- 0.9 among the 12 Hp-/IM-, the 28 Hp+/IM-, and the 17 Hp+/IM+ patients, respectively (P < 0.0001). Specifically, promoter hypermethylation of CDH1, p16, APC, MLH1, and COX2 was found in 68%, 25%, 7%, 0%, and 14% of Hp+/IM- patients and in 71%, 29%, 35%, 12%, and 12% of Hp+/IM+ patients. No significant difference was found among the three groups of patients as far as age, smoking, alcohol, meat and vegetable consumption, and family history of gastric cancer were considered. Twenty-three out of 45 (51%) infected patients underwent the 1-yr follow-up endoscopy: 17 out of 23 (74%) were successfully eradicated. After Hp eradication, CDH1, p16, and APC methylation significantly decreased while COX2 methylation completely disappeared. Conversely, MLH1 methylation did not change significantly in patients with IM. CONCLUSION: Hp infection is associated with promoter methylation of genes which are relevant in the initiation and progression of gastric carcinogenesis. While CDH1 methylation seems to be an early event in Hp gastritis, MLH1 methylation occurs late along with IM. Hp eradication is able to significantly reduce gene methylation thus delaying or reversing Hp-induced gastric carcinogenesis.

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients.

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.

Lack of association between UGT1A7, UGT1A9, ARP, SPINK1 and CFTR gene polymorphisms and pancreatic cancer in Italian patients.

AIM: To investigate simultaneously UGT1A7, UGT1A9, ARP, SPINK and CFTR genes to verify whether genetic polymorphisms predispose to the development of pancreatic cancer (PC). METHODS: Genomic DNA of 61 pancreatic cancer patients and 105 healthy controls (HC) were analyzed. UGT1A7 genotyping was determined by PCR-RFLP analysis. Specific PCR and sequencing were used to analyze genetic variants of UGT1A9, ARP, SPINK1 and CFTR genes. RESULTS: Four different alleles (*1: WT; *2: N129K and R131K; *3: N129K, R131K, and W208R; and *4: W208R) in UGT1A7 and three different alleles (*1: WT; *4: Y242X; and *5: D256N) in UGT1A9 were detected. All UGT1A polymorphisms were observed at similar frequency in PC patients and HC. Seven different alleles in ARP were found in PC patients and HC at similar frequency. The SPINK1 mutations N34S and P55S occurred in five PC patients with a prevalence (4.1%) not significantly different from that observed (2.0%) in HC. The only CFTR DeltaF508 mutation was recognized in three PC patients with a prevalence (4.9%) similar to HC. CONCLUSION: UGT1A7, UGT1A9, ARP, SPINK1 and CFTR gene polymorphisms are not associated with PC in Italian patients.

Comparison of a monoclonal antigen stool test (Hp StAR) with the 13C-urea breath test in monitoring Helicobacter pylori eradication therapy.

AIM: To evaluate the agreement between a mAb-based stool test (HP StAR) and the urea breath test (UBT) in monitoring (H pylori) infection after eradication therapy. METHODS: Patients with discordant results on UBT and Hp StAR underwent endoscopy with biopsies for rapid urease test, culture, and histology to confirm H pylori status. RESULTS: Among 250 patients (50+/-14 years), 240 (96.0%) had concordant UBT and Hp StAR tests with a significant correlation between DOB and A values (R = 0.87; P<0.0001). The remaining 10 (4.0%) patients had discordant tests (positive Hp StAR and negative UBT) with the Hp StAR inaccurate in five cases (false positive) and UBT inaccurate in the other five cases (false negative). The "maximal expected" sensitivity, specificity, +PV, -PV, +LR, and -LR were 91%, 100%, 100%, 97.4%, infinity, and 8.2 respectively, for the UBT, and 100%, 97.4%, 91%, 100%, 38.8, and 0, respectively, for the Hp StAR. Overall accuracy for both tests was 98%. CONCLUSION: Both the UBT and the Hp StAR are equally accurate in monitoring H pylori infection. Nowadays, the choice of the "best" non-invasive H pylori test in the post-treatment setting should be done not only in terms of diagnostic accuracy but also in view of cost and local facilities.

Antisecretory vs. antiproteasic drugs in the prevention of post-ERCP pancreatitis: the evidence-based medicine derived from a meta-analysis study.

Uncertainties still exist about the clinical benefit of pharmacological prevention of post-ERCP pancreatitis by either antisecretory drugs such as somatostatin and its long-acting analogue octreotide, or protease inhibitors such as gabexate mesilate. Recent, large-scale prospective studies have reported a fourfold reduction in acute pancreatitis as compared to a placebo with the prophylactic administration of either gabexate mesilate or somatostatin, whereas octreotide was found to be ineffective. An initial meta-analysis of all available controlled trials on this topic has confirmed these findings. The indiscriminate use of these drugs in all patients is unlikely to be cost-effective, but the selective use of prophylaxis for high-risk patients might be advocated. Moreover, inasmuch as 85% of complications developed within 4 to 6 hours of completing the ERCP, it would be reasonable to infuse drugs only for this limited length of time. A recent prospective trial, carried out on high-risk patients, has surprisingly documented a higher incidence, although a non-significant one, of pancreatitis in patients who received short-term prophylaxis with somatostatin or gabexate mesilate than those given a placebo: 11.5% and 8.1% vs. 6.5%, respectively. In order to explore this discrepancy, the original meta-analysis was updated by including data of this negative trial: heterogeneity among the trials was apparent. A careful scrutiny of the most recent studies has revealed differences in patient population, protocols of drug administration, technique and operator-related risk factors for complications among the trials, which could explain, by themselves, the contrasting results reported by the interventional studies. In conclusion, current literature does not support the prophylactic use of either somatostatin or gabexate mesilate for the prevention of ERCP-related pancreatic damage, even in patients deemed to be at high risk for complications. At present, post-ERCP complications (and pancreatitis) can be prevented efficaciously by appropriate selection of patients, mastering of the technique and operator competence.

Amoxicillin/tetracycline combinations are inadequate as alternative therapies for Helicobacter pylori infection.

BACKGROUND: Triple therapy with proton pump inhibitors or ranitidine bismuth citrate, clarithromycin and either amoxicillin or nitroimidazole derivatives are the present gold standards for cure of Helicobacter pylori infection. However, primary resistance to either clarithromycin or nitroimidazole derivatives is increasing and alternative therapies are needed. AIM: To determine the efficacy and safety of three regimens consisting of amoxicillin and tetracycline or doxycycline combined with either lansoprazole or ranitidine bismuth citrate. METHODS: Two hundred and seventy H. pylori infected patients were randomly given one of the following treatments: amoxicillin 1 g twice a day (b.i.d.) plus tetracycline 500 mg four times a day (q.i.d.) with either lansoprazole 30 mg b.i.d. (group LAT) or ranitidine bismuth citrate 400 mg b.i.d. (group RBCAT) for 7 days and amoxicillin 1 g b.i.d. plus doxycycline 100 mg b.i.d. and lansoprazole 30 mg b.i.d. for 14 days (group LAD). Eradication rate was assessed by UBT at 4-6 weeks after therapy. RESULTS: The three groups (LAT, RBCAT, and LAD) of patients achieved eradication rates of 35% (25-45), 20% (12-29) and 36% (25-46), respectively, on intention-to-treat analysis. Patient compliance was optimal and side-effects minimal in all three groups. CONCLUSIONS: Although the amoxicillin/tetracycline combination is attractive (inexpensive, safe, and with low primary resistance rate), it can not be recommended for H. pylori eradication.