Background and Aims Evidences suggest that the gastric cancer carcinogenesis appears to be explained by a synergistic interaction between Helicobacter pylori (H. pylori) infection, environmental factors, and host factors. Based on a universal eradication of H. pylori in an offshore island (Matzu) with high prevalence of gastric cancer, we first examined the infection rate of H. pylori according to their interleukin-1β (IL-1B) polymorphic genes and lifestyle factors. Secondly, we evaluated the efficacy of clarithromycin-based triple therapy with a levofloxacin-based rescue treatment and tested the hypothesis that the cytochrome (CY) P2C19 and IL-1B polymorphic genotypes would be associated with treatment failure.

Condition	Intervention	Phase
Helicobacter pylori infection	Drug: levofloxacin-based rescue treatment	Phase IV

Further Study Details: 

Primary Outcomes: Successful helicobacter eradication
Secondary Outcomes: Adverse effect
Expected Total Enrollment:  2500

BACKGROUND & AIMS The Matzu islets are located near the north coast of Fukien Province of mainland China and are separate from Taiwan by the Taiwan Strait. The residents in Matzu are reported to have a high incidence of gastric carcinoma, accounting for approximately 25% of all cancers. Age-standardized incidence rate of gastric cancer in Matzu is approximately three-fold compared with nation-wide Taiwan and the case-fatality rate (0.59, the ratio of mortality rate to incidence rate) is significantly higher than cancers having detectable screening tools including colorectal cancer (0.41), cervical cancer (0.22), breast cancer (0.15), and liver cancer (0.56). It is crucial to determine the risk factors for gastric cancer and prevent subsequent gastric cancer progression in this high prevalent area.

Among known risk factors of gastric cancer, Helicobacter pylori (H. pylori) infection and development of gastric cancer is well established.[1] In Correa’s model of gastric carcinogenesis, the gastric mucosa progresses through the stages of chronic active gastritis, glandular atrophy, intestinal metaplasia, and dysplasia before the development of gastric adenocarcinoma.[2] The presence of H. pylori at baseline was associated with an increased risk of progression to dysplasia or gastric cancer, with an odds ratio of 1.8.[3] However, epidemiologic studies have shown that the risk of gastric cancer is also associated with environmental factors, such as salt, nitrates, and low intake of fresh fruits and vegetables.[4][5] Moreover, host susceptibility to H. pylori is also considered an important factor. Genetic polymorphism of interleukin-1β gene (IL-1B) is related with differences of gastric acid suppression in response to H. pylori infection. Polymorphism of IL-1B was reported associated with gastric cancer risk and the vulnerability to H. pylori infection.[6][7] These evidences suggest that the gastric cancer carcinogenesis appears to be explained by a synergistic interaction between H. pylori infection, environmental factors, and host factors.[4] Although it is plausible to believe that eradication of H. pylori infection might prevent gastric cancer, studies supporting this hypothesis remain limited. Wong et al have found that H. pylori eradication significantly decreased the development of gastric cancer only in the subgroup of H. pylori carriers without precancerous lesion.[8] Although an intervention to eradicate H. pylori at their initial stage is theoretically promising, it is constrained by its cost and effectiveness. A proton pump inhibitor (PPI) together with amoxicillin and clarithromycin (PPI/AC regimen) has been treated as the standard first-line regimen for eradication.[9] Among factors influencing the effectiveness of the PPI/AC regimen, genetic polymorphism of cytochrome P2C19 (CYP2C19), a principal enzyme for the metabolism of certain PPIs, is believed to affect therapy.[10] Because the antibiotics (AC) used in treatment regimens are acid sensitive, another polymorphism of IL-1B gene is also considered an effect modifier in the influence of CYP2C19 genotypes.[11] Resistance of H. pylori to clarithromycin is another reason of treatment failure and significantly affects the effectiveness of standard treatment.[12] Recently, levofloxacin, a laevorotatory isomer of ofloxacin, has shown an excellent activity against a variety of Gram-positive and Gram-negative organisms which are resistant to the established agents. Although a levofloxacin-based triple therapy has been shown to achieve an eradication rate of 90-92%,[13] it is uncertain whether the polymorphic genotypes of CYP2C19 and IL-1B would affect the efficacy.

Public policy strategies for the identification of patients at risk for H. pylori-related gastric malignancy are likely to be complex, but testing and treating for infection earlier than later in life is anticipated to be the more beneficial approach. Herein, we have initiated a community-based eradication of H. pylori in an area with high prevalence of gastric cancer since 2004. The aim is to observe whether this intervention could decrease the occurrence of gastric cancer. Besides, we recruited both the residents of Matzu and their migrants to Taiwan, a design trying to elucidate the environmental effect on H. pylori infection and gastric cancer consequence. Based on this ongoing trial, the primary object at present was to test the hypothesis that whether the CYP2C19 and IL-1B polymorphic genotypes and lifestyle factors would influence the prevalence and eradication rate of H. pylori. Secondly, we also determine the efficacy of a levofloxacin-based rescue treatment and evaluate the impact of genotype on the treatment failure.

MATERIALS & METHODS Study Population and Design The target population (aged 30 years of older) included 3,700 residents registered in population list in Matzu and their 2,000 emigrants to Taipei (the capital of Taiwan). We applied a two-stage community-based screening to ascertain pre-cancerous lesion and gastric cancer. The screening design was similar to our previously published work.[14] Figure 1 shows the screening regimens and attendance of the program. The details of the screening protocols were as follows.

Diagnosis of H pylori, Endoscopy, and Treatment The first stage was based on carbon-13 urea breath test (13C-UBT), venous blood sampling, and questionnaire. The information obtained from the questionnaire included demographic data, lifestyle factors, dietary habits, family history, and personal disease. Informed consents were given and ethical approval was obtained from the Ethics Committee of National Taiwan University Hospital.

The second stage was the application of endoscopy to screen whose 13C-UBT was positive, based on the causal relationship between H. pylori infection and gastric cancer.[1][2][3][8] The test had a sensitivity of 98.3% and a specificity of 98.6%, according to the manufacturer’s information.[15] Endoscopic biopsy was routinely performed at corpus and antrum to ascertain histopathological results. Patients would receive a 7-day course of esomeprazole (40mg qd), amoxicilline (500mg bid), and clarithromycin (500mg bid) after endoscopy. 13C-UBT was performed 6 weeks after the end of treatment.

Participants in whom eradication treatment failed were invited to receive an extended triple therapy, which consisted of 10-day course of esomeprazole (40mg qd), amoxicilline (500mg bid), and levofloxacin (500mg qd).[16][17] The 13C-UBT was performed again 6 weeks after the second-line treatment. Subjects with positive results regardless of treatment would receive endoscopic biopsy and culture for drug sensitivity test.

Histologic Assessment Biopsy samples were fixed in 10% buffered formaline, dehydrated, and paraffin embedded. Histologic assessment was performed by two senior histopathologists who were blinded to the treatment and any clinical information related to the patients. Biopsy specimens were graded for the following variables using the modified Sydney classification (Houston): H. pylori density, intensity of acute (polymorphonuclear) infiltrates, intensity of chronic (lymphoplasmacytic) infiltrates, gastric atrophy, intestinal metaplasia, and mucosa-associated lymphoid tissue (MALT). Histologic variables were graded as none, mild, moderate, or marked.[18] Intestinal metaplasia was recognized by the presence of globlet cells and absorptive cells by hematoxylin-eosin stain and periodic acid-Schiff Alcian blue. Gastric atrophy was defined as loss of glandular tissue and fibrous replacement of the laminar propria.

Analysis of CYP2C19 and IL-1B-511 Genotypes For analysis of genotypes, patients’ peripheral blood leukocytes were obtained and DNA was extracted from the cells. Genotyping procedures that identified CYP2C19 wild type gene and the two mutated alleles, CYP2C19m1 in exon 5 and CYP2C19m2 in exon 4, were carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The forward primer was 5’-AAT TAC AAC CAG AGC TTG GC-3’ and the reverse primer 5’-TAT CAC TTT CCA TAA AAG CAAG-3’.[19] PCR products were restricted with Sma I or Bam HI in the PCR buffer, without purification. Digested PCR products were analyzed on 4% agarose gels stained with ethidium bromide. On the basis of their ability to metabolize the PPI, individuals can be classified as extensive metabolizers (homEM: homozygous for the wild type allele), heterozygous extensive metabolizers (hetEM: carrier of only one mutant allele) or poor metabolizers (PM: two mutant alleles).[20] The fragment containing the Ava I polymorphic site at position -511 in the IL-1β gene was amplified using the oligonucleotides 5’-TGG CAT TGA TCT GGT TCA TC-3’ and 5’-GTT TAG GAA TCT TCC CAC TT-3’ as primers.[21] PCR conditions included an initial step at 95°C for 7 min, followed by 35 cycles at 95°C for 45 s, 60°C for 45 s and 72°C for 45 s, and a final elongation at 72°C for 10 min. PCR reaction product was digested with 0.5 unit of Ava I for 5 h at 37°C, which generated two fragments of 190 bp and 115 bp when the C allele was present. The fragments were also assessed by ethidium bromide staining after electrophoresis on a 2% agarose gel.

Statistic Analysis Quantitative data were summarized as mean ± standard deviation (SD) and categorical variables as percentages. Statistical differences in demographic data, lifestyle factors, H. pylori carrier status, and genotypes (IL-1B and CYP2C19) between the residents of Matzu and emigrants to Taiwan were determined with the Student t test or the 2 test. Statistical significance of parameters associated with carrier status of H. pylori in relation to the independent effects of demographic data, lifestyle factors, and genotype status (IL-1B) were assessed with logistic regression analysis.

To determine the efficacy of eradication regimens (both 1st line and 2nd line rescue treatments), intention-to-treat (ITT) and per protocol (PP) analyses were carried out. The ITT analysis included all patients who had taken at least one tablet of the drug. In the PP analysis, patients with poor drug compliance (<70% intake of any study drug) were excluded. Baseline demographic data, lifestyle factors, and eradication rates of H. pylori (ITT and PP) were compared according to their genotypic status with the Student t test or the 2 test. All p values were two-sided; a value of p < 0.05 was considered to be statistical significance.

REFERENCES

1. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991;325(16):1127-31.
2. Correa P. A human model of gastric carcinogenesis. Cancer Res. 1988;48:3554-3560.
3. You WC, Zhang L, Gail MH, Chang YS, Liu WD, Ma JL, Li JY, Jin ML, Hu YR, Yang CS, Blaser MJ, Correa P, Blot WJ, Fraumeni JF Jr, Xu GW. Gastric dysplasia and gastric cancer: Helicobacter pylori, serum vitamin C, and other risk factors. J Natl Cancer Inst. 2000;92(19):1607-12.
4. Tsugane S, Tei Y, Takahashi T, Watanabe S, Sugano K. Salty food intake and risk of Helicobacter pylori infection. Jpn J Cancer Res. 1994;85(5):474-8.
5. Serafini M, Bellocco R, Wolk A, Ekstrom AM. Total antioxidant potential of fruit and vegetables and risk of gastric cancer. Gastroenterology. 2002;123(4):985-91.
6. El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature. 2000;404(6776):398-402.
7. Hamajima N, Matsuo K, Saito T, Tajima K, Okuma K, Yamao K, Tominaga S. Interleukin 1 polymorphisms, lifestyle factors, and Helicobacter pylori infection. Jpn J Cancer Res. 2001;92(4):383-9.
8. Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, Lai KC, Hu WH, Yuen ST, Leung SY, Fong DY, Ho J, Ching CK, Chen JS; China Gastric Cancer Study Group. Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of China: a randomized controlled trial. JAMA. 2004;291(2):187-94.
9. Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report. European Helicobacter Pylori Study Group. Gut. 1997;41(1):8-13.
10. Inaba T, Mizuno M, Kawai K, Yokota K, Oguma K, Miyoshi M, Take S, Okada H, Tsuji T. Randomized open trial for comparison of proton pump inhibitors in triple therapy for Helicobacter pylori infection in relation to CYP2C19 genotype. J Gastroenterol Hepatol. 2002;17(7):748-53.
11. Take S, Mizuno M, Ishiki K, Nagahara Y, Yoshida T, Inaba T, Yamamoto K, Okada H, Yokota K, Oguma K, Shiratori Y. Interleukin-1beta genetic polymorphism influences the effect of cytochrome P 2C19 genotype on the cure rate of 1-week triple therapy for Helicobacter pylori infection. Am J Gastroenterol. 2003;98(11):2403-8.
12. Ducons JA, Santolaria S, Guirao R, Ferrero M, Montoro M, Gomollon F. Impact of clarithromycin resistance on the effectiveness of a regimen for Helicobacter pylori: a prospective study of 1-week lansoprazole, amoxycillin and clarithromycin in active peptic ulcer. Aliment Pharmacol Ther. 1999 ;13(6):775-80.
13. Cammarota G, Cianci R, Cannizzaro O, Cuoco L, Pirozzi G, Gasbarrini A, Armuzzi A, Zocco MA, Santarelli L, Arancio F, Gasbarrini G. Efficacy of two one-week rabeprazole/levofloxacin-based triple therapies for Helicobacter pylori infection. Aliment Pharmacol Ther. 2000;14(10):1339-43.
14. Chen TH, Chiu YH, Luh DL, Yen MF, Wu HM, Chen LS, Tung TH, Huang CC, Chan CC, Shiu MN, Yeh YP, Liou HH, Liao CS, Lai HC, Chiang CP, Peng HL, Tseng CD, Yen MS, Hsu WC, Chen CH; Taiwan Community-Based Integrated Screening Group. Community-based multiple screening model: design, implementation, and analysis of 42,387 participants. Cancer. 2004;100(8):1734-43.
15. Savarino V, Mela GS, Zentilin P, Bisso G, Pivari M, Mansi C, Mele MR, Bilardi C, Vigneri S, Celle G. Comparison of isotope ratio mass spectrometry and nondispersive isotope-selective infrared spectroscopy for 13C-urea breath test. Am J Gastroenterol. 1999;94(5):1203-8.
16. Bilardi C, Dulbecco P, Zentilin P, Reglioni S, Iiritano E, Parodi A, Accornero L, Savarino E, Mansi C, Mamone M, Vigneri S, Savarino V. A 10-day levofloxacin-based therapy in patients with resistant Helicobacter pylori infection: a controlled trial. Clin Gastroenterol Hepatol. 2004;2(11):997-1002.
17. Nista EC, Candelli M, Cremonini F, Cazzato IA, Di Caro S, Gabrielli M, Santarelli L, Zocco MA, Ojetti V, Carloni E, Cammarota G, Gasbarrini G, Gasbarrini A. Levofloxacin-based triple therapy vs. quadruple therapy in second-line Helicobacter pylori treatment: a randomized trial. Aliment Pharmacol Ther. 2003;18(6):627-33.
18. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20(10):1161-81.
19. de Morais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J Biol Chem. 1994;269(22):15419-22.
20. Furuta T, Shirai N, Takashima M, Xiao F, Hanai H, Sugimura H, Ohashi K, Ishizaki T, Kaneko E. Effect of genotypic differences in CYP2C19 on cure rates for Helicobacter pylori infection by triple therapy with a proton pump inhibitor, amoxicillin, and clarithromycin. Clin Pharmacol Ther. 2001;69(3):158-68.
21. Garcia-Gonzalez MA, Lanas A, Savelkoul PH, et al. Association of interleukin 1 gene family polymorphisms with duodenal ulcer disease. Clin Exp Immunol 2003;134:525-531.

Ages Eligible for Study:  30 Years and above,  Genders Eligible for Study:  Both

Accepts Healthy Volunteers

Criteria

Inclusion Criteria:

• Helicobacter pylori infection subjects

Exclusion Criteria:

• Prior gastrectomy

Please refer to this study by ClinicalTrials.gov identifier  NCT00155389

Pan-Chyr Yang, PHD      886-2-2356-2000    pcyang@ha.mc.ntu.edu.tw

Taiwan
      National Taiwan University Hospital, Taiwan, Taipei,  100,  Taiwan; Recruiting

Study chairs or principal investigators

Pan-Chyr Yang, PHD,  Study Chair,  National Taiwan University Hospital   

Study ID Numbers:  940110
Last Updated:  September 9, 2005
Record first received:  September 8, 2005
ClinicalTrials.gov Identifier:  NCT00155389
Health Authority: Taiwan: Department of Health
ClinicalTrials.gov processed this record on 2005-09-13