The initial study included over 3399 people from Linqu County, a rural area of Shandong Province, China, which has one of the highest GC mortality rates in the world (70/10 000 males and 25/10 000 females per year)[21]. In brief, we launched an endoscopic-pathological screening program for GC and precancerous lesions of GC in 3399 residents from 14 randomly selected villages of Linqu County in autumn of 1989 or in spring of 1990. In 1994, a follow-up endoscopic screening was performed in 83% of eligible members. People, diagnosed as normal or superficial gastritis in 1989 or in 1990, were subsequently genotyped for IL-8 and MIF. We analyzed the relation between genotypes and progression of gastric disease. The study design received approval from the Institutional Review Board of Peking University School of Oncology, and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all participants.

Seven biopsies were taken from standard locations in each subject. The procedures and histopathologic criteria have been described elsewhere[22,23]. We reviewed all slides of 372 patients in our study according to the updated Sydney system[24]. Each subject was assigned a global diagnosis based on the most severe diagnosis among the seven biopsies. Independent examinations were performed by three experienced gastroenterologists. If there was any disagreement, the final decision was made based on the majority or subsequent discussion of the case.

After the first examination in 1989, 372 of the 3399 subjects including 172 males and 200 females with a mean age of 42.2 years (range: 24-65 years) were enrolled in this study. All these 372 subjects were initially diagnosed as normal or as superficial gastritis (baseline). After a 56-mo follow-up, theses 372 subjects were subdivided into group I (n = 134), group II (n = 101) and no progression group (n = 137). Lesions in patients of group I were progressed from baseline to severe chronic atrophic gastritis (SCAG). According to the updated Sydney system, recognition of minor degrees of atrophy without intestinal metaplasia in the antrum was difficult, marked degrees of atrophy without intestinal metaplasia in the antrum were selected as the presence of atrophic gastritis. Lesions in patients of group II were progressed from baseline to IM, and lesions in patients of no progression group did no progress from baseline lesion.

In 1994, blood samples were collected and allowed to clot for 30-40 min at room temperature in the dark. Serum was harvested, the clot was frozen immediately at -20°C, and stored at -70°C for 2 or 3 d. During the transfer, dry ice was used.

The detailed serologic assay has been described elsewhere[22]. Serum level of H. pylori-specific IgG and IgA in all samples was measured by enzyme-linked immunosorbent assay (ELISA). Quality-control samples were assayed at Vanderbilt University (Nashville, TN). An optical density ratio (ODR) value > 1.0 and < 1.0 was considered seropositive and negative, respectively.

The presence of H. pylori was further confirmed by immunohistochemistry (IHC). Briefly, paraffin-embedded tissue sections were stained using IHC with an avidin-biotin complex immunoperoxidase kit. Polyclonal rabbit anti-H. pylori (ab7788, Abcam, Cambridge, UK) was used as a primary antibody as previously described[25].

Blood clots were thoroughly washed with Tris-EDTA (TE) buffer containing 50 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA. After centrifugation, pellets were incubated with rotation in a lysis buffer (TE buffer containing 2 g/L SDS and 200 μg/mL proteinase K) overnight at 37°C. Lysate was then extracted with phenol and precipitated with isopropanol. The precipitate was washed with 70% ethanol, dried, and dissolved in TE buffer. Concentration and purity of DNA were determined by spectrophotometry at A_{260 nm} and A_{280 nm}. Then, DNA was aliquoted and stored at -80°C until use.

Polymerase chain reaction (PCR) was performed in a 25 μL reaction mixture containing 100ng of DNA, 0.1 μmol/L of each primer, 0.2 mmol/L of deoxynucleosidetriphosphate, 1.0 U of Taq DNA polymerase (Promega, Madison, WI), and 1 × reaction buffer. The primer sequences of MIF-173, IL-8-251 have been described elsewhere[26]. PCR-based denaturing high-performance liquid chromatography (DHPLC) and single nucleotide polymorphisms (SNP) were further confirmed by direct sequencing.

DHPLC analysis was performed on a transgenomic WAVE system (Transgenomic Inc., Omaha, NE). The detailed genotyping process has been described elsewhere[27]. In brief, PCR products were denatured for 1 min at 94°C and then gradually re-annealed by decreasing the sample temperature from 94°C to 45°C for 30 min to form homo- and/or hetero-duplexes. The PCR products were then applied to the DHPLC column at an optimal oven temperature and eluted with a linear acetonitrile gradient at a flow rate of 0.9 mL/min. The results of DHPLC were further confirmed by DNA sequencing with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA) (Figure 1).

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Figure 1 Denaturing high-performance liquid chromatography (DHPLC) and sequence analysis. A: Interleukin-8 (IL-8)-251 A/T polymorphism; B: Macrophage migration inhibitory factor (MIF)-173 C/G polymorphism.

Data were analyzed using the SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Multiple linear regression analyses were performed with gender, age, smoking, and drinking as explanatory variables to determine which factors influence the progression of gastric lesions. Genotypes of IL-8-251 and MIF-173 loci were also included as explanatory variables when difference in groups I and II and no progression group was detected. Odds ratios (OR) with 95% confidence interval (CI) were computed. P < 0.05 was considered statistically significant.

Multivariate analysis showed that the frequencies of IL-8-251 in groups I and II were significantly different from those in no progression group (Table 2). Compared with IL-8-251 TT genotype, IL-8-251 AA genotype and IL-8-251 A allele carriers exhibited a significantly increased risk for progression from baseline lesions to SCAG (Table 3). The patients with IL-8-251 TA genotype or IL-8-251 A allele carriers had an increased risk for progression from baseline lesions to IM.

Multivariate analysis showed that the MIF-173 GC genotype or MIF-173 C allele carriers were significantly associated with an increased risk for progression from baseline lesions to SCAG and IM (Table 3).

The risk for SCAG in association with IL-8-251 and MIF-173 genotypes was further examined with stratification by H. pylori infection. The OR for development of SCAG in subjects carrying IL-8-251 A allele or with H. pylori infection alone was 2.34 (95% CI: 0.95-2.83) or 3.28 (95% CI: 1.09-9.78), respectively. However, the OR was significantly elevated in subjects carrying the AA genotype and with H. pylori infection (OR = 6.70, 95% CI: 1.29-9.78) (Table 4). There was an interaction between IL-8-251 A allele carriers and H. pylori infection, with a relative risk for development of SCAG due to the interaction of 6.70, and a synergy index of 1.57.

A similar trend to develop SCAG was observed between the MIF-173 C allele carriers and H. pylori infection. The OR of developing SCAG significantly increased in subjects carrying at least one MIF-173 C allele and with H. pylori infection (OR = 6.54, 95% CI: 2.97-14.20) (Table 4). An interaction between the MIF-173 C allele carriers and H. pylori infection was observed (OR = 2.26, synergy index = 3.15).

The association of IM and IL-8-251 with MIF-173 genotypes was further examined with stratification by H. pylori infection. However, the OR for IM in subjects carrying MIF-173 C allele and with H. pylori infection was elevated significantly (OR = 2.93, 95% CI: 1.28-6.60) (Table 5). There was also an interaction between the MIF-173 C allele carriers and H. pylori infection (OR = 2.20, synergy index = 1.25).

Atrophic gastritis and intestinal metaplasia are two important precursory lesions of intestinal type gastric cancer (GC), except for factors of Helicobacter pylori (H. pylori). Host gene polymorphisms also play a very important role in the development of GC.

To date, a few studies are available on the relation between gene polymorphisms of inflammatory cytokines and risk of atrophy and intestinal metaplasia (IM).

In this study, the authors conducted a prospective study using the data obtained during the 56-mo follow-up (including both gastroscopic and histopathological information). The relation between interleukin-8 (IL-8), migration inhibitory factor (MIF) gene polymorphisms and risk of atrophic gastritis and IM was also studied.

Because of the high prevalence of H. pylori infection, antibiotic resistance, and some potential drawbacks in H. pylori eradication therapy (e.g. reflux esophagitis), the result of this study may provide some new evidence for the selection of patients infected with H. pylori and for the prevention of progression of H. pylori-related gastritis.

IL-8, a member of Cys-X-Cys (CXC) chemokine family, is an activator and chemoattractant of neutrophils and lymphocytes, thus playing an important role in the development of gastritis and GC. Macrophage MIF, an important activator of T lymphocytes and macrophages, plays a pivotal role in inflammatory and immune diseases. The expression of MIF is increased after H. pylori infection and related with histological severity of GC and its precursor.

The authors studied the role of different IL-8 and MIF genotypes in the development of precancerous gastric lesions, severe atrophic gastritis or intestinal metaplasia, in a cohort of 372 patients. The study defined H. pylori-infected patients at the risk for GC.