Patients with GC were enrolled in the course of a different study that was approved by the local ethic committee. Written informed consent was obtained. The available material was re-evaluated for the presence of H. pylori infection, including CagA status, and histopathological mucosal alterations. The groups were analyzed according to their histopathological type of carcinoma (i.e. intestinal vs. diffuse type by Laurén) and the location of the primary tumor. Serum samples were assessed for the content of gastrin 17 (G17), pepsinogen 1 (PG1) and pepsinogen 2 (PG2). These analyses have been performed in concordance with the guidelines of the local ethics committee.
Patients' data was verified using the electronic patient documentation system of the Department of Gastroenterology, Hepatology and Infectious Diseases at the University Hospital of Magdeburg, Germany. Patients were included in case of complete records and availability of adequately stored serum samples for serological analysis (-80°C). In total, 118 patients met these criteria.
Only patients with either intestinal (n = 59) or diffuse type tumors (n = 59) according to the Laurén classification were included for further analysis, mixed type tumors were excluded (n = 12). Exclusion of mixed type tumors was done to analyse only clearly distinct tumor entities. Since "Laurén type" was considered as a potential confounding factor for the serological tests, we aimed for a strict comparison between intestinal and diffuse type gastric cancer.
Tumors were classified according to the location of the main tumor mass into carcinomas of the antrum, the corpus and the cardia. Cardia tumors were further subclassified according to the AEG-classification as proposed by Siewert and Stein in 1998 (AEG = adenocarcinoma of the esophagogastric junction) [19
]. Supracardial carcinomas (AEG1) with a high likelihood of the distal esophagus being the site of origin have been excluded, as well as AEG2 with adjacent Barrett's mucosa. Tumors have been stratified into "proximal" and "distal" carcinomas of the stomach according to the location of the main tumor mass as described previously [7
]. Tumors in the region of the esophagogastric junction were clearly classified as "proximal" and antrum-tumors as "distal". The group of corpus-carcinomas was subdivided. Carcinomas with the main tumor mass in the fundus and in the proximal third of the corpus were classified as "proximal", all more distally located corpus carcinomas (lower two thirds) as "distal".
Tumor allocation was assessed either by the primary endoscopy report including endoscopic ultrasound or by the pathological report of the resected specimen in case of gastrectomy. This classification of tumor allocation was chosen by convention of the authors. Tumor allocation was stratified prospectively to any statistical analysis.
Histopathological alterations of the gastric mucosa (IM and glandular atrophy) were assessed according to the updated Sydney system [20
Biopsies had been taken in duplicate from the pre-pyloric antrum and the corpus (lesser and greater curvature). Cardia-derived samples were obtained directly below the Z-Line at the proximal end of the gastric folds. Additional samples were taken from the tumor itself and in some cases from the surrounding mucosa.
Biopsies were processed by routine methods. One section was stained with hematoxilin and eosin, modified Giemsa for detection of H. pylori, and PAS stain.
In patients that were referred to our hospital for gastrectomy with no further endoscopic examination being performed, histology was assessed on the surgical specimen.
Assessment of serum parameters
Serum was prepared from 5-7 ml venous blood by centrifugation at 7.000x g at 4°C for 15 min, aliquoted in three individual cryotubes (each 1-1.5 ml) within 3 hours after taking blood. Samples were stored at -80°C until analysis. The concentration of anti-H. pylori IgG antibodies were analyzed using the Pyloriset EIA-G III (Orion Diagnostica, Finland) according to the manufacturer's instructions. According to the validation of the kit, a positive result was defined as ≥ 30 EIU, a negative result as < 30 EIU. The prevalence of anti-CagA antibodies was investigated using undiluted sera and the Helicobacter pylori Vira blot test kit (Viramed Biotech AG, Lich, Germany) according to the instructions by the manufacturer. Patients with positive CagA-status were classified as "H. pylori positive" even when anti-H. pylori IgG was below the cut-off level also including patients with "a serological scar". It has to be mentioned that by this definition not only patients with actual infection were classified as H. pylori-positive, but also patients with prior eradication therapy or those in which bacteria have disappeared during the progression of histological alterations. Since the so-called "point of no return" from which on eradication cannot prevent further progression of premalignant conditions is not defined yet, we believe that inclusion of all patients with a past history of H. pylori infection is justified.
The analyses of anti-H. pylori IgG antibodies and PG1, PG2 and G17 (Gastrin-17 EIA Test Kit, Pepsinogen-I, Pepsinogen-II EIA Test Kit, Biohit Plc, Finland) was performed at the same aliquot and as described by the manufacturer.
Group comparison was performed using Fisher's exact test, correlation analysis by Spearman's rank correlation test. The t-test for independent samples was applied to assess the influence of age as a confounding factor and to compare the degree of mucosal alterations between groups. Group comparison concerning the serum values of G17, PG1 and PG2, as well as the PG-ratio have been performed by the Mann-Whitney U-test as well as univariate ANOVA for interference analysis. For all tests, a two-sided significance level of P < 0.05 was assumed. Data were analyzed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA).