The p53 gene is the most frequently inactivated tumour suppressor gene identified in human cancers to date (Han et al, 2002). Over 15000 p53 mutations have been documented from tumour and cell line samples (www.iarc.fr), with loss of p53 function most commonly induced through point mutation (Bates and Vousden, 1996). More than 30% of p53 mutations occur at methylated CpG sites in codons 157, 175, 245, 248, 273 and 282 (Chen et al, 1998). Thus, CpG sites frequently act as mutational hot spots, which warrant their investigation to determine their potential role in tumour aetiology. p53 gene mutations appear to be key factors in the development of gastric cancer (Gomyo et al, 1996) having been documented in more than 60% of the reported cancer cases (Tamura et al, 1991). Classically, gastric cancer can be divided into two histological subtypes: diffuse or intestinal (Lauren, 1965). The intestinal type is believed to arise from a sequence of gastritis, intestinal metaplasia and increasing grades of dysplasia. There is controversy regarding the stage at which genetic alterations of the p53 gene occur within the metaplasia–dysplasia sequence (Uchino et al, 1993; Becker et al, 2000). p53 mutations detected in gastritis have been documented by Stemmermann et al (1994) and Kodama et al (1998), while studies by Zheng and YouYoung (1998), Shiao et al (1994) and Ochiai et al (1996) show p53 alterations only in intestinal metaplasia. Mutations of p53 in early gastric cancer have been found by Tohodo et al (1993) and Uchino et al (1993), while Romitti et al (1998), Brito et al (1994) and Joypaul et al (1993) claim that p53 mutations are late events in gastric cancer. However, p53 alterations in these studies have been detected using a variety of methods such as the polymerase chain reaction (PCR), PCR–single-strand conformation polymorphism, direct sequencing and immunohistochemistry. Thus, it appears that the frequency and stage at which p53 alterations are detected may depend on the methods used to detect them (Stemmermann et al, 1994). To investigate the timing of p53 mutations in gastric cancer, we employed the restriction site mutation (RSM) assay (Myers and Parry, 1994). This method detects mutations at restriction enzyme sites in human genes. Fortuitously, five of the eight main p53 mutational hot spots (codons 175, 213, 248, 249 and 282) contain restriction sites and are amenable to RSM analysis in patients with gastritis and intestinal metaplasia. Exhaustive digestion of the target DNA followed by amplification of the enzyme-resistant (mutated) sequences by PCR means that RSM is capable of detecting low levels of mutations (one mutated sequence among 104–105 wild-type sequences) (Jenkins et al, 1999). This, in turn, allows for the molecular selection of mutated samples and makes mutation detection in premalignant samples feasible.
As the bacterium Helicobacter pylori (H. pylori) has been implicated in gastric cancer progression through its induction of inflammatory-mediated reactive oxygen species (ROS), we assessed the H. pylori status of the patients using histology and PCR-based analysis. In addition, the level of ROS present in tissues representative of the different stages of the metaplasia–dysplasia sequence was directly measured in a separate cohort of gastric biopsies using electron spin resonance (ESR) spectroscopy. Electron spin resonance spectroscopy is currently the most sensitive, specific and direct method of measuring free radicals in tissue and body fluids (Ashton et al, 1999; Berliner et al, 2001).