The Genetic and Epigenetic Bases of Gastritis

Gastritis, the inflammation of the mucosal layer of the stomach, is a major clinical entity due to its association with gastric cancer and peptic ulcer disease. The primary cause of gastritis is the infection with the microaerofilic gram negative Helicobacter pylori that during the early phases elicits an acute inflammatory response which eventually evolves to a longstanding chronic gastritis (Ruggiero 2012). In the case of gastric cancer development, chronic gastritis is the first step of the so-called multistep cascade of gastric cancer. This sequence includes the non-atrophic chronic gastritis, multifocal atrophic gastritis, intestinal metapla‐ sia, dysplasia and invasive carcinoma as were described in detail by Correa as the “human model of gastric carcinogenesis” (Fig. 1) (Correa et al. 2007). This multistep model hypothe‐ sizes that the sequence of these lesions reflects a dynamic process from a naive inflammation caused by H. pylori infection to a fully malignant neoplasm of the stomach (Correa et al. 1976; Cuello et al. 1976; Haenszel et al. 1976; Correa et al. 1990). Independent epidemiologi‐ cal studies have confirmed that non-atrophic, atrophic, intestinal metaplasia and dysplasia are all linked through a sequential cause-effect relationship, thus supporting the concept of a human model for gastric carcinogenesis (Ohata et al. 2004). However, the risk of malignant transformation of these lesions is poorly defined. Long-term follow-up studies have shown a risk from 10% to 17% in the case of dysplasia (Saraga et al. 1987; Coma del Corral et al. 1990; Koch et al. 1990; Whiting et al. 2002; Rugge et al. 2003). For intestinal metaplasia, the risk assessment has conflicting results and therefore a limited clinical value (Ramesar et al. 1987; Silva et al. 1990; Rokkas et al. 1991; Conchillo et al. 2001; Vannella et al. 2012). The re‐ cently developed Operative Link for Gastritis Assessment (OLGA) staging system (Rugge et al. 2007), through the evaluation of the extension and site of the atrophic changes, is an at‐ tempt to evaluate the risk of chronic gastritis to progress to intestinal metaplasia and gastric cancer (Rugge et al. 2008; Capelle et al. 2010; Rugge et al. 2010). In this scenario, the identifi‐


Introduction
Gastritis, the inflammation of the mucosal layer of the stomach, is a major clinical entity due to its association with gastric cancer and peptic ulcer disease. The primary cause of gastritis is the infection with the microaerofilic gram negative Helicobacter pylori that during the early phases elicits an acute inflammatory response which eventually evolves to a longstanding chronic gastritis (Ruggiero 2012). In the case of gastric cancer development, chronic gastritis is the first step of the so-called multistep cascade of gastric cancer. This sequence includes the non-atrophic chronic gastritis, multifocal atrophic gastritis, intestinal metaplasia, dysplasia and invasive carcinoma as were described in detail by Correa as the "human model of gastric carcinogenesis" (Fig. 1) (Correa et al. 2007). This multistep model hypothesizes that the sequence of these lesions reflects a dynamic process from a naive inflammation caused by H. pylori infection to a fully malignant neoplasm of the stomach ; Cuello et al. 1976;Haenszel et al. 1976; Correa et al. 1990). Independent epidemiological studies have confirmed that non-atrophic, atrophic, intestinal metaplasia and dysplasia are all linked through a sequential cause-effect relationship, thus supporting the concept of a human model for gastric carcinogenesis (Ohata et al. 2004). However, the risk of malignant transformation of these lesions is poorly defined. Long-term follow-up studies have shown a risk from 10% to 17% in the case of dysplasia (Saraga et al. 1987;Coma del Corral et al. 1990; Koch et al. 1990; Whiting et al. 2002;Rugge et al. 2003). For intestinal metaplasia, the risk assessment has conflicting results and therefore a limited clinical value (Ramesar et al. 1987; Silva et al. 1990; Rokkas et al. 1991;Conchillo et al. 2001;Vannella et al. 2012). The recently developed Operative Link for Gastritis Assessment (OLGA) staging system (Rugge et al. 2007), through the evaluation of the extension and site of the atrophic changes, is an attempt to evaluate the risk of chronic gastritis to progress to intestinal metaplasia and gastric cancer (Rugge et al. 2008;Capelle et al. 2010;Rugge et al. 2010). In this scenario, the identifi-cation of molecular bases in the multistep process of gastric carcinogenesis is highly relevant since it will contribute greatly to the risk assessments of the precursor lesions of gastric cancer. In this chapter we will primarily attempt to summarize and integrate our current knowledge of the genetic as well as epigenetic bases of the dynamic process of chronic gastritis, as well as the other entities of the so-called multistep cascade of gastric cancer.

The genetic bases of gastritis
Human allelic variations at single nucleotide polymorphisms (SNP) are involved at different stages of gastric carcinogenesis. Accordingly, the dynamics of chronic gastritis might be associated with specific allelic variants. These variants, recognized as polymorphisms when occur with a frequency of >1% in the normal population, affects mostly genes of the inflammatory response genes, detoxification enzymes, and cancer-related procesess (Gonzalez et al. 2002).

Polymorphisms in inflammatory response genes
Two clear examples of polymorphisms associated with inflammatory response are interleukin-1 gene cluster and Toll-like receptors (TLRs). As shown in Table 1, Interleukin-1-beta (IL-1B) -31 T genotype and interleukin-1-receptor antagonist (IL1RN) IVS 86 bp VNTR, 2/2 genotype enhance the production of IL-1B, which is associated with an increased risk of hypochlorhydria induced by H. pylori. Because IL-1B is an important pro-inflammatory cytokine and a powerful inhibitor of gastric acid secretion, it might explain the higher risk of development of gastric cancer in chronic gastritis -H. pylori infected patients (El-Omar et al. 2000; Hwang et al. 2002;Yuzhalin 2011). The presence of polymorphisms in the non-coding but regulatory regions may also alter the levels of gene transcription and therefore the susceptibility to gastric cancer. In this sense, single A/T SNP at position -251 from the transcription start site in the promoter region of the IL-8 gene is one of the best examples. The presence of the -251A allele tended to be associated with an increase in IL-8 production and has been reported that this allele is associated with an increased risk for gastric cancer at cardia location (Hull et al. 2000;Xue et al. 2012). It could be extrapolated that IL-8 -251A allele may increase the risk of developing cancer through the elevation of its IL-8 expression. Mediators of the innate immune response, which provide first line of host defense against harmful pathogens are also involved in the dynamics of chronic gastritis. A good example for this type of inflammatory response are the Toll-like receptors (TLRs) (El-Omar et al. 2008). It has been reported that both, the 22-bp nucleotide deletion (-196 to -174 del) in the promoter region of the TLR-2 gene and the +896A/G and +1196C/T polymorphisms (Asp299Gly and Thr399Ile) in the coding region of the TLR-4 gene, promotes a rapidly progressive chronic gastritis (de Oliveira et al. 2012). Since it is well known that not all H. pylori are equally associated with the risk of development of gastric cancer, the combination of bacterial and inflammatory host genotypes have been explored. Figueiredo et al (2002) have shown that the infection with aggressive cagA-positive strains of H. pylori were associated with an increased risk of gastric cancer. In addition, the homozygous IL-1B-511*T carriers (IL-1B-511*T/*T or IL-1B-511*T/*C) and the short allele of IL-1RN (IL-1RN*2/*2) also had an increased susceptibility. However, for each combination of bacterial/host genotype, the odds of having gastric cancer were greatest in those with both bacterial and host high-risk genotypes.

Polymorphisms in genes associated with detoxification enzymes
Members of the cytochrome P-450 superfamily belong to the detoxification enzymes. One of the members of this family is the CYP2E1, a naturally ethanol-inducible enzyme involved in the metabolic activation of low molecular weight compounds such as N-nitrosamines (Boccia et al. 2007). Functional CYP2E1 polymorphisms in the 50-flanking region (PstI, RsaI) alter the transcriptional activity of the gene. A meta-analysis by Boccia et al (2007) suggests that the CYP2E1 PstI/RsaI polymorphism may be a risk factor for gastric cancer in asiatic populations and a synergic relation with tobacco-related detoxification Glutathione S-transferase (GST) genes (see below) may account for a proportion of these cases. GSTs and the polymorphic arylamine N-acetyltransferases (NAT1 and NAT2) are another type of detoxification enzymes that metabolize tobacco-related carcinogens. Zendehdel et al (2009) reported a weak linkage between the GSTP1 Ile105Val polymorphism and the risk for gastric cancer at cardia location (OR = 1.4; 95% CI 0.9-2.1). On the other hand, Katoh et al (2000) found that the NAT1*10 allele should be considered a risk factor among heavy smokers with well-differentiated tumors ( Table 1).

Polymorphisms of cancer-related genes
In cancer-related processes, polymorphisms have been reported in the promoter region of the MET gene, a crucial gene to multiple oncogenic pathways and metastatic behavior (Gherardi et al. 2012). Sequencing of the promoter region of MET revealed some alterations scattered in a proportion of clinical samples of gastric cancer. The most common substitutions were -304C>A and 206C>G. The presence of these SNPs altered the junction sites for putative transcription factors such as Sp1 and AP-1/AP-2. As a consequence, the transcription of MET was constantly being activated (Trzyna et al. 2012). Another example is the polymorphism -160 A at the promoter region of the E-cadherin gene (CDH1), which has been associated with the DNA hypermethylation (see below) of the promoter region (Borges Bdo et al. 2010). In a Japanase study, 117 cases of gastric cancer with H. pylori-induced chronic gastritis were compared with 116 cancer-free yet H. pylori-induced chronic gastritis controls. It was found that the Pro/Pro allele of CDH-1 was associated with an increased risk of developing diffuse-type of gastric cancer compared to the Arg/Arg (Hiyama et al. 2002;Zhou et al. 2007). Finally, other polymorphims of cancer-related genes were reported to show significant associations with gastric cancer risk including EGFR, VEGF and p53 (Tahara et al. 2009). Taken together, data presented here suggest that allelic variations at regulatory or coding regions will affect gene expression patterns and modify the balance towards a more rapid progression of chronic gastritis and other preneoplastic lesions of the multistep cascade of gastric cancer (Fig. 2).   Table 1. Selected single nucleotide polymorphisms (SNP) in inflammatory response genes, detoxification enzymes, and cancer-related processes associated to increase susceptibility of gastric cancer. The sign (-) indicates that the SNP is located in the direction of the promoter region, the sign (+) indicates that the SNP is located in the direction of the coding region, the number indicates the nucleotide positionfrom TSS and the first nucleotide is substituted (/) by the second.  Leung et al. (2006), although they evaluated mucosal biopsies from the antrum and corpus of H. pyloriinfected subjects at the baseline, and after one year of successful H. pylori eradication. In addition, these authors identified a significant reduction in the methylated density of the promoter region of the E-cadherin gene (Leung et al. 2006 (Fig. 4). However, the most interesting finding come after our attempt to integrated protein expression and histopathological assessments. This integration was performed by Significance Analysis of Microarrays (SAM), a multiple testing approach method that has been extensively applied in genomic research (Tusher et al. 2001) and confirmed by logistic regression (Nick et al. 2007). This integrative approach led us to identified that overexpression of p73 was even more significant than severe atrophy and OLGA stage IV in identifying high-risk premalignant gastritis (Carrasco et al. 2010). Therefore, we believe that the identification of the overexpression of p73 might contribute greatly to risk assessment of chronic gastritis to the development of gastric cancer.

Conclusion
Data presented here suggests that genetic, epigenetic by means of DNA methylation might play a role in the dinamics of the progression of chronic gastritis and premalignant cascade to gastric cancer. DNA methylation works in both ways, inactivating or activating tumorrelated genes through the hypermethylation or hypomethylation of the promoter regions of specific genes. The integration of these molecular bases of chronic gastritis with histolopathological assessment by Sydney and OLGA systems will contribute to the better risk assessment for the development of gastric cancer.