This study verified that vacAs1m1 and cag-PAI genes, cagA, cagE and virB11 are important H. pylori markers for gastric cancer development. Also, this study corroborates the importance of cagE and cagA together as cag-PAI marker.

Objective: We aimed to evaluate the inactivation of COX-2, HMLH1 and CDKN2A by promoter methylation and its relationship with the infection by different Helicobacter pylori strains in gastric cancer. Methods: DNA extracted from 76 H. pylori-positive gastric tumor samples was available for promoter methylation identification by methylation-specific PCR and H. pylori subtyping by PCR. Immunohistochemistry was used to determine COX-2, p16^INK4A and HMLH1 expression. Results: A strong negative correlation was found between the expression of these markers and the presence of promoter methylation in their genes. Among cardia tumors, negativity of p16^INK4A was a significant finding. On the other hand, in noncardia tumors, the histological subtypes had different gene expression patterns. In the intestinal subtype, a significant finding was HMLH1 inactivation by methylation, while in the diffuse subtype, CDKN2A inactivation by methylation was the significant finding. Tumors with methylated COX-2 and HMLH1 genes were associated with H. pylori vacA s1 (p = 0.025 and 0.047, respectively), and the nonmethylated tumors were associated with the presence of the gene flaA. Conclusions: These data suggest that the inactivation of these genes by methylation occurs by distinct pathways according to the histological subtype and tumor location and depends on the H. pylori genotype.

Promoter hypermethylation of CDKN2A (p16INK4A protein) is the main mechanism of gene inactivation. However, its association with Helicobacter pylori infection is a controversial issue. Therefore, we examined a series of gastric adenocarcinomas to assess the association between p16INK4A inactivation and H. pylori genotype (vacA, cagA, cagE, virB11 and flaA) according to the location and histological subtype of the tumors. p16INK4A expression and CDKN2A promoter methylation were found in 77 gastric adenocarcinoma samples by immunohistochemistry and methylation-specific PCR, respectively. Helicobacter pylori infection and genotype were determined by PCR. A strong negative correlation between immunostaining and CDKN2A promoter region methylation was found. In diffuse subtype tumors, the inactivation of p16INK4A by promoter methylation was unique in noncardia tumors (p=0.022). In addition, H. pylori-bearing flaA was associated with non-methylation tumors (p=0.008) and H. pylori strain bearing cagA or vacAs1m1 genes but without flaA was associated with methylated tumors (p=0.022 and 0.003, respectively). Inactivation of p16INK4A in intestinal and diffuse subtypes showed distinct carcinogenic pathways, depending on the tumor location. Moreover, the process of methylation of the CDKN2A promoter seems to depend on the H. pylori genotype. The present data suggest that there is a differential influence and relevance of H. pylori genotype in gastric cancer development.

DNA methylation in gastric cancer seems to be influenced by the presence of interleukin polymorphisms and by the H. pylori cagA/vacAs1m1 strains.

TP53 mutations and polymorphisms have been widely related to many cancers as long as these alterations may impair its capacity to induce cell cycle arrest, DNA repair mechanisms, and apoptosis. Although TP53 alterations have been studied in astrocytic tumors, there is a lack of analysis considering specific TP53 mutations and their associations with p53 immunostainning, polymorphisms and their significance among the histological grades. Thus, we analyzed TP53 alterations in exons 2-11, including the codon 72 polymorphism, using DNA sequencing in 96 astrocytic gliomas (18 grade I, 20 grade II, 14 grade III, and 44 grade IV). Also, immunohistochemistry was assessed to evaluate the p53 protein expression. In this study, we found that the higher histological grades were statistically associated with TP53 mutations. Some of these mutations, such as TP53 P98T and TP53 G244S, seemed to be a specific marker for the higher grades, and the TP53 E286K mutation appears to be a World Health Organization grade III-IV progression marker. Also, the TP53 P98T mutation, in exon 4, is very likely to be important on the stabilization of the p53 protein, leading to its immunopositivity and it is potentially associated with the TP53 72Pro/Pro genotype.

CDKN2A promoter hypermethylation has been widely related to many cancers. In astrocytomas, although CDKN2A (p16(INK4A) protein) is often inactivated, there are still some controversial issues regarding the mechanism by which this alteration occurs. Thus, we analyzed a series of astrocytomas to assess the association between CDKN2A expression and methylation of grade I-IV tumors (WHO) and clinicopathological parameters. DNA extracted from formalin-fixed paraffin-embedded material of 93 astrocytic tumors was available for CDKN2A promoter methylation analysis and p16(INK4A) expression by methylation-specific PCR and immunohistochemistry, respectively. A strong negative correlation between nuclear and cytoplasmic immunostaining and CDKN2A promoter methylation was found. Additionally, a significant negative correlation between CDKN2A promoter methylation and age was observed; also, female patients had statistically more CDKN2A methylated promoters (p = 0.036) than men. In conclusion, CDKN2A inactivation by promoter methylation is a frequent event in astrocytomas and it is related to the age and sex of patients.

MTHFR C677T and Helicobacter pylori infection are believed to play critical roles in the DNA methylation process, an epigenetic feature frequently found in gastric cancer. The aim of this study was to verify the associations between the MTHFR C677T polymorphism and the methylation status of three gastric cancer-related genes. The influence of H. pylori strains was also assessed. DNA extracted from 71 gastric tumor samples was available for MTHFR C677T genotyping by PCR-RFLP, promoter methylation identification by MS-PCR and H. pylori detection and posterior subtyping (cagA and vacA genes) by PCR. In the distal tumors, a positive correlation was found between the methylation of CDKN2A and the allele T carriers (r=0.357; p=0.009). Considering the eldest patients (age ≥60 years old), this correlation was even higher (r=0,417; p=0.014). H. pylori infection by highly pathogenic strains (cagA+/vacAs1m1) was also found correlated to promoter methylation of CDKN2A and the allele T carriers in distal tumors (r=0.484; p=0.026). No significant correlation was verified between MTHFR C677T genotype and promoter methylation status when we analyzed the general sample. DNA methylation in CDKN2A associated to the MTHFR 677T carrier is suggested to be a distal tumor characteristic, especially in those 60 years old or older, and it seems to depend on the infection by H. pylori cagA/vacAs1m1 strains.

Evidence suggests that the carcinogenic process guided by Helicobacter pylori is related to the expression of cell cycle and apoptosis proteins as BCL-2, BAX, and MYC. However, the literature is conflicting regarding the expression frequency in the histological subtypes and did not consider cagA gene presence. To investigate the expression of these proteins considering the histological subtypes of gastric cancer associated with H. pylori (cagA), a total of 89 cases were used. H. pylori infection and cagA status were determined by PCR. Immunodetection was performed for MYC, BCL-2, and BAX proteins. H. pylori was found in 95.5% of the patients, among them, 65.8% were cagA(+). Nuclear MYC was detected in 36.4%, BAX in 55.7%, while BCl-2 in just 5%. Nuclear MYC staining was significantly lower in the intestinal than diffuse subtype (p = 0.008) and was related with the presence of H. pylori cagA(+). Additionally, most of the few cases cytoplasmic MYC positive were in the intestinal subtype. In diffuse tumors, although most nuclear MYC positive cases were cagA(+), it was not significant. No difference was observed between BCL-2 or BAX expression considering the presence of cagA gene in the histological subtypes. It seems that MYC could be relevant for the diffuse tumorigenic pathway associated with H. pylori and possibly influenced by the presence of cagA gene, while in intestinal tumors, the tumorigenic pathway does not occur through the MYC expression.