Traps and trumps from adjacent-to-tumor samples in gastric cancer research.

The search for cancer biomarkers is frequently based on comparisons between tumors and adjacent-to-tumor samples. However, even after histological confirmation of been free of cancer cells, these adjacent-to-tumor samples might harbor molecular alterations which are not sufficient to cause them to look like cancer, but can differentiate these cells from normal cells. When comparing them, potential biomarkers are missed, and mainly the opportunity of finding initial aberrations presents in both tumors and adjacent samples, but not in true normal samples from non-cancer patients, resulting in misinterpretations about the carcinogenic process. Nevertheless, collecting adjacent-to-tumor samples brings trumps to be explored. The addition of samples from non-cancer patients opens an opportunity to increase the finds of the molecular cascade of events in the carcinogenic process. Differences between normal samples and adjacent samples might represent the first steps of the carcinogenic process. Adding samples of non-cancer patients to the analysis of molecular alterations relevant to the carcinogenic process opens a new window of opportunities to the discovery of cancer biomarkers and molecular targets.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.

AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines. METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization. RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44). CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

Helicobacter pylori in dental plaque and stomach of patients from Northern Brazil.

AIM: To establish whether virulence factor genes vacA and cagA are present in Helicobacter pylori (H. pylori) retrieved from gastric mucosa and dental plaque in patients with dyspepsia. METHODS: Cumulative dental plaque specimens and gastric biopsies were submitted to histological examination, rapid urease test and polymerase chain reaction (PCR) assays to detect the presence of cagA and vacA polymorphisms. RESULTS: Detection of H. pylori from dental plaque and gastric biopsy samples was greater by PCR compared to histological examination and the rapid urease test. DNA from H. pylori was detected in 96% of gastric mucosa samples and in 72% of dental plaque samples. Sixty-three (89%) of 71 dental plaque samples that were H. pylori-positive also exhibited identical vacA and cagA genotypes in gastric mucosa. The most common genotype was vacAs1bm1 and cagA positive, either in dental plaque or gastric mucosa. These virulent H. pylori isolates were involved in the severity of clinical outcome. CONCLUSION: These pathogenic strains were found simultaneously in dental plaque and gastric mucosa, which suggests that gastric infection is correlated with the presence of H. pylori in the mouth.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon.

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6 percent presented nucleotide substitutions: A2142G (3.3 percent), T2182C (12.9 percent), G2224A (6.45 percent), T2215C (61.3 percent), A2192G (3.3 percent), G2204C (6.4 percent) and T2221C (6.4 percent). Of the mutations identified, four are known mutations related to cases of resistance and 16.1 percent are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Interleukin-1 and TNF-alpha polymorphisms and Helicobacter pylori in a Brazilian Amazon population.

AIM: To study the association between Interleukin-1 (IL-1) and tumor necrosis factor (TNF)-alpha polymorphisms, infection by Helicobacter pylori (H pylori) and the development of gastrointestinal diseases. METHODS: Genomic DNA was extracted from the peripheral blood of 177 patients with various gastrointestinal diseases and from 100 healthy volunteers. The polymorphisms in IL-1beta and TNF-alpha genes were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and those from IL-1RN with PCR. The presence of infection due to H pylori and the presence of the CagA toxin were detected by serology. The histopathological parameters in the gastric biopsies of the patients were according to the Sydney classification. RESULTS: A comparison of the frequencies of the different polymorphisms studied among the patients and the control group demonstrated that the allele IL-1RN*2 was more frequent among patients with gastric ulcers and adenocarcinoma. Carriers of the allele IL-RN*2 and those with reactive serology for anti-CagA IgG had a greater risk of developing peptic ulcer and gastric adenocarcinoma, as well as a higher degree of inflammation and neutrophilic activity in the gastric mucosa. CONCLUSION: Our results indicate a positive association between IL-1RN gene polymorphism and infection by positive H pylori CagA strains and the development of gastric ulcers and adenocarcinoma.

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from north Brazil

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6 percent of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8 percent had subtype s1a, 67.8 percent had subtype s1b, and 17 percent subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4 percent and m2 in 21.2 percent of patients. The cagA gene was detected in 78 percent patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from North Brazil.

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6% of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8% had subtype s1a, 67.8% had subtype s1b, and 17% subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4% and m2 in 21.2% of patients. The cagA gene was detected in 78% patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.

Expressäo dos antígenos ABH e Lewis na gastrite crônica e alteraçöes pré-neoplásica da mucosa gástrica / Expression of ABH and Lewis antigens in chronic gastritis and pre-neoplasic alterations in gastric mucosa

RACIONAL: A aderência do Helicobacter pylori à mucosa gástrica humana é pré-requisito para sua colonizaçäo e o desenvolvimento da gastrite crônica. Os antígenos de grupos sangüíneos, presentes no muco gástrico, säo descritos como prováveis receptores da bactéria neste epitélio. A expressäo alterada destes antígenos está associada ao desenvolvimento do câncer gástrico. OBJETIVOS: Verificar a ocorrência do Helicobacter pylori e a distribuiçäo da expressäo dos antígenos ABH e Lewis correlacionada com as alteraçöes histopatológicas de pacientes com gastrite crônica. PACIENTES E MÉTODOS: Analisaram-se 63 amostras de sangue, saliva e biopsias gástricas de pacientes com gastrite crônica através das técnicas dot-blot-ELISA, imunoperoxidase indireta e coloraçöes do Gram modificado e hematoxilina-eosina. RESULTADOS: Näo foram encontradas associaçöes significativas entre a presença da bactéria e os fenótipos de grupos sangüíneos ABH, Lewis e Secretor. Na maioria dos pacientes, a expressäo dos antígenos ABH e Lewis, estava restrita principalmente ao epitélio foveolar da mucosa gástrica, concordando com a expressäo ao nível salivar. A expressäo inapropriada desses antígenos ocorria sempre na infecçäo pelo Helicobacter pylori e/ou alteraçöes pré-neoplásicas da mucosa gástrica. Em áreas com metaplasia intestinal foi observada a reduçäo da reatividade para os antígenos H e Le b, e principalmente o aumento de Leª. CONCLUSÄO: Alteraçöes no padräo de glicosilaçäo destes antígenos refletem diferentes estágios de diferenciaçäo celular e säo marcadores potenciais na avaliaçäo diagnóstica e prognóstica das patologias gástricas

[Expression of ABH and Lewis antigens in chronic gastritis and pre-neoplasic alterations in gastric mucosa]. / Expressão dos antígenos ABH e Lewis na gastrite crônica e alterações pré- neoplásica da mucosa gástrica.

BACKGROUND: The major cause for chronic gastritis in human is the infection by the Helicobacter pylori. The blood group antigens present at the gastric mucous are described as possible receptor for this bacteria in the epithelium. The alterations in the expression of blood group patterns are associated with the development of gastric cancer. OBJECTIVES: Verify the H. pylori prevalence and examine the immunohistochemical distribution of the ABH and Lewis antigens expression to correlate with histopathological alterations. PATIENTS AND METHODS: From 63 chronic gastritis patients were investigated gastric biopsies, blood and saliva samples by dot-blot-ELISA, indirect immunoperoxidase and hematoxylin-eosin and Gram. RESULTS: No significant association between the presence of the bacteria and the ABH, Lewis and Secretor phenotype was found. For the majority of the patients the antigen expression of the ABH and Lewis blood group was restricted mainly to the foveola epithelium of the gastric mucosa, similar to the saliva. The inappropriate expression of these antigens occurred always in the presence of H. pylori and/or preneoplastic alterations of the gastric mucosa. In areas with intestinal metaplasias we also observed reduced reactivity for the H and Le b antigens and mainly the induced expression of Le . CONCLUSION: Alterations in the pattern of the glycosylation of this antigens are interesting, because they reflect different stages in the cellular differentiation and become potential markers in the diagnostic evaluation and prognosis of gastric pathologies.