Relationship between Fusobacterium nucleatum, inflammatory mediators and microRNAs in colorectal carcinogenesis.

AIM: To examine the effect of Fusobacterium nucleatum (F. nucleatum) on the microenvironment of colonic neoplasms and the expression of inflammatory mediators and microRNAs (miRNAs). METHODS: Levels of F. nucleatum DNA, cytokine gene mRNA (TLR2, TLR4, NFKB1, TNF, IL1B, IL6 and IL8), and potentially interacting miRNAs (miR-21-3p, miR-22-3p, miR-28-5p, miR-34a-5p, miR-135b-5p) were measured by quantitative polymerase chain reaction (qPCR) TaqMan® assays in DNA and/or RNA extracted from the disease and adjacent normal fresh tissues of 27 colorectal adenoma (CRA) and 43 colorectal cancer (CRC) patients. KRAS mutations were detected by direct sequencing and microsatellite instability (MSI) status by multiplex PCR. Cytoscape v3.1.1 was used to construct the postulated miRNA:mRNA interaction network. RESULTS: Overabundance of F. nucleatum in neoplastic tissue compared to matched normal tissue was detected in CRA (51.8%) and more markedly in CRC (72.1%). We observed significantly greater expression of TLR4, IL1B, IL8, and miR-135b in CRA lesions and TLR2, IL1B, IL6, IL8, miR-34a and miR-135b in CRC tumours compared to their respective normal tissues. Only two transcripts for miR-22 and miR-28 were exclusively downregulated in CRC tumour samples. The mRNA expression of IL1B, IL6, IL8 and miR-22 was positively correlated with F. nucleatum quantification in CRC tumours. The mRNA expression of miR-135b and TNF was inversely correlated. The miRNA:mRNA interaction network suggested that the upregulation of miR-34a in CRC proceeds via a TLR2/TLR4-dependent response to F. nucleatum. Finally, KRAS mutations were more frequently observed in CRC samples infected with F. nucleatum and were associated with greater expression of miR-21 in CRA, while IL8 was upregulated in MSI-high CRC. CONCLUSION: Our findings indicate that F. nucleatum is a risk factor for CRC by increasing the expression of inflammatory mediators through a possible miRNA-mediated activation of TLR2/TLR4.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer.

OBJECTIVE: The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. METHODS: We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. RESULTS: High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. CONCLUSION: These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis.

[Echoendoscopy in digestive subepithelial lesions: review of the literature]. / Ecoendoscopia nas lesões subepiteliais do trato digestório: artigo de revisão.

Injury or bulging subepithelial mucosal lesions are covered with normal mucosa, usually asymptomatic. Most are diagnosed in radiology or endoscopy, which may correspond to any layer of the body wall (intramural) or non-belonging to the wall (extramural). This article describes studies for analysis of endoscopic ultrasonography (EUS) as a diagnostic method with high accuracy on the finding of subepithelial lesion. The authors review the literature on the endoscopic ultrasound features of subepithelial lesions and differentiation in intra-or extramural, source layer, echogenicity, vascularity, size and margins, fine needle aspiration (FNA) and needle biopsies of type "trucut" for histological analysis. The EUS has the best combination of accuracy in the diagnosis of gastrointestinal wall layer compromised by lesions or masses, besides studying the echogenicity of the lesion, which helps in differential diagnosis. EUS is safe and detailed, considered the best imaging for definitive diagnosis and therapeutic planning of subepithelial lesions.

Ecoendoscopia nas lesões subepiteliais do trato digestório: artigo de revisão / Echoendoscopy in digestive subepithelial lesions: review of the literature

Lesões subepiteliais ou abaulamentos da mucosa são recobertas por mucosa normal e geralmente assintomáticas. Sua maioria é diagnosticada em exames radiológicos ou de endoscopia digestiva e podem corresponder a qualquer camada da parede do órgão (intramural) ou serem extramurais. Este artigo descreve estudos para análise da ultrassonografia após ecoendoscopia (USE) como método diagnóstico de elevada acurácia diante do achado de lesão subepitelial. Trata-se de trabalho de revisão de literatura sobre as características ecoendoscópicas das lesões subepiteliais e diferenciação em intra ou extramurais, camada de origem, ecogenicidade, vascularização, margens e dimensões, punção aspirativa por agulha fina (PAAF) ou biópsias com agulha do tipo trucut. Ambas as formas são aceitáveis) para análise histológica. A ultrassonografia endoscópica tem melhores índices de acurácia no diagnóstico da camada da parede gastrointestinal comprometida por lesões ou massas, além de estudar a ecogenicidade da lesão. A ultrassonografia endoscópica é um método seguro e detalhado, considerado o melhor exame de imagem para diagnóstico definitivo e programação terapêutica das lesões subepiteliais.

Injury or bulging subepithelial mucosal lesions are covered with normal mucosa, usually asymptomatic. Most are diagnosed in radiology or endoscopy, which may correspond to any layer of the body wall (intramural) or non-belonging to the wall (extramural). This article describes studies for analysis of endoscopic ultrasonography (EUS) as a diagnostic method with high accuracy on the finding of subepithelial lesion. The authors review the literature on the endoscopic ultrasound features of subepithelial lesions and differentiation in intra-or extramural, source layer, echogenicity, vascularity, size and margins, fine needle aspiration (FNA) and needle biopsies of type "trucut" for histological analysis. The EUS has the best combination of accuracy in the diagnosis of gastrointestinal wall layer compromised by lesions or masses, besides studying the echogenicity of the lesion, which helps in differential diagnosis. EUS is safe and detailed, considered the best imaging for definitive diagnosis and therapeutic planning of subepithelial lesions.

Mid-esophagus unresectable cancer treated with a low cost stent. First experience.

BACKGROUND: In the cancer of the esophagus, with recent technologic advances, self-expanding metal stents (SEMS) are at the forefront of the armamentarium for re-establishing luminal patency. Weighed against the numerous advantages of stents are the import conditions and the cost. In light of this, we tested new low cost prostheses having the basic needs and characteristics to aim a significant benefit to poor people having advanced esophageal cancer, in a Brazilian regional public hospital. METHODS: This initial experience included fifteen patients (eleven men and four women, 55 ± 6.17 years old), presenting esophageal cancer, located at the medium third of the thoracic esophagus, extending for 5.5-8 cm long, not suitable for surgical procedure because they had been staged on fourth grade of the disease, two of them having fistula communicating esophagus to respiratory tree. The stents were placed under endoscopic and fluoroscopic guidance, after attempting an esophageal dilatation. An appropriate covered stent was then deployed, twelve of 10 cm and three of 13 cm in length. A chest X-ray was done 2 h after the procedure and a barium swallow was performed within 12 hours. Seven days and monthly until complete a six month follow-up after the procedure the patients were questioned about presence of pain, regurgitation, heartburn, cough, and their alimentary behavior. RESULTS: There were no severe complications and transient mild chest pain resolved until the seventh day after the stent deployment. Chest X-ray demonstrated expansion of the stent in all patients. In 2 cases of fistula, a barium swallow showed its complete sealing. The completion of the proposed follow-up was not achieved in three cases, limited by the patient's death until the third month, due to cancer progression. Recurrent dysphagia to paste food accounted for by tumor overgrowth proximal or distal to the stent and stent migration were not observed in the series. CONCLUSIONS: The new low cost endoprostheses is effective and forthcoming increased experience and prospective trials including questionnaires to analyze quality of life will allow for more informed decisions tailoring to a particular patient situation or to unexpected complications.

[Helicobacter pylori and peptic disease: comparative study of the diagnostic methods]. / Helicobacter pylori e doença péptica: estudo comparativo de métodos diagnósticos.

Prospective endoscopic study of 150 patients revealed chronic gastritis in 109 (72.6%), gastric ulcer in 6 (4%), chronic duodenitis in 9 (6%) and duodenal ulcer in 26 (17.4%). Searching for Helicobacter pylori, positive urease test was observed in 103 (68.67%), histologic evidence in 104 (69.33%) and positive serologic test in 98 (65.33%), without statistical difference. The urease test is recommended in the diary medical practice, for the patients who also will benefit themselves with the endoscopic diagnosis. On the other hand, the serologic test is useful when the endoscopy of the upper digestive tract cannot or must not be realized.

Helicobacter pylori e doença péptica: estudo comparativo de métodos diagnósticos / Helicobacter pylori and peptic disease: comparative study of the diagnostic methods

Foram estudados, prospectivamente, 150 pacientes. Estudo endoscópico revelou gastrite crônica em 109 pacientes (72,6 por cento), úlcera gástrica em 6 (4 por cento), duodenite crônica em 9 (6 por cento) e úlcera duodenal em 26 (17,4 por cento). Quanto à avaliação metodológica para pesquisa do Helicobacter pylori, 103 (68,67 por cento) apresentaram teste da urease positivo, 104 (69,33 por cento), positividade histopatológica e 98 (65,33 por cento), positividade sorológica. Não houve diferença estatística entre os métodos. Pela facilidade de realização, o teste da urease credencia-se como o de melhor indicação nos pacientes que também se beneficiarão com o diagnóstico endoscópico. Caso a endoscopia digestiva alta não possa ou não deva ser realizada, está recomendado o teste sorológico.

Prospective endoscopic study of 150 patients revealed chronic gastritis in 109 (72.6 percent), gastric ulcer in 6 (4 percent), chronic duodenitis in 9 (6 percent) and duodenal ulcer in 26 (17.4 percent). Searching for Helicobacter pylori, positive urease test was observed in 103 (68.67 percent), histologic evidence in 104 (69.33 percent) and positive serologic test in 98 (65.33 percent), without statistical difference. The urease test is recommended in the diary medical practice, for the patients who also will benefit themselves with the endoscopic diagnosis. On the other hand, the serologic test is useful when the endoscopy of the upper digestive tract cannot or must not be realized.

Genetic alterations in benign lesions: chronic gastritis and gastric ulcer.

AIM: To investigate the occurrence of chromosome 3, 7, 8, 9, and 17 aneuploidies, TP53 gene deletion and p53 protein expression in chronic gastritis, atrophic gastritis and gastric ulcer, and their association with H pylori infection. METHODS: Gastric biopsies from normal mucosa (NM, n=10), chronic gastritis (CG, n=38), atrophic gastritis (CAG, n=13) and gastric ulcer (GU, n=21) were studied using fluorescence in situ hybridization (FISH) and immunohistochemical assay. A modified Giemsa staining technique and PCR were used to detect H pylori. An association of the gastric pathologies and aneuploidies with H pylori infection was assessed. RESULTS: Aneuploidies were increasingly found from CG (21%) to CAG (31%) and to GU (62%), involving mainly monosomy and trisomy 7, trisomies 7 and 8, and trisomies 7, 8 and 17, respectively. A significant association was found between H pylori infection and aneuploidies in CAG (P=0.0143) and GU (P=0.0498). No TP53 deletion was found in these gastric lesions, but p53-positive immunoreactivity was detected in 45% (5/11) and 12% (2/17) of CG and GU cases, respectively. However, there was no significant association between p53 expression and H pylori infection. CONCLUSION: The occurrence of aneuploidies in benign lesions evidences chromosomal instability in early stages of gastric carcinogenesis associated with H pylori infection, which may confer proliferative advantage. The increase of p53 protein expression in CG and GU may be due to overproduction of the wild-type protein related to an inflammatory response in mucosa.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and gastric cancer.

AIM: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. METHODS: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (C1) and 150 (C2) controls, respectively. RESULTS: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. CONCLUSION: Our results showed no evidence of a relationship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer.

Aneuploidies, deletion, and overexpression of TP53 gene in intestinal metaplasia of patients without gastric cancer.

Gastric carcinogenesis is attributable to interacting environmental and genetic factors, through a sequence of events including intestinal metaplasia. Using a fluorescence in situ hybridization technique, we investigated the occurrence of aneuploidies of chromosomes 3, 7, 8, 9, and 17, TP53 gene deletion, and expression of p53 in 21 intestinal metaplasia (IM) samples from cancer-free patients and in 20 gastric adenocarcinoma samples. Aneuploidies were found in 71% (15/21) of the IM samples. Trisomy of chromosomes 7 and 9 occurred mainly in complete-type IM; in the incomplete type, trisomy of chromosomes 7 and 8 were more commonly found. The TP53 gene deletion was observed in 60% (3/5) of the IM cases, and immunohistochemistry revealed p53 overexpression in 12% (2/17) of the analyzed IM cases. All gastric adenocarcinoma cases presented higher frequencies of trisomy or tetrasomy of chromosomes 3, 7, 8, 9, and 17. The TP53 deletion was found in all three of the gastric adenocarcinoma analyzed for it, and immunohistochemistry detected overexpression of protein p53 in 80% (12/15) of the analyzed cases. Our study revealed for the first time the presence of aneuploidies of chromosomes 7, 8, 9, and 17 and of TP53 gene deletion and overexpression in IM samples from cancer-free patients. These results suggest that IM and gastric adenocarcinoma may share the same genetic alterations.

Cytogenetic alterations in chagasic achalasia compared to esophageal carcinoma.

Patients with chagasic achalasia (megaesophagus) are liable to have an additional 1.7-20% possibility of developing esophageal squamous cell carcinoma (ESCC). We applied a fluorescence in situ hybridization technique in 20 such patients and found aneuploidies of chromosomes 7, 11, and 17 in 60% (12 of 20 specimens) and deletion of the TP53 gene in 54.5% (6 of 11 specimens; it was only possible to obtain data by FISH technique from 11 of the 20 achalasia patients). The main aneuploidies detected were chromosome 7 monosomy or trisomy (35%) in mid-third megaesophagus cases, and chromosome 17 monosomy or trisomy (25%) in distal-third cases. TP53 gene deletion was more frequent in mid-third (62.5%) than in distal-third megaesophagus cases (40%). In chagasic megaesophagus, no amplification of the cyclin D1 gene (CCND1) was observed. Comparing chagasic megaesophagus to ESCC, we found a higher frequency of aneuploidies in all 10 tumors. The main alterations were trisomy or tetrasomy of chromosomes 17 (90%), 11 (70%), and 7 (70%). Amplification of CCND1 was evidenced as a cluster in 70% of the tumors (22-99% of nuclei), while TP53 gene deletion occurred in 100%. To our knowledge, this is the first cytogenetic analysis of chagasic megaesophagus to show that aneuploidies of chromosomes 7, 11, and 17, and TP53 gene deletion might be related to increased risk for malignancy.

GSTT1, GSTM1 and CYP2E1 genetic polymorphisms in gastric cancer and chronic gastritis in a Brazilian population.

AIM: To test the hypothesis that, in the Southeastern Brazilian population, the GSTT1, GSTM1 and CYP2E1 polymorphisms and putative risk factors are associated with an increased risk for gastric cancer. METHODS: We conducted a study on 100 cases of gastric cancer (GC), 100 cases of chronic gastritis (CG), and 150 controls (C). Deletion of the GSTT1 and GSTM1 genes was assessed by multiplex PCR. CYP2E1/PstI genotyping was performed using a PCR-RFLP assay. RESULTS: No relationship between GSTT1/GSTM1 deletion and the c1/c2 genotype of CYP2E1 was observed among the three groups. However, a significant difference between CG and C was observed, due to a greater number of GSTT1/GSTM1 positive genotypes in the CG group. The GSTT1 null genotype occurred more frequently in Negroid subjects, and the GSTM1 null genotype in Caucasians, while the GSTM1 positive genotype was observed mainly in individuals with chronic gastritis infected with H pylori. CONCLUSION: Our findings indicate that there is no obvious relationship between the GSTT1, GSTM1 and CYP2E1 polymorphisms and gastric cancer.