CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O papel carcinogÃnico de Helicobacter pylori està relacionado à sua capacidade de promover a inflamaÃÃo e, como conseqÃÃncia, a metilaÃÃo do DNA, caracterÃstica epigenÃtica frequentemente associada à carcinogÃnese gÃstrica. Por sua vez, o processo inflamatÃrio pode ser modulado pela presenÃa de alguns dos polimorfismos presentes em genes de interleucinas, bem como pelo genÃtipo bacteriano. Assim, os objetivos desse estudo foram: a) associar o perfil genotÃpico de virulÃncia de H. pylori (quanto aos genes cagA, cagE, vacA e virB11) e o perfil genotÃpico dos polimorfismos de interleucinas prÃ-inflamatÃrias, IL1β -511 C/T, IL1RN, IL6 -174 G/C e TNF -308 G/A com a metilaÃÃo de promotores gÃnicos de CDKN2A, MLH1 e COX-2; b) verificar a associaÃÃo dos polimorfismos da IL6 -174 G/C e TNF -308 G/A com genÃtipo bacteriano no desenvolvimento do cÃncer gÃstrico, considerando os aspectos clinico-patolÃgicos. Para isso, foi extraÃdo DNA a partir de 125 amostras tumorais, coletadas de pacientes submetidos à gastrectomia em hospitais de Fortaleza â Cearà â Brasil. A genotipagem dos polimorfismos foi feita por PCR-RFLP e a anÃlise de metilaÃÃo, por MS-PCR. Os genes de virulÃncia de H. pylori foram analisados por PCR. Em algumas anÃlises, os genÃtipos bacterianos foram agrupados de acordo com os alelos de vacA e a integridade de cag-PAI. Neste estudo, foi verificado que nos tumores da cÃrdia a metilaÃÃo na regiÃo promotora de COX-2 estava associada ao alelo IL1RN*2 (p= 0,015), e o genÃtipo IL-1B -511 T + IL1RN*2 se mostrou importante para a metilaÃÃo desse gene (p=0,013), principalmente na presenÃa de cepas de H. pylori cagA+ (p=0,026) e vacA s1 (p=0,025). A combinaÃÃo genotÃpica IL6 CC+TNFGG parece estar envolvida na nÃo-metilaÃÃo dos promotores dos genes CDKN2A (p=0,046) e MLH1 (p=0,031), mesmo na presenÃa da infecÃÃo por cepa H. pylori cagA+. Considerando os aspectos clinico-patÃlogicos, uma correlaÃÃo positiva foi encontrada entre o gÃnero masculino e pacientes da faixa etÃria >65 anos (r=0,198; p=0,037), na qual esse gÃnero foi predominante (77,5%; p= 0.022). AlÃm disso, tambÃm foi encontrada correlaÃÃo positiva entre o gÃnero feminino e a faixa etÃria de 55-64 (r=+0,217; p=0,021). Quanto ao subtipo histolÃgico, foi visto que tumores difusos estavam correlacionados a pacientes mais jovens (15-44 anos, r=+0,207; p=0,033), enquanto o subtipo intestinal, a pacientes de mais idade (> 65 anos, r=+0,296; p=0,017). Tumores do subtipo difuso foram correlacionados com o gÃnero feminino e aqueles do subtipo intestinal, com o gÃnero masculino (r=+0,226; p=0,019). Quanto aos polimorfismos de interleucinas, o alelo C do polimorfismo IL6 -174G/C foi correlacionado negativamente com pacientes de menor faixa etÃria (r=â0,193; p=0,041), sendo o que pacientes com genÃtipo CC de IL6 foi associado com a infecÃÃo por cepas virulentas (grupo 1c) (r=+0,225; p=0,017); enquanto que pacientes portadores do genÃtipo heterozigoto IL6 -174 GC, com cepas virulentas (grupo 1c) (r=â0,215; p=0,023) e de menor virulÃncia (grupo 2c) (r=+0,204; p=0,031). Os achados desse estudo contribuem com o estabelecimento de um perfil genotÃpico envolvido na metilaÃÃo de alguns genes, no qual pacientes com genÃtipo mais inflamatÃrio e infectados por cepas de H. pylori mais virulentas estÃo associadas a uma maior taxa de metilaÃÃo de alguns genes envolvidos na carcinogÃnese gÃstrica, podendo tambÃm variar de acordo com a localizaÃÃo e o subtipo do tumor. Esse estudo, portanto, oferece uma contribuiÃÃo relevante no que diz respeito à associaÃÃo da cepa de H. pylori com polimorfismos de interleucinas, no qual cepas de maior virulÃncia parecem estar relacionadas com a infecÃÃo de pacientes com genÃtipos menos inflamatÃrios, sendo o contrÃrio tambÃm verdadeiro. / The carcinogenic role of Helicobacter pylori is related to its ability to promote inflammation and, consequently, DNA methylation, epigenetic trait often associated with gastric carcinogenesis. In turn, the inflammation can be modulated by the presence of polymorphisms in some interleukin genes, as well as the bacterial genotype. The objectives of this study were: a) link the genotypic profile of H. pylori virulence (cagA, cagE, vacA and virB11 genes) and genotypic profile of pro-inflammatory interleukins polymorphisms, IL1β -511 C/T, IL1RN, IL6 -174 G/C and TNF-308 G/A with gene promoter methylation of CDKN2A, MLH1, and COX-2 b) verify the association of polymorphisms of IL6 -174 G/C and TNF -308 G/A with bacterial genotype in the gastric cancer development, considering the clinical and pathological aspects. For this, DNA was extracted from 125 tumor samples, collected from patients who underwent gastrectomy at hospitals in Fortaleza â Cearà â Brazil. Polymorphisms genotyping were identified by PCR-RFLP and methylation analysis by MS-PCR. Virulence genes of H. pylori were analyzed by PCR. In some analyzes, the bacterial genotypes were grouped according to the alleles of vacA and integrity of cag-PAI. In this study, we observed that in cardia tumors the methylation of COX-2 promoter region was associated with IL1RN*2 allele (p=0.015), and genotype IL-1B -511T+IL1RN*2 was important to methylation of this gene (p=0.013), especially in the presence of H. pylori cagA+strains (p=0.020) and vacA s1 (p=0.032). The genotype combination IL6 CC+TNFGG seems to be involved in non-methylation of promoters of genes CDKN2A (p=0.046) and MLH1 (p=0.031), even in the presence of infection by strain H. pylori cagA+. Considering the clinical and pathological aspects, a positive correlation was found between males and patients aged >65 years (r=0.198, p=0.037), in which this gender was predominant (77.5%, p=0.022) . In addition, positive correlation was found between female and patients aged 55-64 years (r=+0.217, p=0.021). Regarding the histologic subtype, we found that diffuse tumors were correlated with younger patients (15-44 years, r=+0.207, p=0.033), while the intestinal subtype, with the older patients (>65 years, r=+ 0.296, p=0.017). Tumors of the diffuse subtype were correlated with female gender and those of the intestinal subtype, with males (r=+0.226, p=0.019). Regarding polymorphisms of interleukins, the C allele of IL6 polymorphism -174G/C was negatively correlated with the younger group (r=-0.193, p=0.041), and the patients with CC genotype of IL6 was associated with infection by virulent strains (group 1c) (r=+0.225, p=0.017), whereas patients with the genotype IL6 -174 GC, with virulent strains (group 1c) (r=-0.215, p=0.023) and less virulent (group 2c) (r=+0.204, p=0.031). Our findings contribute to the establishment of a genotypic profile involved in methylation of some genes in which patients with genotype more inflammatory and infected with strains of H. pylori more virulent are associated with a higher rate of methylation of some genes involved in gastric carcinogenesis and may also vary according to location and tumor subtype. This study thus provides an important contribution with regard to the association of the strain of H. pylori polymorphisms interleukins, in which most virulent strains appear to be related to infection of patients with inflammatory genotypes less, and the converse is also true.

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No. of bitstreams: 1 Dissertação - Gleyce dos Santos Barbosa.pdf: 2917376 bytes, checksum: 06d23d301fe537d8d8c6b4a14c4689cf (MD5) Previous issue date: 2012-01-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the northern Brazil, especially in the states of Amazonas and Pará, there is a high incidence of gastric cancer which available treatments are ineffective in most cases. The biflorin, a prenylated ortanaftoquinona obtained from the roots of Capraria biflora L., showed, in previous studies, an inhibition of the tumor growth in vivo and in vitro in several cell lines, without inducing mutagenicity. The ACP02, main cell line used in this work, was established from a primary diffuse gastric adenocarcinoma, removed from the stomach cardia region of a 66-year-old pacient born in Pará. Among the main karyotypes and genetic changes of this lineage are the trisomy of chromosome 8, with amplification of the MYC oncogene, and the deletion of the chromosome 17’s short arm, where the tumor suppressor gene TP53 is located. These characteristics observed in the established line correspond to those observed in the original tumor, what indicates that this line is a good alternative to the study of the human gastric cancer pathophysiology and the drug screening. Considering biflorin’s promising potencial and the necessity to regionalize the study of new anticancer drugs to answer genotype particularities of each group of patients, we evaluated the activity of biflorin on ACP02 through the morphological cell analysis, the cell viability assays, the clonogenic assay, scratch assay, the cell differentiation test by NBT, the evaluation of the MYC status and telomere length by FISH, in 0, 1.0, 2.5 and 5.0 M concentrations, after 24 and 72 hours of treatment. The morphological changes indicate cell death by necrosis, and suggest the occurrence of the differentiation process, due to change in the format of the attached cell. The biflorin showed a cytotoxic activity (IC50 1.92 M) and cytostatic, anticlonogenic and antimotility activities, statistically significant (p<0,05) since 1 M. There was a significant reduction in MYC amplification rate in cells treated with 2.5 e 5 M of the drug (p<0,05), which can explain the occurrence of cellular differentiation process, in view of the physiological role of this gene. This differentiation was confirmed by the results obtained during the NBT test, in which the cells showed the ability to metabolize salt from a treatment of 1.0 M. The telomere length was reduced in cells treated with 5.0 M of biflorina (p<0,05). These results showed that biflorin acts on important targets of anticancer therapy, when used on the gastric line ACP02, at concentrations around 2.5 M, which makes it a promising substance for the treatment of this tumor type. / No norte do Brasil, sobretudo nos Estados do Amazonas e Pará, há alta incidência de câncer gástrico, cujos tratamentos disponíveis são ineficazes, na maioria dos casos. A biflorina, uma ortonaftoquinona prenilada obtida das raízes de Capraria biflora L., demonstrou, em trabalhos prévios, inibir o crescimento tumoral in vivo e in vitro de várias linhagens celulares, sem induzir mutagenicidade. A ACP02, principal linhagem celular utilizada nesse trabalho, foi estabelecida a partir de um adenocarcinoma gástrico difuso primário, retirado da região cárdia estomacal de um paciente de 66 anos oriundo do Pará. Entre as principais alterações cariotípicas e genéticas dessa linhagem estão a trissomia do cromossomo 8, com amplificação do oncogene MYC, e a deleção do braço curto do cromossomo 17, onde estaria localizado o gene supressor de tumor TP53. Essas características observadas na linhagem estabelecida correspondem àquelas observadas no tumor que a deu origem, evidenciando que tal linhagem é uma boa alternativa para o estudo da fisiopatologia do câncer gástrico humano e triagem de drogas. Tendo em vista o potencial promissor da biflorina e a necessidade de regionalizar o estudo de novas drogas anticâncer para atender as particularidades genotípicas de cada grupo de pacientes, avaliamos a atividade da biflorina sobre ACP02 através da análise morfológica das células, dos ensaios de viabilidade celular, do ensaio clonogênico, do teste de motilidade, do teste de diferenciação celular pelo NBT e da avaliação do status de MYC e comprimento dos telômeros por FISH, nas concentrações de 0, 1,0, 2,5 e 5 M, após 24 e 72h de tratamento. As alterações morfológicas indicaram morte celular por necrose, além de sugerirem a ocorrência de processo de diferenciação, devido à alteração no formato da célula aderida. A biflorina apresentou atividade citotóxica (CI50 1,92 M) e atividades citostática, anticlonogênica e inibidora da motilidade in vitro, estatisticamente significativas (p<0,05) desde a concentração de 1 M. Houve redução significativa da taxa de amplificação de MYC, nas células tratadas com 2,5 e 5,0 M da droga (p<0,05), o que pode justificar a ocorrência de processo de diferenciação celular, tendo em vista o papel fisiológico desse gene. Essa diferenciação foi confirmada pelos resultados obtidos durante o teste do NBT, no qual as células apresentaram capacidade de metabolizar o sal a partir do tratamento de 1,0 M. O comprimento do telômero foi reduzido nas células tratadas com 5,0 M de biflorina (p<0,05). Esses resultados demonstram que a biflorina atua sobre importantes alvos da terapia anticâncer, quando utilizada sobre a linhagem gástrica ACP02 em concentrações em torno de 2,5 M, o que a torna uma substância promissora para o tratamento desse tipo de tumor.

Introduction The prognosis in oesophago-gastric cancer is poor with less than 15% patients surviving beyond 5 years after diagnosis. The addition of neoadjuvant therapy has been shown to increase survival in patients suitable for curative surgery. However, the additional gains are modest and the majority of patients do not respond sufficiently from therapy to gain any benefit. There is an urgent need to identify markers that can predict response to neoadjuvant therapy in order provide safer, more effective, individualised treatment regimes. Methods A prospective, multi-centre, collaborative study was undertaken in patients with oesophago-gastric cancer undergoing neoadjuvant therapy and potentially curative surgery. Levels of circulating biomarkers M2-Pyruvate kinase, alkaline phosphatase, CA19-9, CEA and CA 72-4 were measured in patients before and after administering the first cycle of chemotherapy. Binary logistic regression analysis was performed to assess the ability of biomarkers to predict histological response to therapy. Results 165 patients were recruited to the main study. 105 patients had complete histopathological data for analysis. There were 27 responders and 78 non-responders to neoadjuvant therapy. There were no differences in pre-therapy demographic, pathological or treatment factors between the two groups. Responders had less post-operative lymphovascular invasion (P= 0.004) and higher R0 resection rates (P=0.03). Pre-therapy M2-Pyruvate kinase levels were lower in responders compared to non-responders (P=0.037) and levels were able to predict response with each unit increase in the biomarker level being associated with a 4.1% decrease in the likelihood of response (P=0.027). M2-PK levels were not associated with any pre-operative demographic, clinical or pathological factors. Conclusions Pre-therapy dimeric M2-PK levels can predict response to neoadjuvant therapy in patients with oesophago-gastric cancer. The test could be of clinical value for 1 in every 8 patients undergoing the test.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21997号 / 医博第4511号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 妹尾 浩, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13061号 / 論医博第2119号 / 新制||医||1018(附属図書館) / (主査)教授 羽賀 博典, 教授 妹尾 浩, 教授 戸井 雅和 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20988号 / 医博第4334号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 坂井 義治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23568号 / 医博第4782号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background Gastric cancer is one of the most prevalent cancers in the world and the third most common cause of death from cancer. The diagnosis and treatment are often complex and require a multifaceted approach. Hence, appropriate and timely management is essential for better patient outcomes. Our aim was to determine if rural inhabitation affects the mortality of patients with gastric adenocarcinoma. If such an association exists, we propose to ascertain whether this is related to delayed diagnosis, differing tumor characteristics, or treatment inequalities. Methods The Cox model was applied to gastric adenocarcinoma cases diagnosed during 2004-2011 in American residents aged 20+ years in the Surveillance, Epidemiology, and End Results (SEER) program to determine the impact of rurality on mortality. Binary logistic regression was used to compare the odds of not receiving surgical treatment for localized tumors between rural and urban areas. It was also used to measure the association of rurality with stage at diagnosis (non-metastatic vs. metastatic). Results There was a significant association of rurality on 5-year mortality [HR 1.14 (1.09-1.20), p < 0.01]. No significant association was observed between rural-urban residency and stage at diagnosis, with an odds ratio (OR) of 0.95 (0.87-1.03), p = 0.21. The median time from diagnosis to any first-course treatment was one month for both rural and urban counties. Rural residents were far more likely not to receive surgical treatment for localized tumors than their urban counterparts [OR 1.70 (1.41-2.05), p < 0.01]. A greater percentage of rural inhabitants had cardia tumors as compared to urban ones, 39.8% vs. 33.8% respectively. Non-cardia tumors were far less likely not to receive surgical treatment (i.e., more likely to receive surgical treatment) than cardia tumors [OR 0.35 (0.30-0.41), p < 0.01]. Conclusions Rurality is associated with worse gastric adenocarcinoma mortality. This may be due to a lesser probability of receiving surgical treatment for early-stage disease and differences in the primary site of the tumor between rural and urban counties, but not due to differences in stage at presentation. Future research should focus on improving health care access in rural communities.

Posterior reversible encephalopathy syndrome (PRES) is a clinical-radiographic syndrome that presents with neurological manifestations, including seizures, headache, or confusion, and is associated with posterior cerebral white matter edema on imaging. PRES is typically a benign and reversible condition. However, PRES can be fatal or associated with permanent deficits. Numerous conditions are associated with PRES, including hypertensive encephalopathy, renal diseases, and cytotoxic or immunosuppressant drugs. Recently, many case reports described the association between PRES and chemotherapeutic agents. However, trastuzumab-associated PRES is rarely reported. Herein, we report a case of a 51-year-old female with a history of metastatic gastric cancer who developed seizures while being treated with trastuzumab, and neuroimaging confirmed the diagnosis of PRES.

Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.