Orientadores: Carmen Veríssima Ferreira Halder, Ronaldo Aloise Pilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T09:59:14Z (GMT). No. of bitstreams: 1 Pelizzaro-Rocha_KarinJuliane_D.pdf: 18909118 bytes, checksum: fa9cec97e3d81e8fb670dab95bc1bc4d (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: The abstract is available with the full electronic document when available / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Introdução: O acesso biliar ecoguiado é um método de drenagem alternativo à drenagem percutânea transhepática (DPTH) e à cirurgia em pacientes com obstrução biliar distal incurável que falharam drenagem por Colangiopancreatografia Endoscópica Retrógrada (CPRE). Nos casos em que a drenagem ecoguiada anterógrada transpapilar (ou transanastomótica) e o rendez-vous ecoguiado não podem ser realizados como primeira opção, a coledocoduodenostomia (CDT) e a hepaticogastrostomia (HPG) ainda podem ser realizadas em pacientes selecionados. Estas duas vias de drenagem não anatômicas criam uma fístula entra a via biliar e o estômago ou duodeno. Não há dados na literatura que determinem superioridade de uma ou outra técnica. Objetivo: Comparar o sucesso técnico, sucesso clínico e fatores associados entre as duas vias de drenagem em pacientes com obstrução da via biliar distal maligna incurável que não lograram sucesso na drenagem por CPRE ou rendez-vous ecoguiado. Métodos: Entre abril de 2010 e dezembro de 2013, 49 pacientes com obstrução biliar distal maligna incurável que falharam CPRE e rendez-vous ecoguiado foram randomizados para CDT ou HPG. Dados referentes ao sucesso técnico, sucesso clínico, tempo de procedimento, complicações, qualidade de vida e sobrevida foram coletados até três meses após o procedimento. Todos os procedimentos foram realizados em um centro terciário de endoscopia pelo mesmo endoscopista. Próteses biliares parcialmente recobertas (Boston Scientific, Wallflex, 10 mm, 8 cm ou 6 cm) foram utilizadas em todos os pacientes com sucesso técnico. Nos casos de HPG a punção ecoguiada foi intra-hepática no ducto hepático esquerdo. Nos casos de CDT a punção foi extra-hepática no segmento distal não obstruído do colédoco. Após a punção foi realizada colangiografia com introdução de um fio guia hidrofílico de 0,035 polegada. Dilatação com cateter e um dispositivo de needle knife foi realizada para permitir introdução do sistema de disparo da prótese biliar com 8,5 Fr. Resultados: Quarenta e nove procedimentos foram realizados (25 HPG e 24 CDT). Todos os pacientes tinham dilatação da via biliar intra e extra-hepática. A taxa de sucesso técnico foi de 96 % para HPG e de 91% para CDT (p = 0,609). A taxa de sucesso clínico foi de 91% para o grupo HPG e de 77% para o grupo CDT (p = 0,234). No grupo da HPG 5 pacientes (20%) tiveram complicações (3 sangramentos, 2 biliomas e uma bacteremia). No grupo da CDT 3 pacientes (12,5%) tiveram complicações (1 bilioma, 1 sangramento e 1 perfuração). Somente o caso da perfuração necessitou tratamento cirúrgico. As outras complicações foram tratadas clinicamente. O tempo de procedimento médio foi de 47,83 min para a HPG e de 48,88 min para a CDT (p = 0,843). Conclusão: O presente estudo não demonstrou diferença estatisticamente significante em relação ao sucesso técnico, sucesso clínico, complicações e tempo de procedimento entre os dois grupos estudados. Mais estudos são necessários para elucidar o papel de cada via de drenagem / Background: EUS-guided biliary access is an alternative for percutaneous access or surgery in patients with malignant unresectable distal biliary obstruction and failed ERCP. When rendezvous or anterograde transpapillary/transanastomotic intervention fails as primary drainage options, a Choledochoduodenostomy (CDT) or a Hepaticogastrostomy (HGT) can still be performed in selected patients. This procedure creates a new \" \" y I w one route or the other should be recommended. Aim: To compare technical and clinical success and possible associated factors between the two different drainage routes CDT and HGT in patients with distal unresectable malignant biliary obstruction that failed standard ERCP and EUS-guided rendez vouz (RV) maneuver. Methods: Between April/2010 and December/2013 49 consecutive jaundiced patients with distal unresectable malignant biliary obstruction that failed previous ERCP and EUS-guided RV maneuver were elected randomly to undergo either EUS-guided CDT or HGT. Data including indications, clinical and technical success, procedural times and complications with a three-month follow-up were prospectively collected in a database. All procedures were performed in a tertiary center by the same endoscopist. A partially covered SEMS (Boston Scientific, Wallflex, 10 mm, 8 cm or 6 cm) was used in all technically successful procedures. After puncture of left hepatic duct in case of HGT or the distal unobstructed segment of common bile duct in case of CDT a cholangiogram was obtained followed by advancement of a 0,035-inch guide wire into the biliary system. Bougies and wire-guided needle-knife were used to perform track dilation to allow passage of an 8.5 Fr stent delivery system. Results: Forty-nine cases (25 HGT and 24 CDT) were performed. All patients had intra and extra hepatic biliary dilation. Technical success rate was 96 % for HGT and 91% for CDT (p = 0.609). Clinical success rate was 91% for HPG and 77% for CDT (p = 0.234). In the HGT group five patients (20%) had complications (3 bleeding, 2 bilomas and 1 bacteremia). In the CDT group 3 patients (12.5%) had complications (1 biloma, 1 bleeding and 1 perforation). Only the perforation patient required surgery. All other complications were managed clinically. The median procedural time was 47.83 min for HGT and 48.88 min for CDT (p = 0.843). Conclusion: No significant difference was found in regards to technical or clinical success, complications and procedure time between the two drainage routes. More studies are needed to clarify situations in which the CDT or the HGT should be advocated

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008.

Introdução: O acesso biliar ecoguiado é um método de drenagem alternativo à drenagem percutânea transhepática (DPTH) e à cirurgia em pacientes com obstrução biliar distal incurável que falharam drenagem por Colangiopancreatografia Endoscópica Retrógrada (CPRE). Nos casos em que a drenagem ecoguiada anterógrada transpapilar (ou transanastomótica) e o rendez-vous ecoguiado não podem ser realizados como primeira opção, a coledocoduodenostomia (CDT) e a hepaticogastrostomia (HPG) ainda podem ser realizadas em pacientes selecionados. Estas duas vias de drenagem não anatômicas criam uma fístula entra a via biliar e o estômago ou duodeno. Não há dados na literatura que determinem superioridade de uma ou outra técnica. Objetivo: Comparar o sucesso técnico, sucesso clínico e fatores associados entre as duas vias de drenagem em pacientes com obstrução da via biliar distal maligna incurável que não lograram sucesso na drenagem por CPRE ou rendez-vous ecoguiado. Métodos: Entre abril de 2010 e dezembro de 2013, 49 pacientes com obstrução biliar distal maligna incurável que falharam CPRE e rendez-vous ecoguiado foram randomizados para CDT ou HPG. Dados referentes ao sucesso técnico, sucesso clínico, tempo de procedimento, complicações, qualidade de vida e sobrevida foram coletados até três meses após o procedimento. Todos os procedimentos foram realizados em um centro terciário de endoscopia pelo mesmo endoscopista. Próteses biliares parcialmente recobertas (Boston Scientific, Wallflex, 10 mm, 8 cm ou 6 cm) foram utilizadas em todos os pacientes com sucesso técnico. Nos casos de HPG a punção ecoguiada foi intra-hepática no ducto hepático esquerdo. Nos casos de CDT a punção foi extra-hepática no segmento distal não obstruído do colédoco. Após a punção foi realizada colangiografia com introdução de um fio guia hidrofílico de 0,035 polegada. Dilatação com cateter e um dispositivo de needle knife foi realizada para permitir introdução do sistema de disparo da prótese biliar com 8,5 Fr. Resultados: Quarenta e nove procedimentos foram realizados (25 HPG e 24 CDT). Todos os pacientes tinham dilatação da via biliar intra e extra-hepática. A taxa de sucesso técnico foi de 96 % para HPG e de 91% para CDT (p = 0,609). A taxa de sucesso clínico foi de 91% para o grupo HPG e de 77% para o grupo CDT (p = 0,234). No grupo da HPG 5 pacientes (20%) tiveram complicações (3 sangramentos, 2 biliomas e uma bacteremia). No grupo da CDT 3 pacientes (12,5%) tiveram complicações (1 bilioma, 1 sangramento e 1 perfuração). Somente o caso da perfuração necessitou tratamento cirúrgico. As outras complicações foram tratadas clinicamente. O tempo de procedimento médio foi de 47,83 min para a HPG e de 48,88 min para a CDT (p = 0,843). Conclusão: O presente estudo não demonstrou diferença estatisticamente significante em relação ao sucesso técnico, sucesso clínico, complicações e tempo de procedimento entre os dois grupos estudados. Mais estudos são necessários para elucidar o papel de cada via de drenagem / Background: EUS-guided biliary access is an alternative for percutaneous access or surgery in patients with malignant unresectable distal biliary obstruction and failed ERCP. When rendezvous or anterograde transpapillary/transanastomotic intervention fails as primary drainage options, a Choledochoduodenostomy (CDT) or a Hepaticogastrostomy (HGT) can still be performed in selected patients. This procedure creates a new \" \" y I w one route or the other should be recommended. Aim: To compare technical and clinical success and possible associated factors between the two different drainage routes CDT and HGT in patients with distal unresectable malignant biliary obstruction that failed standard ERCP and EUS-guided rendez vouz (RV) maneuver. Methods: Between April/2010 and December/2013 49 consecutive jaundiced patients with distal unresectable malignant biliary obstruction that failed previous ERCP and EUS-guided RV maneuver were elected randomly to undergo either EUS-guided CDT or HGT. Data including indications, clinical and technical success, procedural times and complications with a three-month follow-up were prospectively collected in a database. All procedures were performed in a tertiary center by the same endoscopist. A partially covered SEMS (Boston Scientific, Wallflex, 10 mm, 8 cm or 6 cm) was used in all technically successful procedures. After puncture of left hepatic duct in case of HGT or the distal unobstructed segment of common bile duct in case of CDT a cholangiogram was obtained followed by advancement of a 0,035-inch guide wire into the biliary system. Bougies and wire-guided needle-knife were used to perform track dilation to allow passage of an 8.5 Fr stent delivery system. Results: Forty-nine cases (25 HGT and 24 CDT) were performed. All patients had intra and extra hepatic biliary dilation. Technical success rate was 96 % for HGT and 91% for CDT (p = 0.609). Clinical success rate was 91% for HPG and 77% for CDT (p = 0.234). In the HGT group five patients (20%) had complications (3 bleeding, 2 bilomas and 1 bacteremia). In the CDT group 3 patients (12.5%) had complications (1 biloma, 1 bleeding and 1 perforation). Only the perforation patient required surgery. All other complications were managed clinically. The median procedural time was 47.83 min for HGT and 48.88 min for CDT (p = 0.843). Conclusion: No significant difference was found in regards to technical or clinical success, complications and procedure time between the two drainage routes. More studies are needed to clarify situations in which the CDT or the HGT should be advocated

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

The authors report a case of endometriosis that presented as a cystic mass in the tail of the pancreas, leading to extensive evaluation and ultimately a major surgical resection. The diagnosis was made by histopathological evaluation, revealing endometrial glands and stroma in the wall of the mass with hemorrhagic fluid in the cystic lumen, compatible with pancreatic involvement by an endometrial cyst.

L’objectif principal de cette étude est de déterminer la valeur pronostique de l’infiltrat lymphocytaire dans l’adénocarcinome du pancréas. Les densités des lymphocytes T CD3+, CD4+, CD8+, FOXP3+ et CD45RO+ intratumoraux (T) et péritumoraux (PT) de 111 spécimens ont été mesurées avec des micromatrices tissulaires. Un Index Lymphocytaire (IL) a été créé basé sur les valeurs des CD4+ T, CD8+ PT et le ratio CD3+ T/PT regroupant les patients selon que les tumeurs présentaient aucune (IL---), 1 à 2 (IL+/-) ou les 3 caractéristiques immunitaires favorables (IL+++). La survie médiane des patients atteints d’un cancer du pancréas est significativement différente selon la catégorie d’index lymphocytaire; elle était de 14 mois pour IL---, de 19 mois pour IL +/- et de 29 mois pour IL+++ (p=0,01). L’IL est un facteur indépendant de survie en analyse multivariée ainsi que la différenciation tumorale et l’utilisation d’un traitement adjuvant. L’IL est un facteur pronostique de survie des adénocarcinomes du pancréas réséqués et devrait pouvoir permettre une meilleure classification des patients. / The main objective of this project was to determine the prognostic significance of T-lymphocytes densities in pancreatic adenocarcinoma. Using tissue microarrays, CD3+, CD4+, CD8+, CD45RO+, and FOXP3 T-cells were quantified by immunohistochemistry in the intratumoral (T) and peritumoral (PT) compartments of 111 specimens. An immune score (IS) based on T CD4+ and PT CD8+ counts and T/PT CD3+ ratio grouped patients into IS---, IS+/- and IS+++ if none, 1 or 2, or all 3 of the favorable immune features were present, respectively. Patients have different overall survival (OS) depending on their IS status. Patients IS--- have a median OS of 14 months and of 19 months and 29 months for those IS+/- and IS+++ respectively (log-rank p=0.01). By multivariate analysis, IS was independently associated with survival as were the histological grade and adjuvant therapy. An immune score that combines specific T-cell location and density may have prognostic value in patients with resected pancreatic adenocarcinoma, independently from current pathologic features.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy. / Dissertation