Tu2058 LRP6 Overexpression as a Potential Marker of Early Stage Tumor Progression in Pancreatic...
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Tu2055 Evaluation of Chromosal Aberrations in the Primary Tumor, Lymph Node Metastases and Disseminated Tumor Cells of Patients With Esophageal Cancer: Implications for Anti-Tumoral Therapy? Daniel Vallbohmer, Sarah Schumacher, Stephan E. Baldus, Christian Vay, Andreas Krieg, Jan Schulte am Esch, Wolfram T. Knoefel, Nikolas H. Stoecklein Introduction: Recent analyses uncovered genetic variations between paired samples from primary gastrointestinal tumors, lymph node metastases and disseminated tumor cells (DTCs). These findings might help to explain individually variable responses to standard (neo-)adjuvant therapies and further suggest that multimodality treatment options in gastroin- testinal cancer should be guided by these individual genetic tumor characteristics. Therefore, we assessed the genetic variations in the primary tumor, lymph node metastases and DTCs of patients with esophageal cancer. Patients and Methods: In this translational analysis 86 patients with esophageal cancer undergoing multimodality therapy were included. Initially, we established a protocol for double immunofluorescence labeling for simultaneous visualiza- tion of epithelial cell adhesion molecule (EpCAM) expression on cytokeratin positive cells for the detection of DTCs in bone marrow and lymph nodes. After isolation of positively stained cells, their genomic DNA was globally amplified using the MSE-adapter PCR method. Finally, we applied comparative genomic hybridization (CGH) for the genome-wide screening of DNA-gains/-losses on paired samples from primary tumors, lymph node metastases and DTCs of the study patients. Results: DTCs were detected in 25% of the bone marrow and 38% of the lymph node samples. Interestingly, CGH analysis revealed differences between the numbers of chromosal aberrations in DTCs of the bone marrow compared to the lymph node samples with a higher frequency of aberrations in DTCs in the lymph node samples. In addition, genomic analysis revealed differences in the nature of chromosomal aberrations between primary tumors and corresponding lymph node metastases. Moreover, cluster analysis demonstrated similarities of the aberration spectrum between the DTCs and lymph node metastases while primary tumors showed distinct profiles. Conclusion: Chromosal aberration patterns in lymph node metastases and disseminated tumor cells of patients with esophageal cancer undergoing multimodality therapy are very similar while primary tumors show a different genomic aberration pattern. These individual genetic tumor characteristics might guide future multimodality treatment options in esophageal cancer. Tu2056 Sevofluorane Reduces Liver Damage Secondary to Ischemic/Reperfusion Injury by a Mechanism Not Related to a Preconditioning Effect Fernanda P. Cavalcante, Ana Maria M. Coelho, Marcel C. Machado, Sandra N. Sampietre, Nilza A. Molan, Eleazar Chaib, Luiz C. D'Albuquerque Background/Aim: Previous studies have demonstrated that sevoflurane protects liver from ischemia/reperfusion (I/R) injury however it was not shown yet if this protection is by preconditioning or if it depends on a continuous administration of the anesthetic during the whole I/R period. In the present study we evaluated the mechanism of the protective effect of sevoflurane in ischemia/reperfusion injury Methods: Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral segments. Liver pedicle clamp was removed after 1 hour of partial ischemia. Anesthesia was induced with cetamine and xylazine and rats were intubated and mechanical ventilated. Rats were divided in 3 groups: Group1-Sevo Continued (n=15): sevoflurane was administered during the whole I/R injury time and animals remained intubated during the whole I/R time, Group 2-Sevo 30 minutes (n=15): sevoflurane was administered during 30 minutes and discontinued before liver ischemia, and Group-3 Control (n=15): animals was submitted to I/R and no sevoflurane was administrated. Just as group 2, rats were extubated after reperfusion. Four hours after reperfusion blood was collected for determinations of AST, ALT. Liver tissues were assembled mitochondrial oxidation and phosphorylation and malond- ialdehyde (MDA) content. Pulmonary vascular permeability and myeloperoxidade (MPO) were also determined. Results: Four hours after reperfusion Sevo Continued group presented elevation of AST and ALT serum levels significantly lower than Sevo 30 minutes and Control groups (p<0.05). A significant reduction on liver mitochondrial dysfunction and pulmonary vascular permeability was observed in Sevo Continued group compared to Sevo 30 minutes and Control groups (p<0.05). No differences in liver MDA and pulmonary MPO activity were observed Conclusion: Sevoflurane attenuates liver ischemia/reperfusion injury probably by a mechanism not related to a by preconditioning effect. Tu2057 Anti-Inflammatory Effects of Hypertonic Saline Solution in Pancreatic Ischemia/Reperfusion Injuries Renato S. Godoy, Ana Maria M. Coelho, Sandra N. Sampietre, Nilza A. Molan, Oscar M. Takayanagi, Marcel C. Machado, José Jukemura, Luiz C. D'Albuquerque Background/Aim: Injury caused by ischemia/reperfusion (I/R) may result in pancreatic graft loss in pancreas transplants. Therapeutics strategies to reduce pancreatic I/R injury are extremely important to improve the outcomes of clinical transplantation. We have previously demonstrated that hypertonic saline 7.5% had anti-inflammatory response in acute pancreat- itis and liver ischemia/reperfusion models. The aim of this study was to evaluate the effects of hypertonic saline solution 7.5% in I/R pancreatic. Methods: Pancreatic ischemia was performed in Wistar rats during one hour by clamping the splenic vessels under mechanical ventilation. The vascular clamp was removed 1 hour after ischemia and pancreatic revascu- larization was achieved, followed by 4h or 24h of reperfusion. The animals divided into 3 groups: Group NT (n=20): subjected to pancreatic I/R without treatment; Group NS (n= 20): subjected to I/R and treated with normal saline solution (NaCl 0.9%), 15 minutes before reperfusion; Group HTS (n=20): subjected to I/R pancreatic and treated with hypertonic saline solution (NaCl 7.5%), 15 minutes before reperfusion. Four and twenty four hours after reperfusion blood were collected for determinations of amylase, TNF-α, IL-6, and IL-10, creatinine, urea. Pancreatic malondialdehyde (MDA) content was also performed. After 24hours of reperfusion pulmonary tissues were assembled for myeloperoxidade (MPO) S-1107 SSAT Abstracts analyses. Results: There was a decrease of inflammatory cytokines in the Group HTS compared with control, NT and NS groups. It was observed a significant decrease in serum urea and creatinine in the animals treated with normal (NS) and hypertonic saline (HTS) compared to not treated animals (NT). The serum amylase levels and the determination of pancreatic MDA showed no significant differences between groups with I/R. Conclusions: Hypertonic saline solution decreases the systemic inflammatory response by cytokines reduction (TNF- α, IL-6, and IL-10)in pancreatic I/R injury. Further studies will be necessary to prove the clinical benefits in patients subject to pancreatic transplantation. Tu2058 LRP6 Overexpression as a Potential Marker of Early Stage Tumor Progression in Pancreatic Ductal Adenocarcinoma Nicolas Zea, William C. Conway, John S. Bolton, Nancy K. Davis, Cruz Velasco, Paul B. Fossier, Jovanny Zabaleta Introduction: The Wnt-β-Catenin signaling pathway, in particular the canonical pathway, has been implicated in pancreatic ductal adenocarcinoma (PDAC) development. Since muta- tions in the key intracellular components of this pathway are rare in PDAC, understanding the molecular mechanisms by which the signaling pathway is aberrantly activated, and how it influences tumor behavior, is of utmost importance. In this study, we hypothesized that over-expression of components upstream of the signaling pathway, in particular the Wnt signaling co-receptor LRP6, are involved in PDAC tumorigenesis. Methods: Twelve lymph node negative (LN-) and twelve lymph node positive (LN+) paraffin embedded tumor tissues were randomly selected to perform screening gene identification via gene chip microarray analysis. Once genes of interest were identified by fold-change, 61 tumor samples were obtained and then subcategorized in terms of lymph node status, survival time, and grade of differentiation and used to validate the results using real-time PCR (RT-PCR). Results: 20,817 genes were investigated with the microarray analysis. Using gene chip microarray software, we removed the background and used scatter graphs to select those genes with at least 2-fold difference (up or down) between LN- and LN+. Further selection by p value (p<0.05) identified 957 genes significantly different between the two groups. The LRP6 gene expression showed a 2.46-fold increase in the LN- when compared to LN+ samples (1192.9 vs 485). RT-PCR for LRP6 in LN- (n=29) and LN+ (n=32) confirmed results of the microarray (p-value=0.00044). In addition, LRP6 showed a trend of over-expression towards tumors of lower grades of differentiation (table 1). In terms of survival time, no statistical significance was found between LN- and LN+. Conclusions: The Wnt signaling co-receptor LRP6 is one of the most upstream genes involved in the Wnt-β-Catenin signaling pathway. Our data shows that LRP6 is significantly over-expressed in patients with negative nodal status, as well as portraying a tendency of over-expression in lower differentiation grades of pancreatic tumor. Our results reflect an over-expression of LRP6 early in the series of tumorigenesis events and depict the importance of further studies to understand its relationship to tumor behavior and prognosis. Difference within tumor samples regarding grade of differentiation Tu2059 A Neurokinin-1 Receptor (NK1R) Antagonist (NK-1RA) That Reduces Postoperative Adhesions Reduces the Adhesion Related Chemokines Cxcl1(Kc) and Cxcl2 (MIP-2) and Their Receptor, CXCR2 Hisashi Kosaka, Michael R. Cassidy, Arthur F. Stucchi, James M. Becker Introduction: Postoperative adhesions occur in 90-100% of patients after abdominopelvic surgery. We previously showed that adhesiogenesis is associated with leukocyte migration into the peritoneum and that the NK1R plays an important role in adhesiogenesis. The aim of this study was to characterize the temporal changes and the effects of a NK-1RA on the gene expression of the leukocyte chemoattractants CXCL1 and CXCL2 during adhesiogenesis. Methods: Adhesions were induced in mice using our previously published cecal cauterization model. Wild-type (WT) mice (n =7/group) were administered saline (WT+S) or a NK-1RA (WT+NK) (25mg/kg) intraperitoneally at surgery. At 0, 3, and 6-hrs post-operatively, cecal adhesion tissue was measured for CXCL1, CXCL2 and CXCR2 mRNA levels by real-time- PCR. Adhesions were measured on POD 7 (n=7/group) in WT, WT+NK and CXCR2 knockout (KO) mice. RESULTS: In WT+S mice, CXCL1 and CXCL2 mRNA levels increased at 3-hrs post-operatively compared to non-operated controls (380.8±143 vs 0.04±0.08; p<0.05; 198.3±105 vs 0.07±0.05; p<0.05) while CXCR2 mRNA increased at 6-hrs (16.5±2.9 vs 0.17±0.06; p<0.05). Administration of the NK-1RA significantly reduced mRNA levels of CXCL1 and CXCL2 3hrs post-operatively compared to controls (380.8±143 vs 89.9±23.7; p<0.05; 198.3±105 vs 22.7±6.2; p<0.05) while CXCR2 mRNA levels were reduced by 64% (16.5±2.8 vs 5.9±1.6; p<0.05) at 6hrs. Adhesion formation was reduced in both WT+NK and CXCR2 KO mice by 55% (4.8±0.16 vs 2.2±0.19; p<0.05) and 56% (5.0±0.0 vs 2.2±0.2; p<0.05), respectively compared to WT+S. CONCLUSIONS: These data suggest that the NK- 1R mediates leukocyte migration into the peritoneum indicating a new role for the NK-1RA in adhesiogenesis. SSAT Abstracts
Transcript of Tu2058 LRP6 Overexpression as a Potential Marker of Early Stage Tumor Progression in Pancreatic...
Tu2055
Evaluation of Chromosal Aberrations in the Primary Tumor, Lymph NodeMetastases and Disseminated Tumor Cells of Patients With EsophagealCancer: Implications for Anti-Tumoral Therapy?Daniel Vallbohmer, Sarah Schumacher, Stephan E. Baldus, Christian Vay, Andreas Krieg,Jan Schulte am Esch, Wolfram T. Knoefel, Nikolas H. Stoecklein
Introduction: Recent analyses uncovered genetic variations between paired samples fromprimary gastrointestinal tumors, lymph node metastases and disseminated tumor cells(DTCs). These findings might help to explain individually variable responses to standard(neo-)adjuvant therapies and further suggest thatmultimodality treatment options in gastroin-testinal cancer should be guided by these individual genetic tumor characteristics. Therefore,we assessed the genetic variations in the primary tumor, lymph node metastases and DTCsof patients with esophageal cancer. Patients and Methods: In this translational analysis 86patients with esophageal cancer undergoing multimodality therapy were included. Initially,we established a protocol for double immunofluorescence labeling for simultaneous visualiza-tion of epithelial cell adhesion molecule (EpCAM) expression on cytokeratin positive cellsfor the detection of DTCs in bone marrow and lymph nodes. After isolation of positivelystained cells, their genomic DNA was globally amplified using the MSE-adapter PCR method.Finally, we applied comparative genomic hybridization (CGH) for the genome-wide screeningof DNA-gains/-losses on paired samples from primary tumors, lymph node metastases andDTCs of the study patients. Results: DTCs were detected in 25% of the bone marrow and38% of the lymph node samples. Interestingly, CGH analysis revealed differences betweenthe numbers of chromosal aberrations in DTCs of the bone marrow compared to the lymphnode samples with a higher frequency of aberrations in DTCs in the lymph node samples.In addition, genomic analysis revealed differences in the nature of chromosomal aberrationsbetween primary tumors and corresponding lymph node metastases. Moreover, clusteranalysis demonstrated similarities of the aberration spectrum between the DTCs and lymphnode metastases while primary tumors showed distinct profiles. Conclusion: Chromosalaberration patterns in lymph node metastases and disseminated tumor cells of patients withesophageal cancer undergoing multimodality therapy are very similar while primary tumorsshow a different genomic aberration pattern. These individual genetic tumor characteristicsmight guide future multimodality treatment options in esophageal cancer.
Tu2056
Sevofluorane Reduces Liver Damage Secondary to Ischemic/Reperfusion Injuryby a Mechanism Not Related to a Preconditioning EffectFernanda P. Cavalcante, Ana Maria M. Coelho, Marcel C. Machado, Sandra N. Sampietre,Nilza A. Molan, Eleazar Chaib, Luiz C. D'Albuquerque
Background/Aim: Previous studies have demonstrated that sevoflurane protects liver fromischemia/reperfusion (I/R) injury however it was not shown yet if this protection is bypreconditioning or if it depends on a continuous administration of the anesthetic duringthe whole I/R period. In the present study we evaluated the mechanism of the protectiveeffect of sevoflurane in ischemia/reperfusion injury Methods: Wistar male rats underwentpartial liver ischemia performed by clamping the pedicle from medium and left anteriorlateral segments. Liver pedicle clamp was removed after 1 hour of partial ischemia. Anesthesiawas induced with cetamine and xylazine and rats were intubated and mechanical ventilated.Rats were divided in 3 groups: Group1-Sevo Continued (n=15): sevoflurane was administeredduring the whole I/R injury time and animals remained intubated during the whole I/Rtime, Group 2-Sevo 30 minutes (n=15): sevoflurane was administered during 30 minutesand discontinued before liver ischemia, and Group-3 Control (n=15): animals was submittedto I/R and no sevoflurane was administrated. Just as group 2, rats were extubated afterreperfusion. Four hours after reperfusion blood was collected for determinations of AST,ALT. Liver tissues were assembledmitochondrial oxidation and phosphorylation andmalond-ialdehyde (MDA) content. Pulmonary vascular permeability and myeloperoxidade (MPO)were also determined. Results: Four hours after reperfusion Sevo Continued group presentedelevation of AST and ALT serum levels significantly lower than Sevo 30 minutes and Controlgroups (p<0.05). A significant reduction on liver mitochondrial dysfunction and pulmonaryvascular permeability was observed in Sevo Continued group compared to Sevo 30 minutesand Control groups (p<0.05). No differences in liver MDA and pulmonary MPO activitywere observed Conclusion: Sevoflurane attenuates liver ischemia/reperfusion injury probablyby a mechanism not related to a by preconditioning effect.
Tu2057
Anti-Inflammatory Effects of Hypertonic Saline Solution in PancreaticIschemia/Reperfusion InjuriesRenato S. Godoy, Ana Maria M. Coelho, Sandra N. Sampietre, Nilza A. Molan, Oscar M.Takayanagi, Marcel C. Machado, José Jukemura, Luiz C. D'Albuquerque
Background/Aim: Injury caused by ischemia/reperfusion (I/R) may result in pancreatic graftloss in pancreas transplants. Therapeutics strategies to reduce pancreatic I/R injury areextremely important to improve the outcomes of clinical transplantation. We have previouslydemonstrated that hypertonic saline 7.5% had anti-inflammatory response in acute pancreat-itis and liver ischemia/reperfusion models. The aim of this study was to evaluate the effectsof hypertonic saline solution 7.5% in I/R pancreatic. Methods: Pancreatic ischemia wasperformed in Wistar rats during one hour by clamping the splenic vessels under mechanicalventilation. The vascular clamp was removed 1 hour after ischemia and pancreatic revascu-larization was achieved, followed by 4h or 24h of reperfusion. The animals divided into 3groups: Group NT (n=20): subjected to pancreatic I/R without treatment; Group NS (n=20): subjected to I/R and treated with normal saline solution (NaCl 0.9%), 15 minutes beforereperfusion; GroupHTS (n=20): subjected to I/R pancreatic and treated with hypertonic salinesolution (NaCl 7.5%), 15 minutes before reperfusion. Four and twenty four hours afterreperfusion blood were collected for determinations of amylase, TNF-α, IL-6, and IL-10,creatinine, urea. Pancreatic malondialdehyde (MDA) content was also performed. After24hours of reperfusion pulmonary tissues were assembled for myeloperoxidade (MPO)
S-1107 SSAT Abstracts
analyses. Results: There was a decrease of inflammatory cytokines in the GroupHTS comparedwith control, NT and NS groups. It was observed a significant decrease in serum urea andcreatinine in the animals treated with normal (NS) and hypertonic saline (HTS) comparedto not treated animals (NT). The serum amylase levels and the determination of pancreaticMDA showed no significant differences between groups with I/R. Conclusions: Hypertonicsaline solution decreases the systemic inflammatory response by cytokines reduction (TNF-α, IL-6, and IL-10)in pancreatic I/R injury. Further studies will be necessary to prove theclinical benefits in patients subject to pancreatic transplantation.
Tu2058
LRP6 Overexpression as a Potential Marker of Early Stage Tumor Progressionin Pancreatic Ductal AdenocarcinomaNicolas Zea, William C. Conway, John S. Bolton, Nancy K. Davis, Cruz Velasco, Paul B.Fossier, Jovanny Zabaleta
Introduction: The Wnt-β-Catenin signaling pathway, in particular the canonical pathway,has been implicated in pancreatic ductal adenocarcinoma (PDAC) development. Since muta-tions in the key intracellular components of this pathway are rare in PDAC, understandingthe molecular mechanisms by which the signaling pathway is aberrantly activated, and howit influences tumor behavior, is of utmost importance. In this study, we hypothesized thatover-expression of components upstream of the signaling pathway, in particular the Wntsignaling co-receptor LRP6, are involved in PDAC tumorigenesis. Methods: Twelve lymphnode negative (LN-) and twelve lymph node positive (LN+) paraffin embedded tumor tissueswere randomly selected to perform screening gene identification via gene chip microarrayanalysis. Once genes of interest were identified by fold-change, 61 tumor samples wereobtained and then subcategorized in terms of lymph node status, survival time, and gradeof differentiation and used to validate the results using real-time PCR (RT-PCR). Results:20,817 genes were investigated with the microarray analysis. Using gene chip microarraysoftware, we removed the background and used scatter graphs to select those genes withat least 2-fold difference (up or down) between LN- and LN+. Further selection by p value(p<0.05) identified 957 genes significantly different between the two groups. The LRP6 geneexpression showed a 2.46-fold increase in the LN- when compared to LN+ samples (1192.9vs 485). RT-PCR for LRP6 in LN- (n=29) and LN+ (n=32) confirmed results of the microarray(p-value=0.00044). In addition, LRP6 showed a trend of over-expression towards tumorsof lower grades of differentiation (table 1). In terms of survival time, no statistical significancewas found between LN- and LN+. Conclusions: The Wnt signaling co-receptor LRP6 is oneof the most upstream genes involved in the Wnt-β-Catenin signaling pathway. Our datashows that LRP6 is significantly over-expressed in patients with negative nodal status, aswell as portraying a tendency of over-expression in lower differentiation grades of pancreatictumor. Our results reflect an over-expression of LRP6 early in the series of tumorigenesisevents and depict the importance of further studies to understand its relationship to tumorbehavior and prognosis.Difference within tumor samples regarding grade of differentiation
Tu2059
A Neurokinin-1 Receptor (NK1R) Antagonist (NK-1RA) That ReducesPostoperative Adhesions Reduces the Adhesion Related ChemokinesCxcl1(Kc) and Cxcl2 (MIP-2) and Their Receptor, CXCR2Hisashi Kosaka, Michael R. Cassidy, Arthur F. Stucchi, James M. Becker
Introduction: Postoperative adhesions occur in 90-100% of patients after abdominopelvicsurgery. We previously showed that adhesiogenesis is associated with leukocyte migrationinto the peritoneum and that the NK1R plays an important role in adhesiogenesis. The aimof this study was to characterize the temporal changes and the effects of a NK-1RA on thegene expression of the leukocyte chemoattractants CXCL1 and CXCL2 during adhesiogenesis.Methods: Adhesions were induced in mice using our previously published cecal cauterizationmodel. Wild-type (WT) mice (n =7/group) were administered saline (WT+S) or a NK-1RA(WT+NK) (25mg/kg) intraperitoneally at surgery. At 0, 3, and 6-hrs post-operatively, cecaladhesion tissue was measured for CXCL1, CXCL2 and CXCR2 mRNA levels by real-time-PCR. Adhesions weremeasured on POD7 (n=7/group) inWT,WT+NK and CXCR2 knockout(KO) mice. RESULTS: In WT+S mice, CXCL1 and CXCL2 mRNA levels increased at 3-hrspost-operatively compared to non-operated controls (380.8±143 vs 0.04±0.08; p<0.05;198.3±105 vs 0.07±0.05; p<0.05) while CXCR2 mRNA increased at 6-hrs (16.5±2.9 vs0.17±0.06; p<0.05). Administration of the NK-1RA significantly reduced mRNA levels ofCXCL1 and CXCL2 3hrs post-operatively compared to controls (380.8±143 vs 89.9±23.7;p<0.05; 198.3±105 vs 22.7±6.2; p<0.05) while CXCR2 mRNA levels were reduced by 64%(16.5±2.8 vs 5.9±1.6; p<0.05) at 6hrs. Adhesion formation was reduced in both WT+NKand CXCR2 KO mice by 55% (4.8±0.16 vs 2.2±0.19; p<0.05) and 56% (5.0±0.0 vs 2.2±0.2;p<0.05), respectively compared to WT+S. CONCLUSIONS: These data suggest that the NK-1R mediates leukocyte migration into the peritoneum indicating a new role for the NK-1RAin adhesiogenesis.
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