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174 Expression of Oncogenic H-RASG12v Does Not Induce Malignant Transformation or Senescence in Barrett's Epithelial Cells
the pathogenesis of Barrett's-associated dysplasia and others that appear to be novel candidates for further investigation.
Reduced Epidermal Growth Factor (EGF) Expression Is Associated with An Increased Risk for the Reflux-Induced Cascade Causing Barrett's Esophagus and Esophageal Adenocarcinoma Vivianda Menke, Peter D. Siersema, Leon M. Moons, Katinka P. van Zoest, Raymond G. Pot, Bettina E. Hansen, Herman van Dekken, Johannes G. Kusters, Ernst J. Kuipers
*p<0,05;**, p<0,01 Table 1. Gene Ontology for differentially (p <0.05) expressed genes
Introduction: Barrett's esophagus (BE) is a chronic inflammatory metaplastic condition of the esophagus, often preceded by reflux esophagitis (RE). BE is the main risk factor for the development of esophageal adenocarcinoma (EAC). Little is known about the esophageal resistance against continuous bile and acid stimuli during RE and BE. Binding of epidermal growth factor (EGF) to its receptor (EGFR) promotes a variety of processes involved in mucosal protection and repair following injury, and EGFR overexpression has been associated with gastrointestinal tumors. Aim: To establish the effect of the EGF (+61 A/G) gene polymorphism on local expression levels and progression in the RE-BE-EAC cascade. Methods: DNA was obtained from 871 unrelated Dutch Caucasians consisting of 198 healthy controls (mean age 56 ± 15y; 58% male), 298 patients (mean age 55 ± 14y; 55% male) with endoscopically confirmed RE, 246 patients (mean age 61 ± 12y; 68% male) with BE, with a segment of ≥2 cm specialized intestinal metaplasia, and 129 patients (mean age 63 ± 10y; 82% male) with EAC. EGF genotypes (+61 A/G) were determined by modified TAQMAN assays. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (CI) for variant genotypes. Immunohistochemistry for EGF and EGFR, to exclude an indirect effect of EGF on its receptor, was performed on esophageal biopsies from a subgroup of BE patients. Multivariate analysis was used to calculate p-values for variant expression. Results: EGF G/G homozygosity was increased in RE (OR=2.6; 95% CI:1.3-5.2), BE (OR= 3.0; 95% CI:1.5-6.2), and EAC (OR=4.1; 95% CI:1.7-9.7) compared to healthy controls. Local esophageal EGF expression was significantly lower in BE patients with G/G homozygosity compared to those with A/G heterozygosity (p=0.008) and A/A homozygosity (p=0.002). We found that EGFR was strongly expressed in all BE tissues, independent of EGF genotypes or EGF expression levels in BE tissue. Conclusion: The EGF G/G polymorphism is associated with reduced EGF expression and an increased risk of developing RE, BE and EAC. This indicates that reduced EGF expression is a limiting factor in mucosal protection against esophageal tumor development.
159 Comprehensive Self-Management for Irritable Bowel Syndrome Works Whether Delivered By Telephone or in-Person Monica Jarrett, Kevin C. Cain, Vicky Hertig, Robert L. Burr, Sheldon N. Rosen, Margaret Heitkemper Objective. Determine the effectiveness of a comprehensive self-management (CSM) intervention delivered via telephone (T-CSM) or in-person (IP-CSM) as compared to usual care (UC) for adults with IBS. Methods. Potential participants were recruited through community advertisements and screened for eligibility criteria during the baseline period. 188 participants were randomized to T-CSM, IP-CSM or UC. Both CSM interventions were delivered weekly over 9 weeks, and addressed nutritional, cognitive and behavioral strategies for managing IBS symptoms. Outcomes were measured at baseline and 3, 6, and 12 months follow-up times. Two primary outcome variables were analyzed: a) IBS symptom severity (IBS-SS) from a daily diary, measured as the percent of days with at least one IBS symptom (abdominal pain, bloating, constipation, diarrhea, intestinal gas, urgency) being moderate or worse, and b) IBS-specific quality of life (QOL) measured on a 0-100 scale. Mixed-model analyses were used to analyze data from the three follow-up times, controlling for baseline values of IBS symptom severity, QOL, and psychological distress as covariates. Results. The two primary outcome variables showed highly significant treatment differences with both T-CSM and IP-CSM having better outcomes than UC (p <.003). There were no significant differences between T-CSM and IP-CSM. The treatment effect was strongest at 3 months: The percent of subjects whose IBS-SS decreased by at least 15 percentage points was 59% and 70% for T-CSM and IP-CSM, compared to 26% for UC; the percent of subjects whose QOL improved by at least 10 points was 40% and 42% for T-CSM and IP-CSM, compared to 18% for UC. Treatment effect at 12 months was still strong for IBS-SS (62% and 58% improved for TCSM and IP-CSM, versus 27% or UC) but was somewhat weaker for QOL due mainly to improvement in the UC group (27% and 37% improvement for T-CSM and IP-CSM versus 27% for UC). Many of the secondary outcome variables showed similar results, with T-CSM and IP-CSM both better than UC, and the two delivery modalities being about equally effective. Conclusions. Comprehensive self-management therapy leads to improvement in IBS symptoms and quality of life, and this improvement is still maintained at 12 months. Delivery of the therapy by telephone appears to be just as effective as in-person delivery. Since some subjects expressed a preference for in-person, while others expressed a preference for telephone, perhaps patients could be offered a choice of delivery modality.
174 Expression of Oncogenic H-RASG12v Does Not Induce Malignant Transformation or Senescence in Barrett's Epithelial Cells. Xi Zhang, Hui Ying Zhang, Stuart J. Spechler, Rhonda F. Souza Introduction: Although oncogenes are known to promote cell growth, they also can activate tumor suppressor pathways that induce senescence, an irreversible state of growth arrest. To become malignant, cells must acquire the ability to avoid senescence. In esophageal squamous cells, oncogenes have been shown to induce senescence by activating the p16 tumor suppressor pathway. In human fibroblasts deficient in p16, in contrast, oncogenes induce malignant transformation. In Barrett's metaplasia, p16 inactivation occurs early and frequently. Therefore, we hypothesized that oncogenes should transform p16-deficient Barrett's cells, and we tested that hypothesis using a telomerase-immortalized, non-neoplastic Barrett's epithelial (BAR-T) cell line. Methods: BAR-T cells, which are deficient in p16 but not in p53, were stably infected with the retroviral vector pBabe-zeocin containing oncogenic H-RasGV12; pBabe-zeocin without the ras insert served as a control. Expression levels of HRas, phospho-MEK1/2, phospho-ERK1/2, p53, and p21 were determined by Western blotting using β-actin as a control. Growth rates were determined by population doubling times. Cell contact inhibition and anchorage-independent growth in soft agar were used to determine neoplastic transformation; SEG-1 esophageal adenocarcinoma cells served as a positive control. Results: Infection with pBabe-H-RasG12V substantially increased H-ras expression in BAR-T cells compared to uninfected and empty vector-infected control cells. Two clones with the highest levels of H-ras expression (R1 and R7) were selected for further analyses. Expression levels of phospho-MEK1/2 and ERK1/2 were increased in those clones, indicating that they maintained functional Ras activity. Compared with empty-vector controls, H-ras expression did not increase p53 or p21 expression levels. There were no significant differences in population doubling times among H-Ras-containing clones (37.6 ± .7 hours SEM clone R1; 35 ± .5 hours clone R7) and empty vector-infected controls (36.5 ± .8 hours). HRas-containing cells demonstrated growth inhibition upon contact and lacked anchorageindependent growth after 3 weeks in soft agar unlike SEG-1 cells, which developed numerous colonies in soft agar. Conclusions: Expression of H-RasG12V does not induce senescence or malignant transformation in p16-deficient Barrett's epithelial cells. These findings suggest that oncogene expression and avoidance of oncogene-induced senescence are not sufficient for the malignant transformation of Barrett's epithelial cells. Alterations in additional pathways appear to be necessary for cancer development in Barrett's esophagus.
172 Novel Potential Markers of High Grade Dysplasia in Barrett's Esophagus Identified By cDNA Microarray Analysis of Laser Capture Microdissected Archival Tissue Edmond Sabo, Steven F. Moss, Patricia Meitner, Rose Tavares, John Gao, Christopher Corless, Gregory Y. Lauwers, Murray B. Resnick Background: The molecular pathogenesis of the transition from intestinal metaplasia to cancer in Barrett's esophagus (BE) is poorly defined. Identifying genes differentially expressed in BE without dysplasia (IM) from those expressed in high grade dysplasia (HGD) should be of value in improving our understanding of this transition and their differential expression might be of diagnostic and prognostic utility. Aim: To determine the differential transcriptome of HGD compared with non-dysplastic BE through gene microarray analysis of epithelial cells microdissected from archival formalin fixed tissue specimens. Methods: Laser capture microdissection (LCM) was used to isolate epithelial cells from surrounding inflammatory and stromal cells. Epithelial cell mRNA was extracted from areas of IM and HGD in the same patients from archival tissue sections. mRNA was reverse transcribed and applied onto Affymetrix cDNA microarray chips customized for mRNA from paraffinized, formalinexposed tissue. Differentially expressed genes were identified by the significance analysis of microarrays method and a false detection rate of <20%. For a subset of these genes, differential gene expression was verified by real-time PCR and immunohistochemistry in a further 10 IM and HGD cases. Results: There were 168 genes over-expressed by at least two-fold in HGD versus IM. 72 genes were under-expressed by at least two-fold in HGD. Among the over-expressed genes are several previously demonstrated to be increased in HGD as well as novel candidate genes involved in neoplastic progression including lipocalin 2, S-100A9 (calgranulin B, a component of calprotectin), matrix metallopeptidase 12 secernin 1 and topoisomerase II A. Genes decreased in HGD compared with IM include mucins 3A, 5A and 5B, trefoil factors 1 and 2, meprin A, and alanyl-aminopeptidase. Real-time PCR validated the changes in expression of 8/8 genes examined, including lipocalin 2, S-100A9, secernin 1, trefoil factor 1, meprin A and alanyl-aminopeptidase. Immunohistochemistry confirmed altered expression at the protein level for topoisomerase IIA, S-100A9 and lipocalin . Conclusions: This is the first study to identify by LCM genes differentially expressed in HGD compared with IM in the same patients. This was achieved by accessing esophageal epithelial cells from archival paraffin embedded specimens using customized Affymetrix cDNA microarrays. The differentially expressed genes identified include several previously implicated in
175 Sequential Chromosomal Events Leading to HER-2 Proto-Oncogene Amplification in Barrett's Esophagus Rygiel M. Agnieszka, Francesca Milano, Fiebo J. ten Kate, Brenda Elzer, Paul Fockens, Jacques J. Bergman, Maikel P. Peppelenbosch, Kausilia K. Krishnadath BACKROUND: Her-2 proto-oncogene amplification is an important marker in Barrett's esophagus associated esophageal adenocarcinoma (EAC), correlating with poor prognosis of EAC patients. However, studies showing the sequential genetic events preceding Her-2 amplification in Barrett's esophagus (BE) in the no dysplasia-dysplasia-adenocarcinomasequence are lacking. AIM: The aim was to study sequential chromosomal events that may lead to Her-2 locus amplification in BE and EAC. METHODS: Her-2 locus (17q11.2) and
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Reduced Epidermal Growth Factor (EGF) Expression Is Associated with An Increased Risk for the Reflux-Induced Cascade Causing Barrett's Esophagus and Esophageal Adenocarcinoma Vivianda Menke, Peter D. Siersema, Leon M. Moons, Katinka P. van Zoest, Raymond G. Pot, Bettina E. Hansen, Herman van Dekken, Johannes G. Kusters, Ernst J. Kuipers
*p<0,05;**, p<0,01 Table 1. Gene Ontology for differentially (p <0.05) expressed genes
Introduction: Barrett's esophagus (BE) is a chronic inflammatory metaplastic condition of the esophagus, often preceded by reflux esophagitis (RE). BE is the main risk factor for the development of esophageal adenocarcinoma (EAC). Little is known about the esophageal resistance against continuous bile and acid stimuli during RE and BE. Binding of epidermal growth factor (EGF) to its receptor (EGFR) promotes a variety of processes involved in mucosal protection and repair following injury, and EGFR overexpression has been associated with gastrointestinal tumors. Aim: To establish the effect of the EGF (+61 A/G) gene polymorphism on local expression levels and progression in the RE-BE-EAC cascade. Methods: DNA was obtained from 871 unrelated Dutch Caucasians consisting of 198 healthy controls (mean age 56 ± 15y; 58% male), 298 patients (mean age 55 ± 14y; 55% male) with endoscopically confirmed RE, 246 patients (mean age 61 ± 12y; 68% male) with BE, with a segment of ≥2 cm specialized intestinal metaplasia, and 129 patients (mean age 63 ± 10y; 82% male) with EAC. EGF genotypes (+61 A/G) were determined by modified TAQMAN assays. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (CI) for variant genotypes. Immunohistochemistry for EGF and EGFR, to exclude an indirect effect of EGF on its receptor, was performed on esophageal biopsies from a subgroup of BE patients. Multivariate analysis was used to calculate p-values for variant expression. Results: EGF G/G homozygosity was increased in RE (OR=2.6; 95% CI:1.3-5.2), BE (OR= 3.0; 95% CI:1.5-6.2), and EAC (OR=4.1; 95% CI:1.7-9.7) compared to healthy controls. Local esophageal EGF expression was significantly lower in BE patients with G/G homozygosity compared to those with A/G heterozygosity (p=0.008) and A/A homozygosity (p=0.002). We found that EGFR was strongly expressed in all BE tissues, independent of EGF genotypes or EGF expression levels in BE tissue. Conclusion: The EGF G/G polymorphism is associated with reduced EGF expression and an increased risk of developing RE, BE and EAC. This indicates that reduced EGF expression is a limiting factor in mucosal protection against esophageal tumor development.
159 Comprehensive Self-Management for Irritable Bowel Syndrome Works Whether Delivered By Telephone or in-Person Monica Jarrett, Kevin C. Cain, Vicky Hertig, Robert L. Burr, Sheldon N. Rosen, Margaret Heitkemper Objective. Determine the effectiveness of a comprehensive self-management (CSM) intervention delivered via telephone (T-CSM) or in-person (IP-CSM) as compared to usual care (UC) for adults with IBS. Methods. Potential participants were recruited through community advertisements and screened for eligibility criteria during the baseline period. 188 participants were randomized to T-CSM, IP-CSM or UC. Both CSM interventions were delivered weekly over 9 weeks, and addressed nutritional, cognitive and behavioral strategies for managing IBS symptoms. Outcomes were measured at baseline and 3, 6, and 12 months follow-up times. Two primary outcome variables were analyzed: a) IBS symptom severity (IBS-SS) from a daily diary, measured as the percent of days with at least one IBS symptom (abdominal pain, bloating, constipation, diarrhea, intestinal gas, urgency) being moderate or worse, and b) IBS-specific quality of life (QOL) measured on a 0-100 scale. Mixed-model analyses were used to analyze data from the three follow-up times, controlling for baseline values of IBS symptom severity, QOL, and psychological distress as covariates. Results. The two primary outcome variables showed highly significant treatment differences with both T-CSM and IP-CSM having better outcomes than UC (p <.003). There were no significant differences between T-CSM and IP-CSM. The treatment effect was strongest at 3 months: The percent of subjects whose IBS-SS decreased by at least 15 percentage points was 59% and 70% for T-CSM and IP-CSM, compared to 26% for UC; the percent of subjects whose QOL improved by at least 10 points was 40% and 42% for T-CSM and IP-CSM, compared to 18% for UC. Treatment effect at 12 months was still strong for IBS-SS (62% and 58% improved for TCSM and IP-CSM, versus 27% or UC) but was somewhat weaker for QOL due mainly to improvement in the UC group (27% and 37% improvement for T-CSM and IP-CSM versus 27% for UC). Many of the secondary outcome variables showed similar results, with T-CSM and IP-CSM both better than UC, and the two delivery modalities being about equally effective. Conclusions. Comprehensive self-management therapy leads to improvement in IBS symptoms and quality of life, and this improvement is still maintained at 12 months. Delivery of the therapy by telephone appears to be just as effective as in-person delivery. Since some subjects expressed a preference for in-person, while others expressed a preference for telephone, perhaps patients could be offered a choice of delivery modality.
174 Expression of Oncogenic H-RASG12v Does Not Induce Malignant Transformation or Senescence in Barrett's Epithelial Cells. Xi Zhang, Hui Ying Zhang, Stuart J. Spechler, Rhonda F. Souza Introduction: Although oncogenes are known to promote cell growth, they also can activate tumor suppressor pathways that induce senescence, an irreversible state of growth arrest. To become malignant, cells must acquire the ability to avoid senescence. In esophageal squamous cells, oncogenes have been shown to induce senescence by activating the p16 tumor suppressor pathway. In human fibroblasts deficient in p16, in contrast, oncogenes induce malignant transformation. In Barrett's metaplasia, p16 inactivation occurs early and frequently. Therefore, we hypothesized that oncogenes should transform p16-deficient Barrett's cells, and we tested that hypothesis using a telomerase-immortalized, non-neoplastic Barrett's epithelial (BAR-T) cell line. Methods: BAR-T cells, which are deficient in p16 but not in p53, were stably infected with the retroviral vector pBabe-zeocin containing oncogenic H-RasGV12; pBabe-zeocin without the ras insert served as a control. Expression levels of HRas, phospho-MEK1/2, phospho-ERK1/2, p53, and p21 were determined by Western blotting using β-actin as a control. Growth rates were determined by population doubling times. Cell contact inhibition and anchorage-independent growth in soft agar were used to determine neoplastic transformation; SEG-1 esophageal adenocarcinoma cells served as a positive control. Results: Infection with pBabe-H-RasG12V substantially increased H-ras expression in BAR-T cells compared to uninfected and empty vector-infected control cells. Two clones with the highest levels of H-ras expression (R1 and R7) were selected for further analyses. Expression levels of phospho-MEK1/2 and ERK1/2 were increased in those clones, indicating that they maintained functional Ras activity. Compared with empty-vector controls, H-ras expression did not increase p53 or p21 expression levels. There were no significant differences in population doubling times among H-Ras-containing clones (37.6 ± .7 hours SEM clone R1; 35 ± .5 hours clone R7) and empty vector-infected controls (36.5 ± .8 hours). HRas-containing cells demonstrated growth inhibition upon contact and lacked anchorageindependent growth after 3 weeks in soft agar unlike SEG-1 cells, which developed numerous colonies in soft agar. Conclusions: Expression of H-RasG12V does not induce senescence or malignant transformation in p16-deficient Barrett's epithelial cells. These findings suggest that oncogene expression and avoidance of oncogene-induced senescence are not sufficient for the malignant transformation of Barrett's epithelial cells. Alterations in additional pathways appear to be necessary for cancer development in Barrett's esophagus.
172 Novel Potential Markers of High Grade Dysplasia in Barrett's Esophagus Identified By cDNA Microarray Analysis of Laser Capture Microdissected Archival Tissue Edmond Sabo, Steven F. Moss, Patricia Meitner, Rose Tavares, John Gao, Christopher Corless, Gregory Y. Lauwers, Murray B. Resnick Background: The molecular pathogenesis of the transition from intestinal metaplasia to cancer in Barrett's esophagus (BE) is poorly defined. Identifying genes differentially expressed in BE without dysplasia (IM) from those expressed in high grade dysplasia (HGD) should be of value in improving our understanding of this transition and their differential expression might be of diagnostic and prognostic utility. Aim: To determine the differential transcriptome of HGD compared with non-dysplastic BE through gene microarray analysis of epithelial cells microdissected from archival formalin fixed tissue specimens. Methods: Laser capture microdissection (LCM) was used to isolate epithelial cells from surrounding inflammatory and stromal cells. Epithelial cell mRNA was extracted from areas of IM and HGD in the same patients from archival tissue sections. mRNA was reverse transcribed and applied onto Affymetrix cDNA microarray chips customized for mRNA from paraffinized, formalinexposed tissue. Differentially expressed genes were identified by the significance analysis of microarrays method and a false detection rate of <20%. For a subset of these genes, differential gene expression was verified by real-time PCR and immunohistochemistry in a further 10 IM and HGD cases. Results: There were 168 genes over-expressed by at least two-fold in HGD versus IM. 72 genes were under-expressed by at least two-fold in HGD. Among the over-expressed genes are several previously demonstrated to be increased in HGD as well as novel candidate genes involved in neoplastic progression including lipocalin 2, S-100A9 (calgranulin B, a component of calprotectin), matrix metallopeptidase 12 secernin 1 and topoisomerase II A. Genes decreased in HGD compared with IM include mucins 3A, 5A and 5B, trefoil factors 1 and 2, meprin A, and alanyl-aminopeptidase. Real-time PCR validated the changes in expression of 8/8 genes examined, including lipocalin 2, S-100A9, secernin 1, trefoil factor 1, meprin A and alanyl-aminopeptidase. Immunohistochemistry confirmed altered expression at the protein level for topoisomerase IIA, S-100A9 and lipocalin . Conclusions: This is the first study to identify by LCM genes differentially expressed in HGD compared with IM in the same patients. This was achieved by accessing esophageal epithelial cells from archival paraffin embedded specimens using customized Affymetrix cDNA microarrays. The differentially expressed genes identified include several previously implicated in
175 Sequential Chromosomal Events Leading to HER-2 Proto-Oncogene Amplification in Barrett's Esophagus Rygiel M. Agnieszka, Francesca Milano, Fiebo J. ten Kate, Brenda Elzer, Paul Fockens, Jacques J. Bergman, Maikel P. Peppelenbosch, Kausilia K. Krishnadath BACKROUND: Her-2 proto-oncogene amplification is an important marker in Barrett's esophagus associated esophageal adenocarcinoma (EAC), correlating with poor prognosis of EAC patients. However, studies showing the sequential genetic events preceding Her-2 amplification in Barrett's esophagus (BE) in the no dysplasia-dysplasia-adenocarcinomasequence are lacking. AIM: The aim was to study sequential chromosomal events that may lead to Her-2 locus amplification in BE and EAC. METHODS: Her-2 locus (17q11.2) and
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