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M1962 Development of Multilayerd Epithelium (MLE) After Photodynamic Therapy: Is MLE a Precursor to Barrett's Esophagus?
AGA Abstracts
at 24 hours using propidium iodide. Cell senescence was determined by using the Senescent Cell Histochemistry Kit (Sigma CS0030) that determines SA B-galactosidase activity at pH 6.0 by blue staining epithelial cells. Senescence was assessed 24 hours after PDT. Senescence was also confirmed by DAPI staining of nuclear SAB. RESULTS: Using ALA PDT, we found that cell death was 4%,5%,5%,6%,7%,37%, 91%, and 88% at concentrations of 0, 2.5,5, 10, 25, 50, 100, and 250 ug/ml in cells that were p16-. The results in cells that were p16+ were 4%, 4%, 5%, 6%, 8%, 43%, 92%, and 88% which was not different than tin cells that were p16- (p>0.5). Using SP PDT cell death was 4%,24%,44%,73%,99%,100%,100%, and 100% using dosages of 0,0.25,0.5, 1, 2.5,5,10, and 25 ug/ml in p16- cells. In cells that were p16+, cell death was 4%, 27%,45%,75%,99%,100%,100%,100%. That was not different from p16- cells (p>0.4). All studies were repeated in triplicate. 24 hours after PDT with 0.5 ug of sodium porfimer, the senescent population in p16- cells was 49% and 69% in p16+ cells, (p<0.05). CONCLUSIONS: Barrett's cells, regardless of p16 status, are equally susceptible to ALA or sodium porfimer PDT In Vitro. However, p16+ cells undergo senescence significantly more that p16- cells after PDT. The loss of p16 does not allow as much senescence to occur and may be a mechanism of resistance to PDT.
M1963 Influence of Acid Reflux On Stromal Activation in Barrett's Esophagus Ganapathy A. Prasad, Navtej Buttar, Tsung-Teh Wu, Lori S. Lutzke, Kelly T. Dunagan, Lynn S. Borkenhagen, Kenneth K. Wang Background:Chronic acid reflux causes chronic inflammation, which is associated with activated fibroblasts, which secrete growth factors promoting carcinogenesis. Acid reflux by inducing COX-2 activity may activate fibroblasts. We aimed to examine the influence of acid reflux on fibroblast activation in BE and investigate underlying mechanisms. Methods: We studied 31 patients with LSBE:17 with persistent acid reflux;14 with controlled acid reflux (defined by 24 h pH studies).Fibroblast activation was defined as >/= 50% of subepithelial fibroblasts staining for vimentin and alpha SMA on immunohistochemistry(assessed by a pathologist blinded to status of acid reflux control).To understand mechanisms of acid induced fibroblast activation,we assessed 1)influence of extracellular acid on intracellular fibroblast pH using dual excitation fluorescence ratio imaging 2)COX2 expression (by RT PCR) in BE fibroblasts following varying durations and concentrations of acid exposure. Results: We found that 11/17 patients(65 %)with acid reflux had evidence of myofibroblasts,compared to 5/14 (36%)without acid reflux (p=0.05).Intracellular fibroblast pH decreased following exposure to acid (Figure 1).Figure 2 shows the induction of COX-2 expression in BE fibroblasts following acid exposure. Conclusions: Acid reflux promotes fibroblast activation and induces COX-2 expression in fibroblasts in BE. Further phenotypic characterization of fibroblasts following acid exposure using RT-PCR for alpha SMA and EM is being undertaken. Intracellular fibroblast pH following extracellular acid exposure (pH4.5)
M1961 Autoregulation of Cdx1 and Cdx2 in Development of Barrett's Epithelium Hideaki Kazumori, Mika Yuki, Yoshinori Komazawa, Toshihiro Shizuku, Yoshikazu Kinoshita Background & Aims: The mechanism of transformation to intestinal metaplasia in Barrett's esophagus has not been clarified. We previously reported that bile acids activate the Cdx2 promoter via NF-kB and stimulate the production of Cdx2 protein in esophageal keratinocytes with a resulting production of intestinal type mucin (Gut 55:16-25, 2006). In addition to Cdx2, Cdx1 also plays an important role in the development of Barret's esophagus. Therefore, we studied the direct effects of bile acids on the expression of Cdx1, as well as the precise mechanisms of Cdx1 and Cdx2 expressions in cultured esophageal squamous epithelial cells. Methods: A rat model of Barrett's esophagus was produced by anastomosing the esophagus and jejunum. The expressions of Cdx1 and Cdx2 were investigated by immunohistochemistry, while the response of bile acids to Cdx1 gene expression was studied, using a Cdx1 promoter luciferase assay. Further, esophageal squamous epithelial cells were transfected with a Cdx1 or Cdx2 expression vector, after which their possible transformation to intestinal-type epithelial cells was investigated. Results: Esophago-jejunal anastomoses formed intestinal goblet cell metaplasia in rat esophagus specimens, and the metaplastic epithelium strongly expressed both Cdx1 and Cdx2. Further, when the effects of the bile acids mixture on the expression of Cdx1 were examined, Cdx1 promoter activity was found to be increased in a dose-dependent manner. In cultured esophageal epithelial cells, transfection of the Cdx1 expression vector increased Cdx1 promoter activity, while that of the Cdx2 expression vector increased Cdx2 promoter activity. Conclusions: We found that bile acids stimulate the expressions of both Cdx1 and Cdx2 in esophageal epithelial cells. Autoregulation of the expressions of those proteins has an important role in the development of Barrett's epithelium.
Using BCECF @ 440/495nm
M1962 Development of Multilayerd Epithelium (MLE) After Photodynamic Therapy: Is MLE a Precursor to Barrett's Esophagus? Fumihiro Ogawa, Amitabh Srivastava, Norman S. Nishioka, William P. Puricelli, Gregory Y. Lauwers, Mari Mino-Kenudson Background: Photodynamic therapy (PDT) using porfimer sodium is increasingly used to ablate Barrett's esophagus (BE) and BE-related dysplasia/superficial adenocarcinoma, which then leads to restoration of squamous epithelium, over a variable follow-up period. MLE is a distinctive hybrid-type epithelium with squamous and columnar characteristics; has been observed in association with shorter lengths of BE and is believed to be a precursor of BE. However, MLE has also been reported in post-PDT biopsies. The aim of this study was to determine the prevalence and significance of MLE in pre- and post-PDT biopsies. Methods: The study cohort consisted of 22 BE patients (mean age: 68 yr, M:F = 18:3, mean BE length: 6.9 cm) with high-grade dysplasia (n=11), intramucosal adenocarcinoma (n=9) and invasive adenocarcinoma (n=2) treated with PDT. All patients were on high-dose proton pump inhibitor therapy. Four quadrant every 2 cm biopsies were taken pre and post PDT from the site of the original BE segment. All the biopsies performed within 3 months pre PDT and multiple follow-up biopsies post PDT from the 22 patients were evaluated. The presence of MLE pre and post PDT was correlated with several clinicopathological features. Results: MLE was more frequently observed after (11 patients [50.0%], 24/193 biopsies [12.4%]) than before PDT (2 patients [9.1%], 2/31 biopsies [6.5%]) (p<0.001). MLE was noted only in biopsy levels within 2 cm of GE junction and was often associated with esophageal mucosal glands and/or squamous islands both pre and post PDT. MLE was seen in biopsies performed at an average of 13 months after PDT (range: 1 - 34 months). The mean length of original BE was longer in patients who developed post-PDT MLE (MLE+ group) than in those who did not (MLE- group) (8.4 cm vs. 5.4 cm, respectively, p< 0.01). There was no difference in gender, mean age, and the grade of neoplasia between the 2 groups. At the end of follow-up (mean: 44 months, range: 12 - 117 months), neoplasia persisted in 5 patients (3 in the MLE+ group and 2 in the MLE- group) and residual BE was observed in 8 patients (4 in both groups). Conclusion: MLE often develops after PDT and is significantly associated with longer lengths of the original BE segment. The distal location of MLE and its close proximity to esophageal gland ducts is seen in both pre and post PDT. The presence of post-PDT MLE does not correlate with persistence of BE and/or neoplasia. The results suggest that MLE is a marker of an “unstable” epithelial state and can be seen both during squamous-columnar metaplastic transformation in BE and during columnar-squamous epithelial restoration in response to therapy.
AGA Abstracts
M1964 Superficial Ki67 Expression May More Accurately Measure Proliferation in Barrett's Associated Intestinal Metaplasia and High-Grade Dysplasia Ajay Bansal, Sharad C. Mathur, Sachin B. Wani, Krishna Pondugula, Amit Rastogi, Prateek Sharma Background: DNA microarray gene expression analysis shows that proliferation is a characteristic conserved across multiple tumor types. Ki67 is a widely used proliferation marker that has been successfully applied in assessment of proliferation in chemoprevention trials involving breast and prostate cancer. The role of proliferation as measured by Ki67 in Barrett's esophagus (BE) is controversial, in part due to Ki67 measurement across the entire Barrett's epithelium. Fitzgerald and colleagues have shown that superficial assessment of proliferation markers may be more accurate. We hypothesized that superficial rather than deeper Ki67 expression will more accurately identify proliferation in our population of Barrett's Esophagus. Methods: In a pilot study, biopsies obtained from BE patients undergoing surveillance were collected in Bouin's solution and processed in a standardized manner for H&E staining. Thereafter, Ki67 immunohistochemical staining was performed on 4µm tissue sections with appropriate positive and negative controls. Only diffuse nuclear staining was considered positive. The superficial (surface) Ki67 staining was compared with the deeper staining (defined as the lower 1/3 of crypt). Student's t test was used for statistical analysis and a p value<0.05 was considered significant. Results: Biopsies obtained from 10 BE patients were evaluated. The mean age was 64 years (range: 54-73), all males and Caucasians. Mean BE length was 3 cm (range: 2-6 cm). The mean superficial Ki67 expression in the biopsies with intestinal metaplasia was 1±2% compared to 35±30% in those with high-grade dysplasia (p=0.03). In comparison, the mean deeper Ki67 expression in the biopsies with intestinal metaplasia was 10±6% compared to 6±3% to those with high-grade dysplasia (p=0.22). Conclusion: Superficial but not deeper Ki67 staining adequately discriminated intestinal metaplasia from high-grade dysplasia and may be more accurate to evaluate proliferation in Barrett's Esophagus. If confirmed in larger studies, this data will help design chemoprevention and other biomarker based trials evaluating proliferation in Barrett's Esophagus and associated dysplasia.
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at 24 hours using propidium iodide. Cell senescence was determined by using the Senescent Cell Histochemistry Kit (Sigma CS0030) that determines SA B-galactosidase activity at pH 6.0 by blue staining epithelial cells. Senescence was assessed 24 hours after PDT. Senescence was also confirmed by DAPI staining of nuclear SAB. RESULTS: Using ALA PDT, we found that cell death was 4%,5%,5%,6%,7%,37%, 91%, and 88% at concentrations of 0, 2.5,5, 10, 25, 50, 100, and 250 ug/ml in cells that were p16-. The results in cells that were p16+ were 4%, 4%, 5%, 6%, 8%, 43%, 92%, and 88% which was not different than tin cells that were p16- (p>0.5). Using SP PDT cell death was 4%,24%,44%,73%,99%,100%,100%, and 100% using dosages of 0,0.25,0.5, 1, 2.5,5,10, and 25 ug/ml in p16- cells. In cells that were p16+, cell death was 4%, 27%,45%,75%,99%,100%,100%,100%. That was not different from p16- cells (p>0.4). All studies were repeated in triplicate. 24 hours after PDT with 0.5 ug of sodium porfimer, the senescent population in p16- cells was 49% and 69% in p16+ cells, (p<0.05). CONCLUSIONS: Barrett's cells, regardless of p16 status, are equally susceptible to ALA or sodium porfimer PDT In Vitro. However, p16+ cells undergo senescence significantly more that p16- cells after PDT. The loss of p16 does not allow as much senescence to occur and may be a mechanism of resistance to PDT.
M1963 Influence of Acid Reflux On Stromal Activation in Barrett's Esophagus Ganapathy A. Prasad, Navtej Buttar, Tsung-Teh Wu, Lori S. Lutzke, Kelly T. Dunagan, Lynn S. Borkenhagen, Kenneth K. Wang Background:Chronic acid reflux causes chronic inflammation, which is associated with activated fibroblasts, which secrete growth factors promoting carcinogenesis. Acid reflux by inducing COX-2 activity may activate fibroblasts. We aimed to examine the influence of acid reflux on fibroblast activation in BE and investigate underlying mechanisms. Methods: We studied 31 patients with LSBE:17 with persistent acid reflux;14 with controlled acid reflux (defined by 24 h pH studies).Fibroblast activation was defined as >/= 50% of subepithelial fibroblasts staining for vimentin and alpha SMA on immunohistochemistry(assessed by a pathologist blinded to status of acid reflux control).To understand mechanisms of acid induced fibroblast activation,we assessed 1)influence of extracellular acid on intracellular fibroblast pH using dual excitation fluorescence ratio imaging 2)COX2 expression (by RT PCR) in BE fibroblasts following varying durations and concentrations of acid exposure. Results: We found that 11/17 patients(65 %)with acid reflux had evidence of myofibroblasts,compared to 5/14 (36%)without acid reflux (p=0.05).Intracellular fibroblast pH decreased following exposure to acid (Figure 1).Figure 2 shows the induction of COX-2 expression in BE fibroblasts following acid exposure. Conclusions: Acid reflux promotes fibroblast activation and induces COX-2 expression in fibroblasts in BE. Further phenotypic characterization of fibroblasts following acid exposure using RT-PCR for alpha SMA and EM is being undertaken. Intracellular fibroblast pH following extracellular acid exposure (pH4.5)
M1961 Autoregulation of Cdx1 and Cdx2 in Development of Barrett's Epithelium Hideaki Kazumori, Mika Yuki, Yoshinori Komazawa, Toshihiro Shizuku, Yoshikazu Kinoshita Background & Aims: The mechanism of transformation to intestinal metaplasia in Barrett's esophagus has not been clarified. We previously reported that bile acids activate the Cdx2 promoter via NF-kB and stimulate the production of Cdx2 protein in esophageal keratinocytes with a resulting production of intestinal type mucin (Gut 55:16-25, 2006). In addition to Cdx2, Cdx1 also plays an important role in the development of Barret's esophagus. Therefore, we studied the direct effects of bile acids on the expression of Cdx1, as well as the precise mechanisms of Cdx1 and Cdx2 expressions in cultured esophageal squamous epithelial cells. Methods: A rat model of Barrett's esophagus was produced by anastomosing the esophagus and jejunum. The expressions of Cdx1 and Cdx2 were investigated by immunohistochemistry, while the response of bile acids to Cdx1 gene expression was studied, using a Cdx1 promoter luciferase assay. Further, esophageal squamous epithelial cells were transfected with a Cdx1 or Cdx2 expression vector, after which their possible transformation to intestinal-type epithelial cells was investigated. Results: Esophago-jejunal anastomoses formed intestinal goblet cell metaplasia in rat esophagus specimens, and the metaplastic epithelium strongly expressed both Cdx1 and Cdx2. Further, when the effects of the bile acids mixture on the expression of Cdx1 were examined, Cdx1 promoter activity was found to be increased in a dose-dependent manner. In cultured esophageal epithelial cells, transfection of the Cdx1 expression vector increased Cdx1 promoter activity, while that of the Cdx2 expression vector increased Cdx2 promoter activity. Conclusions: We found that bile acids stimulate the expressions of both Cdx1 and Cdx2 in esophageal epithelial cells. Autoregulation of the expressions of those proteins has an important role in the development of Barrett's epithelium.
Using BCECF @ 440/495nm
M1962 Development of Multilayerd Epithelium (MLE) After Photodynamic Therapy: Is MLE a Precursor to Barrett's Esophagus? Fumihiro Ogawa, Amitabh Srivastava, Norman S. Nishioka, William P. Puricelli, Gregory Y. Lauwers, Mari Mino-Kenudson Background: Photodynamic therapy (PDT) using porfimer sodium is increasingly used to ablate Barrett's esophagus (BE) and BE-related dysplasia/superficial adenocarcinoma, which then leads to restoration of squamous epithelium, over a variable follow-up period. MLE is a distinctive hybrid-type epithelium with squamous and columnar characteristics; has been observed in association with shorter lengths of BE and is believed to be a precursor of BE. However, MLE has also been reported in post-PDT biopsies. The aim of this study was to determine the prevalence and significance of MLE in pre- and post-PDT biopsies. Methods: The study cohort consisted of 22 BE patients (mean age: 68 yr, M:F = 18:3, mean BE length: 6.9 cm) with high-grade dysplasia (n=11), intramucosal adenocarcinoma (n=9) and invasive adenocarcinoma (n=2) treated with PDT. All patients were on high-dose proton pump inhibitor therapy. Four quadrant every 2 cm biopsies were taken pre and post PDT from the site of the original BE segment. All the biopsies performed within 3 months pre PDT and multiple follow-up biopsies post PDT from the 22 patients were evaluated. The presence of MLE pre and post PDT was correlated with several clinicopathological features. Results: MLE was more frequently observed after (11 patients [50.0%], 24/193 biopsies [12.4%]) than before PDT (2 patients [9.1%], 2/31 biopsies [6.5%]) (p<0.001). MLE was noted only in biopsy levels within 2 cm of GE junction and was often associated with esophageal mucosal glands and/or squamous islands both pre and post PDT. MLE was seen in biopsies performed at an average of 13 months after PDT (range: 1 - 34 months). The mean length of original BE was longer in patients who developed post-PDT MLE (MLE+ group) than in those who did not (MLE- group) (8.4 cm vs. 5.4 cm, respectively, p< 0.01). There was no difference in gender, mean age, and the grade of neoplasia between the 2 groups. At the end of follow-up (mean: 44 months, range: 12 - 117 months), neoplasia persisted in 5 patients (3 in the MLE+ group and 2 in the MLE- group) and residual BE was observed in 8 patients (4 in both groups). Conclusion: MLE often develops after PDT and is significantly associated with longer lengths of the original BE segment. The distal location of MLE and its close proximity to esophageal gland ducts is seen in both pre and post PDT. The presence of post-PDT MLE does not correlate with persistence of BE and/or neoplasia. The results suggest that MLE is a marker of an “unstable” epithelial state and can be seen both during squamous-columnar metaplastic transformation in BE and during columnar-squamous epithelial restoration in response to therapy.
AGA Abstracts
M1964 Superficial Ki67 Expression May More Accurately Measure Proliferation in Barrett's Associated Intestinal Metaplasia and High-Grade Dysplasia Ajay Bansal, Sharad C. Mathur, Sachin B. Wani, Krishna Pondugula, Amit Rastogi, Prateek Sharma Background: DNA microarray gene expression analysis shows that proliferation is a characteristic conserved across multiple tumor types. Ki67 is a widely used proliferation marker that has been successfully applied in assessment of proliferation in chemoprevention trials involving breast and prostate cancer. The role of proliferation as measured by Ki67 in Barrett's esophagus (BE) is controversial, in part due to Ki67 measurement across the entire Barrett's epithelium. Fitzgerald and colleagues have shown that superficial assessment of proliferation markers may be more accurate. We hypothesized that superficial rather than deeper Ki67 expression will more accurately identify proliferation in our population of Barrett's Esophagus. Methods: In a pilot study, biopsies obtained from BE patients undergoing surveillance were collected in Bouin's solution and processed in a standardized manner for H&E staining. Thereafter, Ki67 immunohistochemical staining was performed on 4µm tissue sections with appropriate positive and negative controls. Only diffuse nuclear staining was considered positive. The superficial (surface) Ki67 staining was compared with the deeper staining (defined as the lower 1/3 of crypt). Student's t test was used for statistical analysis and a p value<0.05 was considered significant. Results: Biopsies obtained from 10 BE patients were evaluated. The mean age was 64 years (range: 54-73), all males and Caucasians. Mean BE length was 3 cm (range: 2-6 cm). The mean superficial Ki67 expression in the biopsies with intestinal metaplasia was 1±2% compared to 35±30% in those with high-grade dysplasia (p=0.03). In comparison, the mean deeper Ki67 expression in the biopsies with intestinal metaplasia was 10±6% compared to 6±3% to those with high-grade dysplasia (p=0.22). Conclusion: Superficial but not deeper Ki67 staining adequately discriminated intestinal metaplasia from high-grade dysplasia and may be more accurate to evaluate proliferation in Barrett's Esophagus. If confirmed in larger studies, this data will help design chemoprevention and other biomarker based trials evaluating proliferation in Barrett's Esophagus and associated dysplasia.
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