- Email: [email protected]
Sa1835 Nanocytomics as a Novel Modality for Determining Transcriptional Activity
AGA Abstracts
region was acquired in 20 seconds. The prevalence, size and location of SSIM glands relative to the squamocolumnar junction (SCJ) were quantified and compared between Pre-CE-IM and Post-CE-IM groups. RESULTS: 3D-OCT provided 80x - 100x larger field-of-view compared to biopsy and reliably (in 100% cases) sufficient imaging depth, into the muscularis mucosa, for detecting SSIM. In comparison, only 40% to 80% biopsies reached the lamina propria as reported in recent studies. SSIM was found in 72% (13/18) patients in the PreCE-IM group, and 63% (10/16) patients in the Post-CE-IM group. The number of SSIM per patient in the Post-CE-IM group [7.1 (9.3)] was significantly lower compared to the Pre-CE-IM group [34.4 (44.6); p = 0.02]. SSIM gland size (p = 0.69) and distribution (p = 0.54) were not significantly different before and after CE-IM. CONCLUSIONS: The prevalence of SSIM in patients who responded to treatment after RFA is higher than previously reported using four-quadrant biopsy. Compared to other advanced imaging modalities only imaging surface [narrow band imaging (NBI), chromoendoscopy] or small focal areas [confocal, elastic scattering spectroscopy (ESS), etc.], 3D-OCT provides both subsurface and broad area of view uniquely suited to potentially evaluate patients before and after ablation treatment. ACKNOWLEDGEMENT: NIH 5R01-CA075289-14, K99-EB010071-01A1, and AFOSR FA9550-10-1-0063 and FA9550-10-1-0551. Table 1
Figure 1. A) The number of SSIM per patient was significantly lower in the Post-CE-IM group compared to the Pre-CE-IM group (p = 0.02). No significant difference was observed in either B) the gland size (p = 0.69) or C) the gland distribution (p = 0.54) between the two groups.
A: HT29; B: ARID1a deficient HT29; Upper panel is high-resolution images, and lower panel is the heat map. + means transcriptional stimulation - means starved media
Sa1835 Sa1836
Nanocytomics as a Novel Modality for Determining Transcriptional Activity Ashish K. Tiwari, Hariharan Subramanian, Tina P. Gibson, Mart DeLaCruz, Ramesh K. Wali, Vadim Backman, Hemant K. Roy
A New Approach to Image and Enhance Diagnosis of EoE: Radiolabeled Contrast Agents Hedieh Saffari, Gerald J. Gleich, Leonard Pease, Kathryn Peterson
Introduction: Perturbation of the transcriptional status of a cell is a hallmark feature of all biological processes. However, it has been difficult to develop a robust tool to estimate transcriptional activity of cells. Typically, alterations in gene expression are coupled with changes in high order chromatin which are beyond the resolution of conventional light microscopy. Our group has developed a novel optical technique, partial wave spectroscopy (PWS) microscopy, which has allowed us to quantify nanoscale architecture by measuring disorder strength (Ld) (PNAS 2008, Gastro 2011). We herein investigate whether PWS could indeed help delineate the differential transcriptional status of microscopically similar appearing cells by measuring nanoarchitectural changes during transcription. Methods: PWS studies were performed on human colon cancer cell line, HT29, after altering their transcriptional activity by exposing them to different growth conditions (starvation vs. standard media) and treating with transcriptional modulators (Epidermal growth factor, EGF; and Phorbol-ester, PMA). The resulting phenotypes were analyzed with light microscopy and flow-cytometry. Transcription was assessed by global gene expression microarray. Similar experiments were performed with HT29 cells stably transfected with a specific shRNA against ARID1a, a component of high-order chromatin remodeling complex, Swi/Snf. Results: PWS studies revealed that treatment with transcriptional activators, EGF and PMA, significantly increased nuclear Ld, whereas starvation (transcriptional silencing) significantly reduced nuclear Ld (figure 1, table 1). Additionally, short-duration treatments/starvation led to comparable changes in Ld, suggesting its correlation with early events during transcription, and not translational activity. Moreover, ARID1a-deficient HT29 cells (shRNA knock-downs) demonstrated only insignificant changes in Ld after similar treatments, further implicating high-order chromatin reorganization in determining Ld. These findings were corroborated with flow cytometry observations of expected proliferative and arrested phenotypes in different growth conditions with HT29 cells, but minimum phenotypic response under similar circumstances from ARID1a deficient HT29 cells (table 1). These findings were corroborated with transcription profile assessment through global gene expression analysis. Conclusions: We herein demonstrate a remarkable application of this optical technology, PWS, in estimating the transcriptional status of a cell by assessing the heterogeneity (through Ld measurement) resulting from the reorganization of high order chromatin. Ld measured with PWS thus could potentially become an excellent surrogate marker of transcription, and be used for the study of life processes ranging from development to differentiation to carcinogenesis. Cell cycle distribution after treatment (phenotype assessment)
Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). However, the heterogeneity of eosinophil involvement presents a diagnostic challenge to the gastroenterologist as current diagnosis requires invasive EGD with a minimum of 5 biopsies from the esophagus, representing less than 2% of the esophageal surface. To understand this disease, improved methods to characterize the eosinophil density are required. Aim: To use radiolabeled contrast agents to image the spatial distribution of density of eosinophils, the key attribute of EoE. We hypothesize that radiolabeled agents (such as antibodies) that bind to MBP-1 can be used to determine the presence of eosinophils using single-photon emission computed tomography (SPECT) imaging. This is part of our long term goal to develop new strategies to enhance EoE diagnosis and understand disease etiology. Methods: The radiolabeling agent comprises a binding ligand that binds specifically (such as an antibody) or preferentially binds to MBP-1, a key eosinophil protein, and Tc99m as a chelated radionuclide. Fresh frozen biopsy tissues from normal and EoE patients were incubated with the radiolabeled agent, washed in saline buffer, and imaged using SPECT. Eosinophil density following H&E staining was correlated with SPECT intensity. Results: Radiolabeled EoE biopsy tissues demonstrated significantly higher SPECT intensity than radiolabeled control (i.e., eosinophil free) tissue (see figure in which the top and bottom two spots are from EoE patients and the other spot represents the control). Discussion: These proof-of-principle experiments clearly show that eosinophil enriched tissues can be distinguished from eosinophil free tissues using the radiolabeled contrast agents and SPECT detection. This finding is important because it provides a new avenue for clinical detection of EoE. Using this strategy, physicians will have unprecedented insight into the specific involvement of eosinophils within the esophagus of EoE patients, which not only which will improve detection and management of the disease as well as create a better opportunity to understand the disease pathogenesis. Sa1837 Is There Any Agreement Between Endoscopists and Pathologists for the Interpretation of Probe-Based Confocal Laser Endomicroscopy (P-CLE) Findings? Ji Young Bang, Leona Council, Shyam Varadarajulu, Jessica Trevino, Mel Wilcox, Jayapal Ramesh, Shajan Peter Background: Although probe-based confocal laser endomicroscopy (p-CLE) is increasingly used, no prior study has compared the interpretation of findings between endoscopists and pathologists. Methods: All p-CLE were undertaken concurrently with endoscopic procedures by four endoscopists who rendered immediate assessment at the time of the procedure. The same p-CLE videos were then viewed offline by an expert GI pathologist who was blinded to the endoscopists' findings. All endoscopists and the pathologist had undergone prior training in p-CLE interpretation. Histopathology was considered as the gold standard for definitive diagnosis. The main objectives were to determine the sensitivity, specificity and accuracy of p-CLE for diagnosis of dysplastic and neoplastic GI lesions, and to elucidate interobserver agreement between endoscopists and pathologist for p-CLE findings. Results: Of 66 patients, 40 (60.6%) had lesions in the esophagus, 7 (10.6%) in the stomach, 15 (22.7%) in the biliary tract, 3 (4.5%) in the ampulla and 1 (1.5%) in the colon. The overall sensitivity, specificity and accuracy for diagnosing dysplastic/neoplastic lesions using p-CLE were higher for endoscopists than pathologist at 87.0 vs. 69.6%, 80.0 vs. 40.0% and 84.8
S-337
AGA Abstracts
region was acquired in 20 seconds. The prevalence, size and location of SSIM glands relative to the squamocolumnar junction (SCJ) were quantified and compared between Pre-CE-IM and Post-CE-IM groups. RESULTS: 3D-OCT provided 80x - 100x larger field-of-view compared to biopsy and reliably (in 100% cases) sufficient imaging depth, into the muscularis mucosa, for detecting SSIM. In comparison, only 40% to 80% biopsies reached the lamina propria as reported in recent studies. SSIM was found in 72% (13/18) patients in the PreCE-IM group, and 63% (10/16) patients in the Post-CE-IM group. The number of SSIM per patient in the Post-CE-IM group [7.1 (9.3)] was significantly lower compared to the Pre-CE-IM group [34.4 (44.6); p = 0.02]. SSIM gland size (p = 0.69) and distribution (p = 0.54) were not significantly different before and after CE-IM. CONCLUSIONS: The prevalence of SSIM in patients who responded to treatment after RFA is higher than previously reported using four-quadrant biopsy. Compared to other advanced imaging modalities only imaging surface [narrow band imaging (NBI), chromoendoscopy] or small focal areas [confocal, elastic scattering spectroscopy (ESS), etc.], 3D-OCT provides both subsurface and broad area of view uniquely suited to potentially evaluate patients before and after ablation treatment. ACKNOWLEDGEMENT: NIH 5R01-CA075289-14, K99-EB010071-01A1, and AFOSR FA9550-10-1-0063 and FA9550-10-1-0551. Table 1
Figure 1. A) The number of SSIM per patient was significantly lower in the Post-CE-IM group compared to the Pre-CE-IM group (p = 0.02). No significant difference was observed in either B) the gland size (p = 0.69) or C) the gland distribution (p = 0.54) between the two groups.
A: HT29; B: ARID1a deficient HT29; Upper panel is high-resolution images, and lower panel is the heat map. + means transcriptional stimulation - means starved media
Sa1835 Sa1836
Nanocytomics as a Novel Modality for Determining Transcriptional Activity Ashish K. Tiwari, Hariharan Subramanian, Tina P. Gibson, Mart DeLaCruz, Ramesh K. Wali, Vadim Backman, Hemant K. Roy
A New Approach to Image and Enhance Diagnosis of EoE: Radiolabeled Contrast Agents Hedieh Saffari, Gerald J. Gleich, Leonard Pease, Kathryn Peterson
Introduction: Perturbation of the transcriptional status of a cell is a hallmark feature of all biological processes. However, it has been difficult to develop a robust tool to estimate transcriptional activity of cells. Typically, alterations in gene expression are coupled with changes in high order chromatin which are beyond the resolution of conventional light microscopy. Our group has developed a novel optical technique, partial wave spectroscopy (PWS) microscopy, which has allowed us to quantify nanoscale architecture by measuring disorder strength (Ld) (PNAS 2008, Gastro 2011). We herein investigate whether PWS could indeed help delineate the differential transcriptional status of microscopically similar appearing cells by measuring nanoarchitectural changes during transcription. Methods: PWS studies were performed on human colon cancer cell line, HT29, after altering their transcriptional activity by exposing them to different growth conditions (starvation vs. standard media) and treating with transcriptional modulators (Epidermal growth factor, EGF; and Phorbol-ester, PMA). The resulting phenotypes were analyzed with light microscopy and flow-cytometry. Transcription was assessed by global gene expression microarray. Similar experiments were performed with HT29 cells stably transfected with a specific shRNA against ARID1a, a component of high-order chromatin remodeling complex, Swi/Snf. Results: PWS studies revealed that treatment with transcriptional activators, EGF and PMA, significantly increased nuclear Ld, whereas starvation (transcriptional silencing) significantly reduced nuclear Ld (figure 1, table 1). Additionally, short-duration treatments/starvation led to comparable changes in Ld, suggesting its correlation with early events during transcription, and not translational activity. Moreover, ARID1a-deficient HT29 cells (shRNA knock-downs) demonstrated only insignificant changes in Ld after similar treatments, further implicating high-order chromatin reorganization in determining Ld. These findings were corroborated with flow cytometry observations of expected proliferative and arrested phenotypes in different growth conditions with HT29 cells, but minimum phenotypic response under similar circumstances from ARID1a deficient HT29 cells (table 1). These findings were corroborated with transcription profile assessment through global gene expression analysis. Conclusions: We herein demonstrate a remarkable application of this optical technology, PWS, in estimating the transcriptional status of a cell by assessing the heterogeneity (through Ld measurement) resulting from the reorganization of high order chromatin. Ld measured with PWS thus could potentially become an excellent surrogate marker of transcription, and be used for the study of life processes ranging from development to differentiation to carcinogenesis. Cell cycle distribution after treatment (phenotype assessment)
Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). However, the heterogeneity of eosinophil involvement presents a diagnostic challenge to the gastroenterologist as current diagnosis requires invasive EGD with a minimum of 5 biopsies from the esophagus, representing less than 2% of the esophageal surface. To understand this disease, improved methods to characterize the eosinophil density are required. Aim: To use radiolabeled contrast agents to image the spatial distribution of density of eosinophils, the key attribute of EoE. We hypothesize that radiolabeled agents (such as antibodies) that bind to MBP-1 can be used to determine the presence of eosinophils using single-photon emission computed tomography (SPECT) imaging. This is part of our long term goal to develop new strategies to enhance EoE diagnosis and understand disease etiology. Methods: The radiolabeling agent comprises a binding ligand that binds specifically (such as an antibody) or preferentially binds to MBP-1, a key eosinophil protein, and Tc99m as a chelated radionuclide. Fresh frozen biopsy tissues from normal and EoE patients were incubated with the radiolabeled agent, washed in saline buffer, and imaged using SPECT. Eosinophil density following H&E staining was correlated with SPECT intensity. Results: Radiolabeled EoE biopsy tissues demonstrated significantly higher SPECT intensity than radiolabeled control (i.e., eosinophil free) tissue (see figure in which the top and bottom two spots are from EoE patients and the other spot represents the control). Discussion: These proof-of-principle experiments clearly show that eosinophil enriched tissues can be distinguished from eosinophil free tissues using the radiolabeled contrast agents and SPECT detection. This finding is important because it provides a new avenue for clinical detection of EoE. Using this strategy, physicians will have unprecedented insight into the specific involvement of eosinophils within the esophagus of EoE patients, which not only which will improve detection and management of the disease as well as create a better opportunity to understand the disease pathogenesis. Sa1837 Is There Any Agreement Between Endoscopists and Pathologists for the Interpretation of Probe-Based Confocal Laser Endomicroscopy (P-CLE) Findings? Ji Young Bang, Leona Council, Shyam Varadarajulu, Jessica Trevino, Mel Wilcox, Jayapal Ramesh, Shajan Peter Background: Although probe-based confocal laser endomicroscopy (p-CLE) is increasingly used, no prior study has compared the interpretation of findings between endoscopists and pathologists. Methods: All p-CLE were undertaken concurrently with endoscopic procedures by four endoscopists who rendered immediate assessment at the time of the procedure. The same p-CLE videos were then viewed offline by an expert GI pathologist who was blinded to the endoscopists' findings. All endoscopists and the pathologist had undergone prior training in p-CLE interpretation. Histopathology was considered as the gold standard for definitive diagnosis. The main objectives were to determine the sensitivity, specificity and accuracy of p-CLE for diagnosis of dysplastic and neoplastic GI lesions, and to elucidate interobserver agreement between endoscopists and pathologist for p-CLE findings. Results: Of 66 patients, 40 (60.6%) had lesions in the esophagus, 7 (10.6%) in the stomach, 15 (22.7%) in the biliary tract, 3 (4.5%) in the ampulla and 1 (1.5%) in the colon. The overall sensitivity, specificity and accuracy for diagnosing dysplastic/neoplastic lesions using p-CLE were higher for endoscopists than pathologist at 87.0 vs. 69.6%, 80.0 vs. 40.0% and 84.8
S-337
AGA Abstracts