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137a Inter-Observer Agreement for Manometry Classification of Individual Swallows and Diagnoses Using High-Resolution Manometry (HRM) With Esophageal Pressure Topography (EPT): Results of Web-Based Studies With High Participation
AGA Abstracts
Singly positive cells (mCherry or eGFP) were more predominant, suggesting either the Lgr5EGFP-IRES-CreERT2 transgene is intermittently expressed or the cells are lost by another mechanism. In support of this conclusion, EGFP (Lgr5)+ cells appeared during the imaging period in crypts that were previously only mCherry+. We also induced H2B-mCherry expression In Vitro with 4OHT introduction into the culture medium. No mCherry cells were noted initially, however mCherry+ cells began appearing within 20 hours, all in EGFP (Lgr5+) cells. By the end of the culture period, as the number of mCherry positive cells increased, some lost EGFP expression, possibly due to cell differentiation. Additional studies are underway to clarify this observation. Conclusion: Live imaging of enteroids reveals significant cellular and organoid movements, recapitulating In Vivo processes. Labeling of stem cells with H2B-mCherry using different Cre drivers will enable lineage tracing to be observed in real-time, helping establish relationships between different stem cell populations.
registered members of the hrmconsensus.org website were invited to take part. Institution and experience with HRM were recorded. A sample of 147 individual swallows (study A) and 40 diagnostic studies (study B) was reviewed and classified using a drop down menu that utilized the published system. The on-line platform provided a generic EPT format with fixed pressure scale and time base. Contours facilitated analysis of intra-bolus and contractile pressure; however no data summary was provided. The sequence of swallows was fixed for each user, but randomized between users to avoid sequence bias. Users were blinded to other participant's entries. During the study users were free to reclassify and post comments; however, once completed classifications could not be changed. Results: (A) All 147 individual swallows were assessed by 18 users (13 institutions). High levels of agreement (≤2/18 dissenters) were present for normal peristalsis and achalasia but lower levels for peristaltic and intra-bolus pressure (IBP) abnormalities. (B) All 40 diagnostic studies were assessed by 36 users (28 institutions). Overall inter-observer agreement was fair (kappa 0.42) being higher (kappa >0.5) for aperistalsis and achalasia, and lower (kappa <0.4) for peristaltic abnormalities (table). Users with experience of >400 HRM/EPT studies showed somewhat better agreement (n=9; kappa 0.46) and agreement was good for users in the US institution that developed the classification system (n=4; kappa 0.57). Analysis and review of comments revealed that disagreements in both studies were not random but due to (i) multiple abnormalities in single swallows (e.g. wide break in contractile front and spasm, hypertensive contraction and raised IBP in outflow obstruction) (ii) limited functionality of on-line software / lack of a data summary that confounded user's ability to distinguish closely related diagnoses (figure), especially those that depend on precise pressure measurement (e.g. normal vs. outlet obstruction). Conclusion: This on-line study is the largest assessment of interobserver agreement performed for manometric studies. Overall inter-observer agreement for HRM/EPT of individual swallows and diagnostic studies was fair to moderate, increasing with experience of this technology and the classification system. Analysis of swallows with low agreement indicate that “pattern recognition” of HRM/EPT data was not adequate and highlighted the value of objective metrics in the diagnosis of esophageal dysmotility. Inter-observer agreement: Diagnosis
137 Peptide Selection for ERBB2 Molecular Imaging in Esophageal Adenocarcinoma Xiaoming Zhou, Bishnu P. Joshi, David G. Beer, Thomas D. Wang Background: The alarming increase in incidence of esophageal adenocarcinoma (EAC) and its 5-year mortality rate demand the development of more effective early detection strategies. Molecular changes associated with genetic alterations well precede histopathological abnormalities in tumor development, and their detection on imaging may be useful as an adjunct for early diagnostic evaluation. Cell surface targets are particularly appealing in this regard because of their easy accessibility. ErbB2 and other cell surface genes have been found to be amplified in EAC, providing excellent targets for molecular imaging. Phage display technology offers an unbiased approach for selecting target-specific peptide probes, which can be labeled with fluorescent dyes and administered topically for endoscopic imaging to help the physician guide tissue biopsy, establish diagnosis, and formulate individualized therapy. Aims: We aim to develop an ErbB2-specific peptide probe for molecular imaging both In Vitro and In Vivo Methods: We established a stable Chinese Hamster Ovary (CHO) cell line over-expressing extracellular domain (ECD) of ErbB2 fused with 6-His tag on the C-terminus. The recombinant protein was purified from serum-free culture media with cobalt affinity chromatography. The 7-mer M13 phage library was biopanned against purified recombinant ErbB2-ECD proteins. 4 rounds of biopanning were performed using a decreasing concentration (80, 60, 40, and 20 mg/ml) of ErbB2-ECD protein in successive rounds to increase binding specificity. Selective binding of cloned phage/peptide to ErbB2 was validated on bound phage counts, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and fluorescence microscopy. Results: We achieved a purity >95% for the recombinant ErbB2ECD protein as analyzed by SDS-PAGE. After completion of the 4th round of biopanning, from a total of 50 plaques 15 clones (30%) contain an identical DNA sequence, corresponding to a 7-mer peptide sequence HKRPRNN. ELISA and phage counting results showed that this cloned phage preferentially bound to ErbB2 proteins as compared to BSA (p<0.05) as shown in Fig. 1A. Phage counts also demonstrated that the cloned phage preferentially bound to ErbB2 over-expressing cells (EAC cell line Flo1) as compared to the vector reconstituted control cells (p<0.01) as shown in Fig. 1B. Moreover, FITC-labeled synthetic peptide also showed preferential binding to ErbB2 over-expressed cells as demonstrated on flow cytometry and fluorescence microscopy. We are in the process of validating the peptide on biopsy specimens obtained from EAC patients. Conclusion: We achieved selection of one 7-mer peptide which showed preferential binding to ErbB2 protein as well as to ErbB2 over-expressing cells, laying the foundation for further investigation into the feasibility of this peptide for ErbB2 imaging in EAC patients.
Neighbourhood Diagram. Area of circles is proportional to the number of ratings for the diagnosis. Classes close to each other are often confounded (low kappa). Those far apart are distinct (high kappa). 137a
137b
Inter-Observer Agreement for Manometry Classification of Individual Swallows and Diagnoses Using High-Resolution Manometry (HRM) With Esophageal Pressure Topography (EPT): Results of Web-Based Studies With High Participation Mark R. Fox, John E. Pandolfino, Jafar Jafari, Dieter Menne, Ana T. Abreu, Auke Bogte, Alessandra Elvevi, Antonio Ruiz de Leon, Boudewijn F. Kessing, Lubomyr Boris, Jose M. Remes Troche, C. Prakash Gyawali, Candice L. Wilshire, Dorata Wasko-Czopnik, Francois Mion, Heiko Fruehauf, Frank Zerbib, Oliver Gellersen, Henriette Heinrich, Julio Perez de la Serna, Matthias M. Sauter, Gerrit J. Hemmink, Marcin Banasiuk, Miguel A. Valdovinos, Uscinowicz Miroslawa, Monika A. Kwiatek, Jeff Wright, Pim W. Weijenborg, Rami Sweis, Sabine Roman, Rukiye Vardar, Sudip K. Ghosh, Radu Tutuian, Wout O. Rohof, Albert J. Bredenoord
The Natural History of Patients With Normal and Borderline Motor Function on High-Resolution Manometry Karthik Ravi, Laurel Friesen, Rachel B. Issaka, Peter J. Kahrilas, John E. Pandolfino Background: High resolution manometry (HRM) has expanded recognition of esophageal motor abnormalities and the Chicago classification scheme provides a framework for interpretation of these findings. However, there is a paucity of data regarding outcomes in patients defined as having normal or borderline motor abnormalities Aim: To determine the natural history of patients with borderline motor abnormalities on esophageal HRM. Methods: HRM tracings from Northwestern Memorial Hospital between July of 2004 and October of 2005 were reviewed and interpreted utilizing the Chicago classification. Eligible patients were contacted for a phone survey utilizing the impact dysphagia questionnaire (IDQ). Results: A total of 504 patients were identified. After excluding those with achalasia (n=64), EGJ outflow obstruction (n=35), abnormal motor function [absent peristalsis, DES, hypercontactile] (n=18), previous esophagogastric surgery (n=48), technically limited studies (n=21), previous myotomy or dilation (n=11), patients under the age of 18 years old (n=6), and those with an esophageal stricture (n=4) a total of 297 patients were included. To date, 126
Introduction and Aim: Recently the HRM Working Group presented a classification system for esophageal motility disorders using HRM / EPT (Pandolfino, NGM 2009). This study reports inter-observer agreement for classification of individual HRM/EPT water swallows (study A) and diagnoses based on 10 swallows (study B) using this system. Methods: All
AGA Abstracts
S-34
Singly positive cells (mCherry or eGFP) were more predominant, suggesting either the Lgr5EGFP-IRES-CreERT2 transgene is intermittently expressed or the cells are lost by another mechanism. In support of this conclusion, EGFP (Lgr5)+ cells appeared during the imaging period in crypts that were previously only mCherry+. We also induced H2B-mCherry expression In Vitro with 4OHT introduction into the culture medium. No mCherry cells were noted initially, however mCherry+ cells began appearing within 20 hours, all in EGFP (Lgr5+) cells. By the end of the culture period, as the number of mCherry positive cells increased, some lost EGFP expression, possibly due to cell differentiation. Additional studies are underway to clarify this observation. Conclusion: Live imaging of enteroids reveals significant cellular and organoid movements, recapitulating In Vivo processes. Labeling of stem cells with H2B-mCherry using different Cre drivers will enable lineage tracing to be observed in real-time, helping establish relationships between different stem cell populations.
registered members of the hrmconsensus.org website were invited to take part. Institution and experience with HRM were recorded. A sample of 147 individual swallows (study A) and 40 diagnostic studies (study B) was reviewed and classified using a drop down menu that utilized the published system. The on-line platform provided a generic EPT format with fixed pressure scale and time base. Contours facilitated analysis of intra-bolus and contractile pressure; however no data summary was provided. The sequence of swallows was fixed for each user, but randomized between users to avoid sequence bias. Users were blinded to other participant's entries. During the study users were free to reclassify and post comments; however, once completed classifications could not be changed. Results: (A) All 147 individual swallows were assessed by 18 users (13 institutions). High levels of agreement (≤2/18 dissenters) were present for normal peristalsis and achalasia but lower levels for peristaltic and intra-bolus pressure (IBP) abnormalities. (B) All 40 diagnostic studies were assessed by 36 users (28 institutions). Overall inter-observer agreement was fair (kappa 0.42) being higher (kappa >0.5) for aperistalsis and achalasia, and lower (kappa <0.4) for peristaltic abnormalities (table). Users with experience of >400 HRM/EPT studies showed somewhat better agreement (n=9; kappa 0.46) and agreement was good for users in the US institution that developed the classification system (n=4; kappa 0.57). Analysis and review of comments revealed that disagreements in both studies were not random but due to (i) multiple abnormalities in single swallows (e.g. wide break in contractile front and spasm, hypertensive contraction and raised IBP in outflow obstruction) (ii) limited functionality of on-line software / lack of a data summary that confounded user's ability to distinguish closely related diagnoses (figure), especially those that depend on precise pressure measurement (e.g. normal vs. outlet obstruction). Conclusion: This on-line study is the largest assessment of interobserver agreement performed for manometric studies. Overall inter-observer agreement for HRM/EPT of individual swallows and diagnostic studies was fair to moderate, increasing with experience of this technology and the classification system. Analysis of swallows with low agreement indicate that “pattern recognition” of HRM/EPT data was not adequate and highlighted the value of objective metrics in the diagnosis of esophageal dysmotility. Inter-observer agreement: Diagnosis
137 Peptide Selection for ERBB2 Molecular Imaging in Esophageal Adenocarcinoma Xiaoming Zhou, Bishnu P. Joshi, David G. Beer, Thomas D. Wang Background: The alarming increase in incidence of esophageal adenocarcinoma (EAC) and its 5-year mortality rate demand the development of more effective early detection strategies. Molecular changes associated with genetic alterations well precede histopathological abnormalities in tumor development, and their detection on imaging may be useful as an adjunct for early diagnostic evaluation. Cell surface targets are particularly appealing in this regard because of their easy accessibility. ErbB2 and other cell surface genes have been found to be amplified in EAC, providing excellent targets for molecular imaging. Phage display technology offers an unbiased approach for selecting target-specific peptide probes, which can be labeled with fluorescent dyes and administered topically for endoscopic imaging to help the physician guide tissue biopsy, establish diagnosis, and formulate individualized therapy. Aims: We aim to develop an ErbB2-specific peptide probe for molecular imaging both In Vitro and In Vivo Methods: We established a stable Chinese Hamster Ovary (CHO) cell line over-expressing extracellular domain (ECD) of ErbB2 fused with 6-His tag on the C-terminus. The recombinant protein was purified from serum-free culture media with cobalt affinity chromatography. The 7-mer M13 phage library was biopanned against purified recombinant ErbB2-ECD proteins. 4 rounds of biopanning were performed using a decreasing concentration (80, 60, 40, and 20 mg/ml) of ErbB2-ECD protein in successive rounds to increase binding specificity. Selective binding of cloned phage/peptide to ErbB2 was validated on bound phage counts, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and fluorescence microscopy. Results: We achieved a purity >95% for the recombinant ErbB2ECD protein as analyzed by SDS-PAGE. After completion of the 4th round of biopanning, from a total of 50 plaques 15 clones (30%) contain an identical DNA sequence, corresponding to a 7-mer peptide sequence HKRPRNN. ELISA and phage counting results showed that this cloned phage preferentially bound to ErbB2 proteins as compared to BSA (p<0.05) as shown in Fig. 1A. Phage counts also demonstrated that the cloned phage preferentially bound to ErbB2 over-expressing cells (EAC cell line Flo1) as compared to the vector reconstituted control cells (p<0.01) as shown in Fig. 1B. Moreover, FITC-labeled synthetic peptide also showed preferential binding to ErbB2 over-expressed cells as demonstrated on flow cytometry and fluorescence microscopy. We are in the process of validating the peptide on biopsy specimens obtained from EAC patients. Conclusion: We achieved selection of one 7-mer peptide which showed preferential binding to ErbB2 protein as well as to ErbB2 over-expressing cells, laying the foundation for further investigation into the feasibility of this peptide for ErbB2 imaging in EAC patients.
Neighbourhood Diagram. Area of circles is proportional to the number of ratings for the diagnosis. Classes close to each other are often confounded (low kappa). Those far apart are distinct (high kappa). 137a
137b
Inter-Observer Agreement for Manometry Classification of Individual Swallows and Diagnoses Using High-Resolution Manometry (HRM) With Esophageal Pressure Topography (EPT): Results of Web-Based Studies With High Participation Mark R. Fox, John E. Pandolfino, Jafar Jafari, Dieter Menne, Ana T. Abreu, Auke Bogte, Alessandra Elvevi, Antonio Ruiz de Leon, Boudewijn F. Kessing, Lubomyr Boris, Jose M. Remes Troche, C. Prakash Gyawali, Candice L. Wilshire, Dorata Wasko-Czopnik, Francois Mion, Heiko Fruehauf, Frank Zerbib, Oliver Gellersen, Henriette Heinrich, Julio Perez de la Serna, Matthias M. Sauter, Gerrit J. Hemmink, Marcin Banasiuk, Miguel A. Valdovinos, Uscinowicz Miroslawa, Monika A. Kwiatek, Jeff Wright, Pim W. Weijenborg, Rami Sweis, Sabine Roman, Rukiye Vardar, Sudip K. Ghosh, Radu Tutuian, Wout O. Rohof, Albert J. Bredenoord
The Natural History of Patients With Normal and Borderline Motor Function on High-Resolution Manometry Karthik Ravi, Laurel Friesen, Rachel B. Issaka, Peter J. Kahrilas, John E. Pandolfino Background: High resolution manometry (HRM) has expanded recognition of esophageal motor abnormalities and the Chicago classification scheme provides a framework for interpretation of these findings. However, there is a paucity of data regarding outcomes in patients defined as having normal or borderline motor abnormalities Aim: To determine the natural history of patients with borderline motor abnormalities on esophageal HRM. Methods: HRM tracings from Northwestern Memorial Hospital between July of 2004 and October of 2005 were reviewed and interpreted utilizing the Chicago classification. Eligible patients were contacted for a phone survey utilizing the impact dysphagia questionnaire (IDQ). Results: A total of 504 patients were identified. After excluding those with achalasia (n=64), EGJ outflow obstruction (n=35), abnormal motor function [absent peristalsis, DES, hypercontactile] (n=18), previous esophagogastric surgery (n=48), technically limited studies (n=21), previous myotomy or dilation (n=11), patients under the age of 18 years old (n=6), and those with an esophageal stricture (n=4) a total of 297 patients were included. To date, 126
Introduction and Aim: Recently the HRM Working Group presented a classification system for esophageal motility disorders using HRM / EPT (Pandolfino, NGM 2009). This study reports inter-observer agreement for classification of individual HRM/EPT water swallows (study A) and diagnoses based on 10 swallows (study B) using this system. Methods: All
AGA Abstracts
S-34