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Bile Proteomic Profiles Differentiates Cholangiocarcinoma From Primary Sclerosing Cholangitis and Choledocholithiasis
patients with CC complicating PSC were identified. Conclusion: Bile proteomic analysis discriminates benign conditions from CC accurately. This method may become a diagnostic tool in future as it offers a new possibility to diagnose malignant bile duct disease and thus enables efficient therapy particularly in patients with PSC. 322 Carbohydrate Intake: A Risk Factor for Biliary Sludge and Stones During Pregnancy Alan C. Wong, Cynthia W. Ko PURPOSE: Female gender is a risk factor for gallstones and pregnancy is a high-risk period for gallstone development. Gallstone disease is one of the most common non-obstetrical causes of morbidity both during and after pregnancy. High carbohydrate intake has been shown to cause insulin resistance and derangement of serum lipid profiles, conditions thought to favor gallstone formation. The aim of this study was to examine the effect of dietary carbohydrate intake on the formation of biliary sludge and stones during pregnancy. METHODS: This prospective study included 3070 pregnant adult women (avg age 25.2) who underwent gallbladder ultrasound during each trimester of pregnancy and at 4-6 weeks postpartum. All women had at least 2 study ultrasounds for comparison. Women with stones on their first ultrasound or with prior cholecystectomy were excluded. Those with new sludge, new stones, or progression of baseline sludge to stones were considered to have incident gallbladder disease. A semi-quantitative food frequency questionnaire was completed by subjects in the early 3rd trimester. Average daily intake of dietary factors was computed using standard food composition data. Multivariate logistic regression adjusting for age, prepregnancy body mass index, weight gain during pregnancy, parity, Hispanic origin, smoking, history of diabetes, and intake of alcohol, caffeine, total calories, protein, fat, fiber, cholesterol, and fatty acids (saturated, monounsaturated, polyunsaturated, trans) was done to assess the risk of incident gallbladder disease across quartiles of intake of total carbohydrate and individual carbohydrates (starch, sucrose, galactose, fructose, lactose, and maltose). RESULTS: By 4-6 weeks postpartum, the cumulative incidence of new sludge/stones or progression of baseline sludge to stones was 10.2%. The risk of incident gallbladder disease during pregnancy was significantly higher among women in the highest quartile of total carbohydrate intake when compared to those in the lowest quartile (Table). High intake of starch or fructose was associated with increased risk even after additional adjustment for total carbohydrate intake, while galactose was associated with decreased risk. CONCLUSIONS: High consumption of total carbohydrate, starch, and fructose may increase the risk of developing biliary sludge and stones during pregnancy. Dietary modification during pregnancy may reduce this risk. Table. Carbohydrate consumption and the risk of incident gallstone disease*
320 Validation of a Patient Reported Outcome Instrument, the Reflux Symptom Questionnaire Electronic Diary (Resq-ED), in Patients With a Partial Response to PPI Therapy Nimish B. Vakil, Debra G. Silberg, Karin Björck, Katarina Halling, Jean Paty, Maria Karlsson, Hans Denison, Anna Rydén Introduction. About 20-30% of patients with gastroesophageal reflux disease (GERD) have persistent symptoms on proton pump inhibitors (PPIs). No patient reported outcome (PRO) instrument has been developed and documented fit for purpose in clinical trials in patients with a partial response to PPI therapy. Following the FDA guidance on PROs, we aimed to develop and validate the Reflux Symptom Questionnaire electronic Diary (RESQ-eD) for use in this setting. Methods. Psychometric evaluation was conducted in a validation study (NCT00703534) and confirmed in a subsequent dose-finding study (NCT01005251). Both studies had a screening phase followed by 4 weeks' treatment with the reflux inhibitor lesogaberan or placebo as add-on to PPI. Eligible patients had a history of GERD for ≥6 months, and heartburn and/or regurgitation of ≥mild/moderate intensity on ≥3 days in the 7 days before the study. Patients completed the RESQ-eD twice daily (morning and evening) and the Overall Treatment Evaluation (OTE) after 2 and 4 weeks of treatment. In the validation study, patients also completed the Gastrointestinal Symptom Rating Scale (GSRS). Results. In total, 478 patients were randomized in the validation study (mean age: 49 years; 41% men) and 661 in the dose-finding study (mean age: 48 years; 43% men). Patient interviews in the validation study endorsed the relevance and comprehensibility of all RESQ-eD items. Principal component analysis supported a 4-domain structure (Heartburn; Regurgitation; Hoarseness, cough, difficulty swallowing; Burping). Cronbach's α for interitem correlation was in the range 0.89-0.96 in the validation study and 0.85-0.93 in the dose-finding study, indicating high internal consistency reliability. Test-retest reliability was fair to excellent, in the range 0.65-0.85 in the validation study and 0.77-0.85 in the dose finding study. Convergent validity was supported in the validation study by high correlations of RESQ-eD Burping with GSRS Belching, and all other RESQ-eD domains with the GSRS Reflux domain. Low correlations between all RESQ-eD domains and the GSRS Diarrhoea and Constipation domains supported discriminant validity. Graphical depiction of effect size by OTE classification at the end of treatment showed a good ability to capture change in intensity on all RESQ-eD domains in both studies. OTE was also related to changes in the frequency of days with no, at most very mild and at most mild symptoms in the validation study. Conclusions. The RESQ-eD was deemed to be valid, reliable, responsive to change and fit for purpose in clinical trials in partial responders to PPI therapy. This is, to our knowledge, the first GERD instrument developed using the FDA guidance on PROs. In addition to heartburn and regurgitation, partial responders to PPI consider cough, hoarseness, dysphagia and burping to be part of their symptom experience.
*Highest quartile of intake compared to lowest quartile 323 Rapid Postprandial Gallbladder Refilling and Increased Turnover of Bile Prevent Cholesterol Crystallization in Small Heterodimer Partner (SHP) Knockout Mice Helen H. Wang, Piero Portincasa, Min Liu, David Q. Wang The gallbladder is a dynamic organ with complex motor functions under a precise neurohormonal control during fasting and postprandially. Gallbladder emptying and refilling allows the flow of bile into the intestine for micellization and absorption of dietary fat, which are actively regulated by two endocrine pathways CCK and fibroblast growth factor 15 (FGF15). However, it is unclear what the role of gallbladder refilling in cholelithogenesis is. Our aim was to explore whether rapid postprandial gallbladder refilling and increased turnover of bile prevent cholesterol crystallization and gallstones. Methods: Biliary lipid secretion and gallstone studies were performed in male SHP (-/-) and (+/+) mice (n=8 per group) before and during feeding a lithogenic diet (1% cholesterol, 0.5% cholic acid and 15% butterfat) for 56 days. Dynamic gallbladder emptying and refilling functions were measured by physical methods. Intestinal bile salt (BS) concentrations were measured biochemically. Gene expression levels were determined by real-time PCR. Results: Deletion of the SHP gene resulted in significantly increased hepatic BS synthesis, and increased biliary BS outputs by 2 fold in SHP (-/-) mice than (+/+) mice. Intestinal bile acid pool size was significantly greater in SHP (-/-) mice than (+/+) mice. The increased bile acids are most efficacious ligands of FXR and stimulate expression of intestinal FGF15 through the FXR signaling pathways, being consistent with expanded bile acid pool size in SHP (-/-) mice. At 14 days on the lithogenic diet, fasting gallbladder volume was significantly larger in SHP (+/+) mice than (-/-) mice. Feeding corn oil (i.e., a high-fat meal) induced gallbladder emptying and residual gallbladder volume was significantly larger in SHP (+/+) than that in (-/-) mice. At 30 min after the high-fat meal, gallbladder volume began to increase, indicating that postprandial gallbladder refilling started. This increase began to accelerate after 60 min and cumulative gallbladder refilling volume reached 43 μL at 120 min in SHP (-/-) mice. In contrast, there was minimal postprandial gallbladder refilling volume in SHP (+/+) mice, suggesting sluggish gallbladder kinetics. The mean turnover index was significantly greater in SHP (-/-) mice than (+/+) mice. Whereas gallstones were found in 60% (day 28) and 100% (day 56) of SHP (+/+) mice, no cholesterol crystals or gallstones were ever detected in SHP (-/-) mice. Conclusions: Rapid fluctuations in gallbladder refilling with increased turnover of bile within the gallbladder provide a strong washout effect that is more crucial in eliminating solid cholesterol crystals than gallbladder emptying alone. Our findings suggest that these two parameters
321 Bile Proteomic Profiles Differentiates Cholangiocarcinoma From Primary Sclerosing Cholangitis and Choledocholithiasis Tim O. Lankisch, Jochen Metzger, Ahmed A. Negm, Katja Vosskuhl, Eric Schiffer, Siwy Justyna, Tobias J. Weismüller, Andrea S. Schneider, Kathrin Thedieck, Ralf Baumeister, Petra Zürbig, Eva M. Weissinger, Michael P. Manns, Harald Mischak, Jochen Wedemeyer Introduction: Early detection of malignant biliary tract diseases, especially cholangiocarcinoma (CC) in patients with primary sclerosing cholangitis (PSC), is very difficult and often comes too late to give the patient a therapeutic benefit. We hypothesize that bile proteomic analysis distinguishes CC from non-malignant lesions. Methods: We used capillary electrophoresis mass spectrometry (CE-MS) to identify disease specific peptide patterns in patients with choledocholithiasis (n=16), PSC (n=18) and CC (n=16) in a training set. A model for differentiation of choledocholithiasis from PSC and CC (PSC/CC model) and another model distinguishing CC from PSC (CC model) were subsequently validated in independent cohorts (choledocholithiasis (n=14), PSC (n=18) and CC (n=25)). Peptides were characterized by sequencing. Results: Application of the PSC/CC model in the independent test cohort resulted in correct exclusion of 12/14 bile samples from patients with choledocholithiasis and identification of 40/43 patients with PSC or CC (86% specificity, 93% sensitivity). The corresponding receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.93 (95% CI: 0.82 - 0.98, p=0.0001). The CC model succeeded in an accurate detection of 14/18 bile samples from patients with PSC and 21/25 samples with CC (78% specificity, 84% sensitivity) in the independent cohort resulting in an AUC-value of 0.87 (95% CI: 0.73 - 0.95, p=0.0001) in ROC analysis. Eight out of ten samples of
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assessed by a patient reported standard questionnaire (PAGI-SYM, Patient Assessment of Gastro Intestinal Disorders-Symptom Severity Index) by devoting a score from 0 to 5 for each symptom, before and after the treatment. The study is still ongoing. Results: Thirty six patients were classified as pH-positive patients while 46 of them were allocated to pHnegative groups. With the FX, OM and PL, sub-group analysis showed significant improvement of heartburn/regurgitation severity score in pH-positive patients (p, 0,004, 0.005, 0.035, respectively) and also in those with negative pH monitoring result(p, 0.0001, 0.001, 0.035, respectively). Mean decrement in heartburn/regurgitation score for FX, OM and PL for pH-positive patients was 0.74 ± 0.22, 0.71 ± 0.3, 1.14 ± 0.68 respectively, while for the patients with negative pH monitoring result it was 0.78 ± 0.33, 1.32 ± 0.45, 0.6 ± 0.45, respectively. Interestingly, the mentioned symptom severity score decrements did not differ significantly between 3 medications in either pH positive or pH negative patients (p, 0.35, 0.2, respectively). Conclusion: FX, OM and PL produced a clinically significant improvement in symptom severity in NERD patients. Besides, there was no significant difference in extent of symptom severity improvement among 3 medications. Our findings support that: 1) A considerable portion of response to PPIs and antidepressants in NERD patients may be explained as placebo effect. 2) Acid does not play a major role in development of heartburn and regurgitation in NERD patients and this may be a reason for why most of these patients fail to respond to PPI treatment. 3) Psychological comorbidities are likely to have an important role in symptom development and poor symptomatic response in patients with NERD even in those with positive pH monitoring result.
320 Validation of a Patient Reported Outcome Instrument, the Reflux Symptom Questionnaire Electronic Diary (Resq-ED), in Patients With a Partial Response to PPI Therapy Nimish B. Vakil, Debra G. Silberg, Karin Björck, Katarina Halling, Jean Paty, Maria Karlsson, Hans Denison, Anna Rydén Introduction. About 20-30% of patients with gastroesophageal reflux disease (GERD) have persistent symptoms on proton pump inhibitors (PPIs). No patient reported outcome (PRO) instrument has been developed and documented fit for purpose in clinical trials in patients with a partial response to PPI therapy. Following the FDA guidance on PROs, we aimed to develop and validate the Reflux Symptom Questionnaire electronic Diary (RESQ-eD) for use in this setting. Methods. Psychometric evaluation was conducted in a validation study (NCT00703534) and confirmed in a subsequent dose-finding study (NCT01005251). Both studies had a screening phase followed by 4 weeks' treatment with the reflux inhibitor lesogaberan or placebo as add-on to PPI. Eligible patients had a history of GERD for ≥6 months, and heartburn and/or regurgitation of ≥mild/moderate intensity on ≥3 days in the 7 days before the study. Patients completed the RESQ-eD twice daily (morning and evening) and the Overall Treatment Evaluation (OTE) after 2 and 4 weeks of treatment. In the validation study, patients also completed the Gastrointestinal Symptom Rating Scale (GSRS). Results. In total, 478 patients were randomized in the validation study (mean age: 49 years; 41% men) and 661 in the dose-finding study (mean age: 48 years; 43% men). Patient interviews in the validation study endorsed the relevance and comprehensibility of all RESQ-eD items. Principal component analysis supported a 4-domain structure (Heartburn; Regurgitation; Hoarseness, cough, difficulty swallowing; Burping). Cronbach's α for interitem correlation was in the range 0.89-0.96 in the validation study and 0.85-0.93 in the dose-finding study, indicating high internal consistency reliability. Test-retest reliability was fair to excellent, in the range 0.65-0.85 in the validation study and 0.77-0.85 in the dose finding study. Convergent validity was supported in the validation study by high correlations of RESQ-eD Burping with GSRS Belching, and all other RESQ-eD domains with the GSRS Reflux domain. Low correlations between all RESQ-eD domains and the GSRS Diarrhoea and Constipation domains supported discriminant validity. Graphical depiction of effect size by OTE classification at the end of treatment showed a good ability to capture change in intensity on all RESQ-eD domains in both studies. OTE was also related to changes in the frequency of days with no, at most very mild and at most mild symptoms in the validation study. Conclusions. The RESQ-eD was deemed to be valid, reliable, responsive to change and fit for purpose in clinical trials in partial responders to PPI therapy. This is, to our knowledge, the first GERD instrument developed using the FDA guidance on PROs. In addition to heartburn and regurgitation, partial responders to PPI consider cough, hoarseness, dysphagia and burping to be part of their symptom experience.
*Highest quartile of intake compared to lowest quartile 323 Rapid Postprandial Gallbladder Refilling and Increased Turnover of Bile Prevent Cholesterol Crystallization in Small Heterodimer Partner (SHP) Knockout Mice Helen H. Wang, Piero Portincasa, Min Liu, David Q. Wang The gallbladder is a dynamic organ with complex motor functions under a precise neurohormonal control during fasting and postprandially. Gallbladder emptying and refilling allows the flow of bile into the intestine for micellization and absorption of dietary fat, which are actively regulated by two endocrine pathways CCK and fibroblast growth factor 15 (FGF15). However, it is unclear what the role of gallbladder refilling in cholelithogenesis is. Our aim was to explore whether rapid postprandial gallbladder refilling and increased turnover of bile prevent cholesterol crystallization and gallstones. Methods: Biliary lipid secretion and gallstone studies were performed in male SHP (-/-) and (+/+) mice (n=8 per group) before and during feeding a lithogenic diet (1% cholesterol, 0.5% cholic acid and 15% butterfat) for 56 days. Dynamic gallbladder emptying and refilling functions were measured by physical methods. Intestinal bile salt (BS) concentrations were measured biochemically. Gene expression levels were determined by real-time PCR. Results: Deletion of the SHP gene resulted in significantly increased hepatic BS synthesis, and increased biliary BS outputs by 2 fold in SHP (-/-) mice than (+/+) mice. Intestinal bile acid pool size was significantly greater in SHP (-/-) mice than (+/+) mice. The increased bile acids are most efficacious ligands of FXR and stimulate expression of intestinal FGF15 through the FXR signaling pathways, being consistent with expanded bile acid pool size in SHP (-/-) mice. At 14 days on the lithogenic diet, fasting gallbladder volume was significantly larger in SHP (+/+) mice than (-/-) mice. Feeding corn oil (i.e., a high-fat meal) induced gallbladder emptying and residual gallbladder volume was significantly larger in SHP (+/+) than that in (-/-) mice. At 30 min after the high-fat meal, gallbladder volume began to increase, indicating that postprandial gallbladder refilling started. This increase began to accelerate after 60 min and cumulative gallbladder refilling volume reached 43 μL at 120 min in SHP (-/-) mice. In contrast, there was minimal postprandial gallbladder refilling volume in SHP (+/+) mice, suggesting sluggish gallbladder kinetics. The mean turnover index was significantly greater in SHP (-/-) mice than (+/+) mice. Whereas gallstones were found in 60% (day 28) and 100% (day 56) of SHP (+/+) mice, no cholesterol crystals or gallstones were ever detected in SHP (-/-) mice. Conclusions: Rapid fluctuations in gallbladder refilling with increased turnover of bile within the gallbladder provide a strong washout effect that is more crucial in eliminating solid cholesterol crystals than gallbladder emptying alone. Our findings suggest that these two parameters
321 Bile Proteomic Profiles Differentiates Cholangiocarcinoma From Primary Sclerosing Cholangitis and Choledocholithiasis Tim O. Lankisch, Jochen Metzger, Ahmed A. Negm, Katja Vosskuhl, Eric Schiffer, Siwy Justyna, Tobias J. Weismüller, Andrea S. Schneider, Kathrin Thedieck, Ralf Baumeister, Petra Zürbig, Eva M. Weissinger, Michael P. Manns, Harald Mischak, Jochen Wedemeyer Introduction: Early detection of malignant biliary tract diseases, especially cholangiocarcinoma (CC) in patients with primary sclerosing cholangitis (PSC), is very difficult and often comes too late to give the patient a therapeutic benefit. We hypothesize that bile proteomic analysis distinguishes CC from non-malignant lesions. Methods: We used capillary electrophoresis mass spectrometry (CE-MS) to identify disease specific peptide patterns in patients with choledocholithiasis (n=16), PSC (n=18) and CC (n=16) in a training set. A model for differentiation of choledocholithiasis from PSC and CC (PSC/CC model) and another model distinguishing CC from PSC (CC model) were subsequently validated in independent cohorts (choledocholithiasis (n=14), PSC (n=18) and CC (n=25)). Peptides were characterized by sequencing. Results: Application of the PSC/CC model in the independent test cohort resulted in correct exclusion of 12/14 bile samples from patients with choledocholithiasis and identification of 40/43 patients with PSC or CC (86% specificity, 93% sensitivity). The corresponding receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.93 (95% CI: 0.82 - 0.98, p=0.0001). The CC model succeeded in an accurate detection of 14/18 bile samples from patients with PSC and 21/25 samples with CC (78% specificity, 84% sensitivity) in the independent cohort resulting in an AUC-value of 0.87 (95% CI: 0.73 - 0.95, p=0.0001) in ROC analysis. Eight out of ten samples of
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AGA Abstracts
assessed by a patient reported standard questionnaire (PAGI-SYM, Patient Assessment of Gastro Intestinal Disorders-Symptom Severity Index) by devoting a score from 0 to 5 for each symptom, before and after the treatment. The study is still ongoing. Results: Thirty six patients were classified as pH-positive patients while 46 of them were allocated to pHnegative groups. With the FX, OM and PL, sub-group analysis showed significant improvement of heartburn/regurgitation severity score in pH-positive patients (p, 0,004, 0.005, 0.035, respectively) and also in those with negative pH monitoring result(p, 0.0001, 0.001, 0.035, respectively). Mean decrement in heartburn/regurgitation score for FX, OM and PL for pH-positive patients was 0.74 ± 0.22, 0.71 ± 0.3, 1.14 ± 0.68 respectively, while for the patients with negative pH monitoring result it was 0.78 ± 0.33, 1.32 ± 0.45, 0.6 ± 0.45, respectively. Interestingly, the mentioned symptom severity score decrements did not differ significantly between 3 medications in either pH positive or pH negative patients (p, 0.35, 0.2, respectively). Conclusion: FX, OM and PL produced a clinically significant improvement in symptom severity in NERD patients. Besides, there was no significant difference in extent of symptom severity improvement among 3 medications. Our findings support that: 1) A considerable portion of response to PPIs and antidepressants in NERD patients may be explained as placebo effect. 2) Acid does not play a major role in development of heartburn and regurgitation in NERD patients and this may be a reason for why most of these patients fail to respond to PPI treatment. 3) Psychological comorbidities are likely to have an important role in symptom development and poor symptomatic response in patients with NERD even in those with positive pH monitoring result.