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Correlation Of Gene Haplotypes With Complications After Ileal Pouch Anal Anastomosis
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 20.2. Routine Provocative Leak Testing In Colorectal Surgery As A Quality Improvement Metric. S. Kwon,1 R. Billingham,7 K. D. Bussard,4 J. Frankhouse,5 K. Horvath,1 S. Steele,3 M. Johnson,8 S. McNevin,4 R. Thirby,2 K. Morris,5 M. Whiteford,5 A. Simons,6 L. McIntyre,1 D. R. Flum1; 1University of Washington/Harborview Medical Center, Seattle, WA; 2 Virginia Mason Medical Center, Seattle, WA; 3Madigan Army Medical Center, Tacoma, WA; 4Providence Sacred Heart Medical Center & Childrens Hospital, Spokane, WA; 5Legacy Good Samaritan Medical Center, Portland, OR; 6St. John Medical Center, Longview, WA; 7Swedish Medical Center, Seattle, WA; 8Skagit Valley Hospital, Mount Vernon, WA Introduction: The value of provocative testing of colorectal anastomoses to reduce morbidity and mortality has yet to be determined. Part of the challenge of determining its use as a quality improvement (QI) metric is that leak testing at the time of operation can be done to screen for leak as well as to confirm a suspected leak. If used for confirmation of a suspected leak then leak testing might actually appear to be associated with increased risk of leak. Methods: The Surgical Care and Outcomes Assessment Program (SCOAP) is a Washington State QI benchmarking tool based on chart-abstracted process of care and risk-adjusted outcomes data. We evaluated leak testing, death and/or adverse events requiring reintervention as a composite adverse event (CAE) for patients undergoing elective left-sided colon or rectal resections at 40 SCOAP hospitals from October 1, 2005 to December 31, 2009. To account for use of leak testing among patients where there was a suspected leak, we distinguished procedures that were performed at hospitals where leak testing was ‘‘selective’’ (<90% use) separate from those where leak testing was greater than 90% (routine) in a given calendar quarter. Results: Of 3,449 (58.8614.8 yrs; 55.0% women) patients the CAE rate was 5.5% and in-hospital mortality 0.5%. Provocative leak testing increased (56.0% in Q0 to 75.8% in Q16) and rates of CAE decreased (7.0% in Q0 to 4.6% in Q16) over time (test for trend p-value0.01 for both). Male sex, low albumin levels (<3.0 g/dL), use of an immunosuppressant, increased operating time, and higher comorbidity score were associated with an increased rate of CAE. Among all patients at routine testing hospitals, those undergoing leak testing had a CAE rate of 5.8% compared to 11.1% among untested (p-value¼0.18) with a>75% reduction in risk after adjustment for patient characteristics (OR 0.23, 95% CI 0.06-0.95). For example, leak testing among patients with adequate nutritional status (albumin>3g/dL) was associated with sharp reductions in CAE from 20% to 5.8% when testing was used (p-value¼0.02). Conclusions: Routine leak testing appears to be associated with better outcomes and was part of the reason the SCOAP network reduced the rates of CAE.
211
Operations done at routine centers: all patients
Deaths Composite Adverse Event
All patients (n¼1,267)
Tested group (n¼36)
Untested group (n¼1,230)
P-value
8 (0.6%) 75 (5.9%)
8 (0.7%) 71 (5.8%)
0 (0%) 4 (11.1%)
0.63 0.18
Operations done at routine centers: Patients with albumin level greater than 3 mg/dL
Deaths Composite Adverse Event
All patients (n ¼ 535)
Tested group (n ¼ 520)
Untested group (n ¼ 15)
P value
4 (0.8%) 33(6.2%)
4 (0.8%) 30 (5.8%)
0 (0%) 3 (20.0%)
0.73 0.02
20.3. Correlation Of Gene Haplotypes With Complications After Ileal Pouch Anal Anastomosis. R. Sehgal, A. Berg, J. Polinski, J. P. Hegarty, A. A. Kelly, Z. Lin, L. S. Poritz, W. A. Koltun; Penn State Milton S. Hershey Medical Center, Hershey, PA Introduction: Ileal pouch anal anastomosis (IPAA) is the operative gold standard for the treatment of ulcerative colitis (UC). However, complications such as pouchitis and Crohn’s disease (CD) like phenomena confound its success. Recently, genome wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNP’s) that are associated with inflammatory bowel disease (CD, UC or both). The purpose of this study was to identify SNP’s that correlate with complications after IPAA with the goal of identifying genes of relevance that could focus future research investigations into the pathophysiologic basis of these IPAA complications and possibly assist with preoperative surgical decision making. Methods: 142 IPAA patients were retrospectively classified as: 1) Asymptomatic (no pouchitis or CD like complications for at least 2 yrs after IPAA); 2) CD-like complications (presence of fistuli, pouch inlet stricture, proximal small bowel disease or pouch granulomata, occurring at least 6 months after surgery; 3) mild pouchitis (<4 episodes pouchitis/yr for 2 years, each episode responding to a 2 wk course of a single antibiotic); and 4) severe pouchitis (4 episodes pouchitis/yr for 2 years including the need for long term therapy to maintain remission). Genotyping for 71 SNP’s in 49 genes previously associated with IBD was performed on a customized Illumina BeadXpress VeraCode platform. Statistical assessment of the genetic associations, including multiple hypothesis testing, was evaluated with the Fisher exact test. Results: The most significant associated SNP’s, p values and their respective genes/loci are summarized in the table below: Conclusions: Specific allelic SNP determinants in 9 individual genes/loci appear to correlate with specific complications after IPAA. The majority of these genes have been previously associated with CD and play a role in enteric bacterial recognition and destruction (NOD2, ATG16L1, TLR, CARD 9) and/ or NF-kappa B activation (NOD2, TNFSF15). The highest statistical significance was found in the CD-like and severe pouchitis groups. This could be due to the more clearly defined phenotype in these two groups (as opposed to the more nebulous ‘‘mild pouchitis’’), but
212
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
may also reflect a more Crohn’s-like pathophysiology in these originally diagnosed UC patients. The 10q21 locus which has been associated with CD in several large GWAS studies is a gene desert with little understood function but had the highest correlation with CD like complications and may represent a fertile area for further investigation. Whether identifying such genetic determinants preoperatively could play a role in surgical decision making warrants further investigation.
treated with PGE2 for 10 days showed more epithelium and fewer goblet cells (E) than that of vehicle treated (F), indicating that PGE2 may promote TEC growth by keeping the epithelium in a proliferating stage. Conclusions: We established a reliable in vitro culture system and in vivo approach to study the function of key factors involved in the formation and growth of TESI and TEC from OU. Our observation that PGE2 promotes the growth of mouse TESI and TEC has a potential benefit for generating human tissue engineered intestine in an efficient manner.
20.5. Use of a DPP-IV inhibitor leads to accelerated intestinal adaptation in a mouse model of short bowel syndrome. M. Okawada, H. M. Maria, D. H. Teitelbaum; University of Michigan, Ann Arbor, MI
20.4. PGE2 Promotes The Growth Of Tissue Engineered Small And Large Intestine. Y. Li, A. L. Speer, J. A. Matthews, F. G. Sala, E. R. Barthel, Y. Torashima, T. C. Grikscheit; Children’s Hospital Los Angeles, Los Angeles, CA Introduction: Our objective is to determine if PGE2 promotes the formation and growth of Tissue Engineered-Small Intestine (TESI) and –Colon (TEC). PGE2 regulates multiple biological processes under both normal and pathological conditions. In intestine, PGE2 stimulates epithelial cell proliferation and migration and promotes regeneration of gastrointestinal epithelium after injury. TESI, a potential durable autologous therapy for Short Bowel Syndrome, has been generated in our lab. Methods: Organoid units (OU) from ActinGFP transgenic mice were either cultured in a matrigel system with or without the presence of 100ng/ml PGE2 for 6 days, or implanted into the omentum of NOD/SCID gamma chain deficient mice. The implanted mice were then treated with vehicle or 10 ug PGE2/kg of body weight twice a day until harvest. All TESI and TEC were harvested and analyzed with gross morphology and histology after 10 days or 14 days. Cyclooxygenase 2 protein (COX2), a rate-limiting enzyme involved in PGE2 synthesis, was stained by immunohistochemistry. Results: COX2expressing cells were located only in the regenerating area of TESI (A) and TEC (B); in the in vitro culture system, PGE2 promotes growth of both small intestine OU and colon OU when measuring size (data not shown). In the in vivo model, exogenous PGE2 stimulates epithelial growth of TESI when measuring the epithelial thickness (C and D). TEC
Introduction: Glucagon-like peptide 2 (GLP-2) acts as one of the strongest mediators of small intestinal mucosal epithelial adaptation by stimulation of crypt cell proliferation and inhibition of enterocyte apoptosis. The use of GLP-2 may have a use in accelerating the mucosal adaptive response in short bowel syndrome (SBS). In fact, mammalian organisms up-regulate the production of GLP-2 during this adaptive phase. However, incretin hormones are rapidly inactivated (within 7 minutes) by the enzyme dipeptidyl peptidase-IV (DPP4). Currently, inhibitors of DPP-4 (DPP4-I) are used to inhibit a related incretin, GLP-1, and used for the treatment of diabetes. Based on this, we hypothesized that by selectively inhibiting DPP4, endogenous GLP-2 would be preserved, thereby prolonging the circulating life of incretin hormones, and lead to improved intestinal adaptation in SBS. Methods: 8-week old C57BL/6J mice underwent a 50% proximal small bowel resection after being placed on a liquid diet for the preceding 2 days. Sitagliptin, (DPP4-I), was given orally every eight hours (50mg/kg/dose, n¼6) starting 1 day before surgery. Six mice were given oral phosphate-buffered saline orally as a control (SBS+placebo). Body weight was followed daily. Blood samples were taken daily to determine serum glucose levels. At 3 days, the adaptive response was assessed in the remnant gut by measuring villus height, crypt depth, apoptosis and proliferation (BrdU incorporation). mRNA abundance of jejunal and ileal GLP-2 receptor (GLP-2R) and sodium glucose co-transporter 1(SGLT1) were examined using real time polymerase chain reaction (PCR). Results: Treatment with DPP4-I led to a significant increase in villus height and crypt depth vs. SBS+placebo mice (Table). DPP4-I treatment did not significantly change apoptosis rates, but did significantly increase the rate of epithelial cell proliferation in the crypts compared to SBS+placebo controls(Figure). GLP-2R and SGLT-1 mRNA expression in jejunum and ileum were not significantly changed. Percent body weight loss and the level of peripheral blood glucose did not significantly change between SBS+DPP4-I and SBS +placebo groups, demonstrating the relative safety of DPP4-I in a setting of SBS. Conclusions: This study showed a very novel treatment approach for patients with SBS. Use of a DPP4-I led to an accelerated intestinal adaptive process. This unique approach for patients with SBS has the advantage of capitalizing on a patient’s endogenous GLP-2 expression, with a previously approved FDA drug, and may have clinical applications.
211
Operations done at routine centers: all patients
Deaths Composite Adverse Event
All patients (n¼1,267)
Tested group (n¼36)
Untested group (n¼1,230)
P-value
8 (0.6%) 75 (5.9%)
8 (0.7%) 71 (5.8%)
0 (0%) 4 (11.1%)
0.63 0.18
Operations done at routine centers: Patients with albumin level greater than 3 mg/dL
Deaths Composite Adverse Event
All patients (n ¼ 535)
Tested group (n ¼ 520)
Untested group (n ¼ 15)
P value
4 (0.8%) 33(6.2%)
4 (0.8%) 30 (5.8%)
0 (0%) 3 (20.0%)
0.73 0.02
20.3. Correlation Of Gene Haplotypes With Complications After Ileal Pouch Anal Anastomosis. R. Sehgal, A. Berg, J. Polinski, J. P. Hegarty, A. A. Kelly, Z. Lin, L. S. Poritz, W. A. Koltun; Penn State Milton S. Hershey Medical Center, Hershey, PA Introduction: Ileal pouch anal anastomosis (IPAA) is the operative gold standard for the treatment of ulcerative colitis (UC). However, complications such as pouchitis and Crohn’s disease (CD) like phenomena confound its success. Recently, genome wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNP’s) that are associated with inflammatory bowel disease (CD, UC or both). The purpose of this study was to identify SNP’s that correlate with complications after IPAA with the goal of identifying genes of relevance that could focus future research investigations into the pathophysiologic basis of these IPAA complications and possibly assist with preoperative surgical decision making. Methods: 142 IPAA patients were retrospectively classified as: 1) Asymptomatic (no pouchitis or CD like complications for at least 2 yrs after IPAA); 2) CD-like complications (presence of fistuli, pouch inlet stricture, proximal small bowel disease or pouch granulomata, occurring at least 6 months after surgery; 3) mild pouchitis (<4 episodes pouchitis/yr for 2 years, each episode responding to a 2 wk course of a single antibiotic); and 4) severe pouchitis (4 episodes pouchitis/yr for 2 years including the need for long term therapy to maintain remission). Genotyping for 71 SNP’s in 49 genes previously associated with IBD was performed on a customized Illumina BeadXpress VeraCode platform. Statistical assessment of the genetic associations, including multiple hypothesis testing, was evaluated with the Fisher exact test. Results: The most significant associated SNP’s, p values and their respective genes/loci are summarized in the table below: Conclusions: Specific allelic SNP determinants in 9 individual genes/loci appear to correlate with specific complications after IPAA. The majority of these genes have been previously associated with CD and play a role in enteric bacterial recognition and destruction (NOD2, ATG16L1, TLR, CARD 9) and/ or NF-kappa B activation (NOD2, TNFSF15). The highest statistical significance was found in the CD-like and severe pouchitis groups. This could be due to the more clearly defined phenotype in these two groups (as opposed to the more nebulous ‘‘mild pouchitis’’), but
212
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
may also reflect a more Crohn’s-like pathophysiology in these originally diagnosed UC patients. The 10q21 locus which has been associated with CD in several large GWAS studies is a gene desert with little understood function but had the highest correlation with CD like complications and may represent a fertile area for further investigation. Whether identifying such genetic determinants preoperatively could play a role in surgical decision making warrants further investigation.
treated with PGE2 for 10 days showed more epithelium and fewer goblet cells (E) than that of vehicle treated (F), indicating that PGE2 may promote TEC growth by keeping the epithelium in a proliferating stage. Conclusions: We established a reliable in vitro culture system and in vivo approach to study the function of key factors involved in the formation and growth of TESI and TEC from OU. Our observation that PGE2 promotes the growth of mouse TESI and TEC has a potential benefit for generating human tissue engineered intestine in an efficient manner.
20.5. Use of a DPP-IV inhibitor leads to accelerated intestinal adaptation in a mouse model of short bowel syndrome. M. Okawada, H. M. Maria, D. H. Teitelbaum; University of Michigan, Ann Arbor, MI
20.4. PGE2 Promotes The Growth Of Tissue Engineered Small And Large Intestine. Y. Li, A. L. Speer, J. A. Matthews, F. G. Sala, E. R. Barthel, Y. Torashima, T. C. Grikscheit; Children’s Hospital Los Angeles, Los Angeles, CA Introduction: Our objective is to determine if PGE2 promotes the formation and growth of Tissue Engineered-Small Intestine (TESI) and –Colon (TEC). PGE2 regulates multiple biological processes under both normal and pathological conditions. In intestine, PGE2 stimulates epithelial cell proliferation and migration and promotes regeneration of gastrointestinal epithelium after injury. TESI, a potential durable autologous therapy for Short Bowel Syndrome, has been generated in our lab. Methods: Organoid units (OU) from ActinGFP transgenic mice were either cultured in a matrigel system with or without the presence of 100ng/ml PGE2 for 6 days, or implanted into the omentum of NOD/SCID gamma chain deficient mice. The implanted mice were then treated with vehicle or 10 ug PGE2/kg of body weight twice a day until harvest. All TESI and TEC were harvested and analyzed with gross morphology and histology after 10 days or 14 days. Cyclooxygenase 2 protein (COX2), a rate-limiting enzyme involved in PGE2 synthesis, was stained by immunohistochemistry. Results: COX2expressing cells were located only in the regenerating area of TESI (A) and TEC (B); in the in vitro culture system, PGE2 promotes growth of both small intestine OU and colon OU when measuring size (data not shown). In the in vivo model, exogenous PGE2 stimulates epithelial growth of TESI when measuring the epithelial thickness (C and D). TEC
Introduction: Glucagon-like peptide 2 (GLP-2) acts as one of the strongest mediators of small intestinal mucosal epithelial adaptation by stimulation of crypt cell proliferation and inhibition of enterocyte apoptosis. The use of GLP-2 may have a use in accelerating the mucosal adaptive response in short bowel syndrome (SBS). In fact, mammalian organisms up-regulate the production of GLP-2 during this adaptive phase. However, incretin hormones are rapidly inactivated (within 7 minutes) by the enzyme dipeptidyl peptidase-IV (DPP4). Currently, inhibitors of DPP-4 (DPP4-I) are used to inhibit a related incretin, GLP-1, and used for the treatment of diabetes. Based on this, we hypothesized that by selectively inhibiting DPP4, endogenous GLP-2 would be preserved, thereby prolonging the circulating life of incretin hormones, and lead to improved intestinal adaptation in SBS. Methods: 8-week old C57BL/6J mice underwent a 50% proximal small bowel resection after being placed on a liquid diet for the preceding 2 days. Sitagliptin, (DPP4-I), was given orally every eight hours (50mg/kg/dose, n¼6) starting 1 day before surgery. Six mice were given oral phosphate-buffered saline orally as a control (SBS+placebo). Body weight was followed daily. Blood samples were taken daily to determine serum glucose levels. At 3 days, the adaptive response was assessed in the remnant gut by measuring villus height, crypt depth, apoptosis and proliferation (BrdU incorporation). mRNA abundance of jejunal and ileal GLP-2 receptor (GLP-2R) and sodium glucose co-transporter 1(SGLT1) were examined using real time polymerase chain reaction (PCR). Results: Treatment with DPP4-I led to a significant increase in villus height and crypt depth vs. SBS+placebo mice (Table). DPP4-I treatment did not significantly change apoptosis rates, but did significantly increase the rate of epithelial cell proliferation in the crypts compared to SBS+placebo controls(Figure). GLP-2R and SGLT-1 mRNA expression in jejunum and ileum were not significantly changed. Percent body weight loss and the level of peripheral blood glucose did not significantly change between SBS+DPP4-I and SBS +placebo groups, demonstrating the relative safety of DPP4-I in a setting of SBS. Conclusions: This study showed a very novel treatment approach for patients with SBS. Use of a DPP4-I led to an accelerated intestinal adaptive process. This unique approach for patients with SBS has the advantage of capitalizing on a patient’s endogenous GLP-2 expression, with a previously approved FDA drug, and may have clinical applications.