- Email: [email protected]
Anti-CTLA4 treatment reduces systemic inflammatory cytokines in a murine model of intestinal polyposis
S132
Surgical Forum Abstracts
independently associated with reduced survival on multivariate analysis. CONCLUSIONS: Incomplete initial resection and development of resistance are independent predictors of survival in patients with advanced or recurrent GIST. Selection of patients for resection versus continuing TKI therapy in the setting of recurrence requires a multidisciplinary approach. Reoperation should be reserved for TKI response and for those which complete resection of the recurrence is possible.
Reduced BMP signaling due to BMPR1A germline mutations in juvenile polyposis Fadi S Dahdaleh MD, Jennifer C Carr MD, Donghong Wang BSc, Daniel Calva-Cerqueira MD, James R Howe MD, FACS University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Juvenile polyposis (JP) is characterized by the development of hamartomatous polyps that collectively carry a high risk of malignant transformation. Germline mutations in the gene for bone morphogenetic protein receptor 1A (BMPR1A) and the tumor suppressor SMAD4 predispose to JP. We set out to evaluate the effect of JP BMPR1A mutations on downstream signaling through the bone morphogenetic protein (BMP) pathway. METHODS: Mutations found in JP patients were individually recreated in a wild-type (WT) BMPR1A expression vector using a PCR-based site directed mutagenesis (SDM) approach. SDM vectors were then cotransfected with a BMP responsive element luciferase vector (BRE-Luc) and a Renilla internal control into HEK293T cells. Light units reflecting downstream signaling strength were then quantified after 48 hours. Student’s t-test was used for statistical analysis. RESULTS: Twenty-two SDM JP mutants were created (14 nonsense and 8 missense). After adjusting for transfection efficiency, BMP signaling was found to be altered for 19 mutations, 17 of which were lower than WT. Two missense and 1 nonsense mutation did not lead to a significant change in activity. Compared to WT, the mean decrease in luciferase for nonsense mutations was 73.1% (p⬍0.001) and was 23.7% (p⫽0.223) for missense mutations. CONCLUSIONS: To date, JP BMPR1A mutations have not been systematically studied in an in vitro model or been shown to affect downstream signaling. This study confirms that the BMPR1A mutations found in JP patients lead to loss of BMP signaling, which is the likely mechanism leading to this phenotype.
Anti-CTLA4 treatment reduces systemic inflammatory cytokines in a murine model of intestinal polyposis Joseph D Phillips MD, Nichole R Blatner PhD, Mary F Mulcahy MD, Philipp Beckhove MD, Khashayarsha Khazaie PhD, ScD, David J Bentrem MD, FACS Northwestern University/Feinberg School of Medicine, Chicago, IL, Northwestern University/Robert H. Lurie Comprehensive Cancer Center, Chicago, IL and German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
J Am Coll Surg
INTRODUCTION: The role of inflammation in the progression of intestinal polyps to cancer is well established. Cytotoxic T-lymphocyte antigen 4 (CTLA4) mediates non-antigen specific interactions w/ antigen presenting cells and prevents or down-regulates the activation of T-cells in response to a stimulus. Treatment of patients with anti-CTLA-4 antibody (aCTLA4) has shown some promise in melanoma, prostate and ovarian tumors. We have recently observed a reduction in polyp burden with aCTLA4 in a mouse model of polyposis. Our hypothesis is that this reduction is mirrored by a decrease in systemic inflammatory cytokines. METHODS: Using a well-established murine model of polyposis, APC-delta-468, animals were randomized to receive intraperitoneal injections of either 100 micrograms monoclonal aCTLA4 or PBS. Animals were sacrificed and polyps were counted by gross inspection at 3-weeks and 6-weeks post injection. Peripheral blood was analyzed by multiplex ELISA. RESULTS: A significant reduction in small intestinal polyp counts were observed in the 3-week and 6-week treatment groups compared to controls. Multiplex ELISA revealed significantly lower levels of TNF-alpha, IL-6, IL-1-beta, and IL-17 in the peripheral blood of mice treated with aCTLA. CONCLUSIONS: The reduction of polyp load observed with aCTLA4 treatment correlates with lower levels of systemic markers of inflammation. Thus, aCTLA4 may be useful for treatment of intestinal pre-neoplasia by dampening the inflammation induced progression from polyp to cancer. Further studies are needed to assess the full immunologic affect of aCTLA4 in the treatment of polyposis and colon cancer.
Inhibition of the pro-survival factor MADD (MAP Kinase Activating Death Domain) in human colorectal cancer Ryan M Carr BSc, Tania Pilli MD, Liangchen Li PhD, Bellur Prabhakar PhD, Ajay V Maker MD University of Illinois-Chicago, Chicago, IL INTRODUCTION: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality. Up to 50% of CRC patients will develop metastatic disease. For these patients, there is a need for new therapeutic strategies. TNF-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in select cancer cells. However, many colon cancers are resistant to TRAIL. The prosurvival factor, MADD (a splice variant of IG20) inhibits TRAILinduced apoptosis and may be expressed in human colon cancer. METHODS: Utilizing HeLa cells as positive controls and the three colorectal cancer cell lines HT-29, COLO205 and SW620, we analyzed cells for expression of IG20 by RT-PCR. A lentiviral construct was created to deliver shRNA to colorectal cancer cell lines and knock down expression of IG20. RESULTS: High expression of IG20 was identified in control HeLa cells and the three human colorectal cancer cell lines. Expression was consistently reduced by ⬎50% after 96 hours of transduction in all cell lines tested. There was no effect on IG20 expression in non-
Surgical Forum Abstracts
independently associated with reduced survival on multivariate analysis. CONCLUSIONS: Incomplete initial resection and development of resistance are independent predictors of survival in patients with advanced or recurrent GIST. Selection of patients for resection versus continuing TKI therapy in the setting of recurrence requires a multidisciplinary approach. Reoperation should be reserved for TKI response and for those which complete resection of the recurrence is possible.
Reduced BMP signaling due to BMPR1A germline mutations in juvenile polyposis Fadi S Dahdaleh MD, Jennifer C Carr MD, Donghong Wang BSc, Daniel Calva-Cerqueira MD, James R Howe MD, FACS University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Juvenile polyposis (JP) is characterized by the development of hamartomatous polyps that collectively carry a high risk of malignant transformation. Germline mutations in the gene for bone morphogenetic protein receptor 1A (BMPR1A) and the tumor suppressor SMAD4 predispose to JP. We set out to evaluate the effect of JP BMPR1A mutations on downstream signaling through the bone morphogenetic protein (BMP) pathway. METHODS: Mutations found in JP patients were individually recreated in a wild-type (WT) BMPR1A expression vector using a PCR-based site directed mutagenesis (SDM) approach. SDM vectors were then cotransfected with a BMP responsive element luciferase vector (BRE-Luc) and a Renilla internal control into HEK293T cells. Light units reflecting downstream signaling strength were then quantified after 48 hours. Student’s t-test was used for statistical analysis. RESULTS: Twenty-two SDM JP mutants were created (14 nonsense and 8 missense). After adjusting for transfection efficiency, BMP signaling was found to be altered for 19 mutations, 17 of which were lower than WT. Two missense and 1 nonsense mutation did not lead to a significant change in activity. Compared to WT, the mean decrease in luciferase for nonsense mutations was 73.1% (p⬍0.001) and was 23.7% (p⫽0.223) for missense mutations. CONCLUSIONS: To date, JP BMPR1A mutations have not been systematically studied in an in vitro model or been shown to affect downstream signaling. This study confirms that the BMPR1A mutations found in JP patients lead to loss of BMP signaling, which is the likely mechanism leading to this phenotype.
Anti-CTLA4 treatment reduces systemic inflammatory cytokines in a murine model of intestinal polyposis Joseph D Phillips MD, Nichole R Blatner PhD, Mary F Mulcahy MD, Philipp Beckhove MD, Khashayarsha Khazaie PhD, ScD, David J Bentrem MD, FACS Northwestern University/Feinberg School of Medicine, Chicago, IL, Northwestern University/Robert H. Lurie Comprehensive Cancer Center, Chicago, IL and German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
J Am Coll Surg
INTRODUCTION: The role of inflammation in the progression of intestinal polyps to cancer is well established. Cytotoxic T-lymphocyte antigen 4 (CTLA4) mediates non-antigen specific interactions w/ antigen presenting cells and prevents or down-regulates the activation of T-cells in response to a stimulus. Treatment of patients with anti-CTLA-4 antibody (aCTLA4) has shown some promise in melanoma, prostate and ovarian tumors. We have recently observed a reduction in polyp burden with aCTLA4 in a mouse model of polyposis. Our hypothesis is that this reduction is mirrored by a decrease in systemic inflammatory cytokines. METHODS: Using a well-established murine model of polyposis, APC-delta-468, animals were randomized to receive intraperitoneal injections of either 100 micrograms monoclonal aCTLA4 or PBS. Animals were sacrificed and polyps were counted by gross inspection at 3-weeks and 6-weeks post injection. Peripheral blood was analyzed by multiplex ELISA. RESULTS: A significant reduction in small intestinal polyp counts were observed in the 3-week and 6-week treatment groups compared to controls. Multiplex ELISA revealed significantly lower levels of TNF-alpha, IL-6, IL-1-beta, and IL-17 in the peripheral blood of mice treated with aCTLA. CONCLUSIONS: The reduction of polyp load observed with aCTLA4 treatment correlates with lower levels of systemic markers of inflammation. Thus, aCTLA4 may be useful for treatment of intestinal pre-neoplasia by dampening the inflammation induced progression from polyp to cancer. Further studies are needed to assess the full immunologic affect of aCTLA4 in the treatment of polyposis and colon cancer.
Inhibition of the pro-survival factor MADD (MAP Kinase Activating Death Domain) in human colorectal cancer Ryan M Carr BSc, Tania Pilli MD, Liangchen Li PhD, Bellur Prabhakar PhD, Ajay V Maker MD University of Illinois-Chicago, Chicago, IL INTRODUCTION: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality. Up to 50% of CRC patients will develop metastatic disease. For these patients, there is a need for new therapeutic strategies. TNF-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in select cancer cells. However, many colon cancers are resistant to TRAIL. The prosurvival factor, MADD (a splice variant of IG20) inhibits TRAILinduced apoptosis and may be expressed in human colon cancer. METHODS: Utilizing HeLa cells as positive controls and the three colorectal cancer cell lines HT-29, COLO205 and SW620, we analyzed cells for expression of IG20 by RT-PCR. A lentiviral construct was created to deliver shRNA to colorectal cancer cell lines and knock down expression of IG20. RESULTS: High expression of IG20 was identified in control HeLa cells and the three human colorectal cancer cell lines. Expression was consistently reduced by ⬎50% after 96 hours of transduction in all cell lines tested. There was no effect on IG20 expression in non-