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The RON receptor tyrosine kinase is alternatively spliced in pancreatic ductal adenocarcinoma to create active protein isoforms
Vol. 217, No. 3S, September 2013
mice, pancreatic inflammation was induced by 17 days of cerulein injections with subsequent collection of the pancreas. Pancreatic tissue was then sectioned and stained with chloracetate esterase (CAE) and toluidine blue for mast cells, mouse Ly-6G (Gr-1) antibody for granulocytes, mouse F4/80 antibody for macrophages, and Foxp3 antibody for T-regulatory cells. RESULTS: Snail over-expression in KrasG12D mice significantly increased mast cell and granulocyte infiltration to the pancreatic tumor microenvironment. Snail over-expression in KrasG12D mice did not increase T-regulatory cell or macrophage infiltration to the pancreatic tumor microenvironment. Cerulein-induced pancreatitis in KrasG12D mice increased mast cell infiltration, and Snail over-expression appears to potentiate the effect of cerulein in these mice. CONCLUSIONS: Snail over-expression in KrasG12D mice amplifies the inflammatory response in the pancreatic tumor microenvironment by increasing the infiltration of mast cells and granulocytes. Ongoing experiments are underway to characterize the mechanism by which Snail contributes to increased inflammation. Bmi1 is required for murine pancreatic cancer initiation Filip Bednar, MD, PhD, Meredith A Collins, BS, Wei Yan, MD, PhD, Yaqing Zhang, PhD, Daisuke Nakada, PhD, Diane M Simeone, MD, FACS, Sean J Morrison, PhD, Marina Pasca di Magliano, PhD University of Michigan, Ann Arbor, MI INTRODUCTION: Bmi1, a component of the Polycomb Repressor Complex 1, regulates adult stem cell maintenance and behaves as an oncogene in multiple malignancies. We used genetically engineered mouse models to study its role in the initiation of pancreatic cancer. METHODS: We combined Pdx1-Cre, KrasLSL-G12D/+ (KC) mice and Pdx1-Cre, KrasLSL-G12D/+, Ink4a-/- mice with Bmi1 loxP/loxP mice to generate mouse models of pancreatic cancer with a conditional knockout of Bmi1 (KBC mice). We also bred Bmi1IRES-CreER/+, Rosa26lacZ and Bmi1IRES-CreER/+, KrasLSL-G12D/+ (BK) transgenic mice to study the role of Bmi1+ cells in neoplastic initiation. We performed comprehensive histologic analysis and molecular profiling of the mouse pancreata at 3, 12, and 20 weeks after birth. RESULTS: Bmi1+ cells in the pancreas were observed mostly as individual acinar cells and single cells in the pancreatic islets. Early pancreatic intraepithelial neoplasias (PanINs) were observed in the pancreata of BK and KC mice after the induction of pancreatitis with intraperitoneal injections of caerulein. When Bmi1 was knocked out in KBC mice, no PanIN lesions or early pancreatic adenocarcinomas were observed. Genetic deletion of p16Ink4a did not rescue the inhibition of cancer formation by Bmi1 deletion. CONCLUSIONS: Bmi1 is required for murine pancreatic cancer initiation and involves a p16Ink4a-independent mechanism. Pancreatic Bmi1+ cells have the capacity to serve as the cells of
Surgical Forum Abstracts
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origin for murine pancreatic adenocarcinoma. The results suggest a critical role for epigenetic modification by Bmi1 and the Polycomb Repressor Complexes in the biology of pancreatic ductal adenocarcinoma. Genetic and Epigenetic Changes in CarcinomaAssociated Fibroblasts Derived From Human Colorectal Cancers Amy Mrazek, MD, Laura Porro, MD, Joseph R Carmical, MD, Guillermo Gomez, MD, Aakash Gajjar, MD, FACS, Mark R Hellmich, PhD, Celia Chao, MD, FACS University of Texas Medical Branch, Galveston, TX INTRODUCTION: Cells in the stromal microenvironment facilitate colorectal cancer (CRC) progression and "co-evolve" with the cancer cells. In contrast to the epithelial cancer cells, the adjacent stromal cells are characterized generally by greater overall genomic stability. DNA methylation is an epigenetic mechanism characterized by methyl modifications, regulating heritable gene expression. The contribution of DNA methylation from the surrounding stromal tissue, such as carcinoma-associated fibroblasts (CAF) is not well known. The aim of this study is to correlate changes in methylation with changes in gene expression between normal colon mucosa fibroblasts (NF) and CAF from CRC. METHODS: DNA and RNA were extracted from cultured NF and CAF from CRC resections. Genome-wide methylation analysis from bisulfite-converted DNA was analyzed utilizing the Illumina Human Methylation27 (Illumina; San Diego). Gene expression values between NF and CAF were compared and correlated with methylation patterns. Unsupervised cluster analysis was used to visualize and characterize broad methylation and gene expression patterns in the data. All tests were 2-tailed and P<0.05 was considered significant. RESULTS: Paired methylation and gene expression analyses on 12 NF and 11 CAF colon samples were identified. One-hundred different genes: 46 were hypermethylated and 54 were hypomethylated in CAFs. An example of a gene upregulated in CAF is chemokine CXCL13 and downregulated is tumor suppressor TP73. CONCLUSIONS: Previous studies in such epigentic changes have focused on the cancer cells. We have identified genes not previously associated with CRC-specific hypo- or hyper-methylation that are CAF-specific. Elucidating the methylation status of CAF may help to identify potential biomarkers for risk and prognosis to therapy. The RON receptor tyrosine kinase is alternatively spliced in pancreatic ductal adenocarcinoma to create active protein isoforms Jeffery Chakedis, BS, MD, Randall French, BS, PhD, Michele Babicky, MD, Megan Harper, BS, Haleigh Howard, BS, Dawn Jaquish, BS, Evangeline Mose, BS, Andrew M Lowy, MD, FACS University of California-San Diego, San Diego, CA
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Surgical Forum Abstracts
J Am Coll Surg
INTRODUCTION: The RON tyrosine kinase receptor is overexpressed in the majority of pancreatic ductal adenocarcinomas (PDAC) which promotes pancreatic cancer cell migration, invasion and apoptotic resistance. Previous work has found the RON gene is alternatively spliced to produce protein isoforms which can be both oncogenic and tumor suppressing. Here we report the novel identification of isoforms in PDAC. METHODS: Patient-derived PDAC xenografts (PDXS) were established in the pancreata of NSG mice and low passage tumors were analyzed by Reverse Transcription PCR, western blot, and mass spectrometry. Primers specific for exons 3-8 and 10-18 were created to assess alternative splicing. DNA sequencing was used to analyze the transcripts. RESULTS: 19 PDX’s and 7 PDAC cell lines were analyzed and alternative transcription of the RON gene was identified as noted in the Table. Deletions of exons 5, 6, and 11 were common in all tumors that over expressed wild type RON, and the exon 11 deletion isoform was as common as the wild-type transcript. Two novel isoforms were discovered containing intron 13 and partial deletions of exon 5 and 6. Protein analysis by western blot and mass spectrometry confirmed protein expression and activation. Overexpression of a truncated form of RON (short-form RON) in the HPDE6/E7 cell line caused oncogenic transformation which led to increased invasion.
RON Isoform
Wild type RON Exon 5+6 deletion (ROND160, ROND155) Exon 6 deletion (ROND90) Exon 11 deletion (ROND165, ROND155) Partial exon 10, Partial exon 11 deletion (ROND100) short-form RON (ROND55) c-terminal deletion (ROND170) Intron 13 inclusion
Number of PDX which express isoform, n (%)
Number of cell lines which express isoform, n (%)
18/19 (95)
7/7 (100)
13/19 (68)
6/7 (86)
17/19 (89)
6/7 (86)
17/19 (89)
7/7 (100)
12/19 (63)
6/7 (86)
15/19 (79)
6/7 (86)
17/19 (89) 16/19 (84)
6/7 (86) 5/7 (71)
CONCLUSIONS: In human PDAC the RON gene is alternatively transcribed and spliced to produce variants which are active and can impart a more invasive phenotype. Overexpression of these isoforms has biologic implications and could impact response to therapeutics.
Identification of a regulatory T cell subset that correlates with in vivo and in vitro immune responses in breast cancer patients receiving a CD4-eliciting, HER2 peptide vaccine (AE37) Alfred Francois Trappey, MD, John S Berry, MD, Mohamed Mursal, BS, Yusuf Jama, MS, Athina Zacharia, MS, Rebecca Pham, BS, Eric Margulies, BA, Sathibalan Ponniah, PhD, Elizabeth A Mittendorf, MD, FACS, George E Peoples, MD, FACS San Antonio Military Medical Center, San Antonio, TX INTRODUCTION: CD4+ regulatory T-cells (Treg) prevent autoimmunity, and may be a potential obstacle to immunotherapy. Identifying Treg population/s that correlate with in vivo and in vitro immune responses would be useful for monitoring cancer immunotherapy. We analyzed several Treg phenotypes and compared their levels to in vivo and in vitro assays in a Phase II trial of breast cancer (BCa) patients (pts) receiving a HER2 peptide vaccine (AE37). METHODS: Disease-free, high-risk BCa pts enrolled after completion of standard therapy were randomized to 6 monthly inoculations (R0-R6) of AE37+GM-CSF (PA) or GM-CSF (AA). Delayed-type hypersensitivity (DTH) and blood draws were performed at R0 and R6. Tregs were detected by staining with CD4, CD25 and CD127 antibodies and flow cytometry. In vitro assay was proliferation (prolif) (3H-thymidine incorporation). RESULTS: CD4+ cells with all combinations of CD25 and CD127 expression were analyzed for reduction from R0-R6 in 73 pts (44AA, 29PA). Comparing PAvAA, the only significant difference was in CD4+CD25hi++CD127 low population (AAeR0 1.70.2%vR6 1.60.2%, p¼0.5; PAeR0 1.70.1% vR6 1.40.1%, p¼0.05; R6 PAvR6 AA, p¼0.04). The decrease in this Treg population from R0-R6 also correlated with increased AE37-specific DTH responses (2.9v37.2mm, p<0.01) and CD4 cell prolif (0v3915Dcpm, p<0.01). PA pts without disease recurrence (n¼27) showed a decrease in this population R0-R6 (1.70.2%v1.30.1%, p¼0.05) while those with recurrence (n¼2) did not (2.30.6%v1.91.1%, p¼0.4). CONCLUSIONS: Immune response to the AE37 vaccine monitored by the Treg phenotype CD4+CD25hi++CD127low correlates well with in vivo and in vitro tests. Decrease in this population post-vaccination demonstrates stronger vaccine responses and may confer survival benefit. Influence of thyroid shape determined by Fourier analysis on intra- and postoperative complications in patients operated on for Graves disease Franck Georges Billmann, MD, PhD, FACS, Therezia Bokor-Billmann, MD, Joachim Voigt, MD, Erhard Kiffner, MD Universita¨tsklinikum Freiburg, Freiburg, Germany INTRODUCTION: Total thyroidectomy appears superior to less radical resections in the treatment of Graves disease (GD). The disadvantages of this surgery include intra- and postoperative complications
mice, pancreatic inflammation was induced by 17 days of cerulein injections with subsequent collection of the pancreas. Pancreatic tissue was then sectioned and stained with chloracetate esterase (CAE) and toluidine blue for mast cells, mouse Ly-6G (Gr-1) antibody for granulocytes, mouse F4/80 antibody for macrophages, and Foxp3 antibody for T-regulatory cells. RESULTS: Snail over-expression in KrasG12D mice significantly increased mast cell and granulocyte infiltration to the pancreatic tumor microenvironment. Snail over-expression in KrasG12D mice did not increase T-regulatory cell or macrophage infiltration to the pancreatic tumor microenvironment. Cerulein-induced pancreatitis in KrasG12D mice increased mast cell infiltration, and Snail over-expression appears to potentiate the effect of cerulein in these mice. CONCLUSIONS: Snail over-expression in KrasG12D mice amplifies the inflammatory response in the pancreatic tumor microenvironment by increasing the infiltration of mast cells and granulocytes. Ongoing experiments are underway to characterize the mechanism by which Snail contributes to increased inflammation. Bmi1 is required for murine pancreatic cancer initiation Filip Bednar, MD, PhD, Meredith A Collins, BS, Wei Yan, MD, PhD, Yaqing Zhang, PhD, Daisuke Nakada, PhD, Diane M Simeone, MD, FACS, Sean J Morrison, PhD, Marina Pasca di Magliano, PhD University of Michigan, Ann Arbor, MI INTRODUCTION: Bmi1, a component of the Polycomb Repressor Complex 1, regulates adult stem cell maintenance and behaves as an oncogene in multiple malignancies. We used genetically engineered mouse models to study its role in the initiation of pancreatic cancer. METHODS: We combined Pdx1-Cre, KrasLSL-G12D/+ (KC) mice and Pdx1-Cre, KrasLSL-G12D/+, Ink4a-/- mice with Bmi1 loxP/loxP mice to generate mouse models of pancreatic cancer with a conditional knockout of Bmi1 (KBC mice). We also bred Bmi1IRES-CreER/+, Rosa26lacZ and Bmi1IRES-CreER/+, KrasLSL-G12D/+ (BK) transgenic mice to study the role of Bmi1+ cells in neoplastic initiation. We performed comprehensive histologic analysis and molecular profiling of the mouse pancreata at 3, 12, and 20 weeks after birth. RESULTS: Bmi1+ cells in the pancreas were observed mostly as individual acinar cells and single cells in the pancreatic islets. Early pancreatic intraepithelial neoplasias (PanINs) were observed in the pancreata of BK and KC mice after the induction of pancreatitis with intraperitoneal injections of caerulein. When Bmi1 was knocked out in KBC mice, no PanIN lesions or early pancreatic adenocarcinomas were observed. Genetic deletion of p16Ink4a did not rescue the inhibition of cancer formation by Bmi1 deletion. CONCLUSIONS: Bmi1 is required for murine pancreatic cancer initiation and involves a p16Ink4a-independent mechanism. Pancreatic Bmi1+ cells have the capacity to serve as the cells of
Surgical Forum Abstracts
S133
origin for murine pancreatic adenocarcinoma. The results suggest a critical role for epigenetic modification by Bmi1 and the Polycomb Repressor Complexes in the biology of pancreatic ductal adenocarcinoma. Genetic and Epigenetic Changes in CarcinomaAssociated Fibroblasts Derived From Human Colorectal Cancers Amy Mrazek, MD, Laura Porro, MD, Joseph R Carmical, MD, Guillermo Gomez, MD, Aakash Gajjar, MD, FACS, Mark R Hellmich, PhD, Celia Chao, MD, FACS University of Texas Medical Branch, Galveston, TX INTRODUCTION: Cells in the stromal microenvironment facilitate colorectal cancer (CRC) progression and "co-evolve" with the cancer cells. In contrast to the epithelial cancer cells, the adjacent stromal cells are characterized generally by greater overall genomic stability. DNA methylation is an epigenetic mechanism characterized by methyl modifications, regulating heritable gene expression. The contribution of DNA methylation from the surrounding stromal tissue, such as carcinoma-associated fibroblasts (CAF) is not well known. The aim of this study is to correlate changes in methylation with changes in gene expression between normal colon mucosa fibroblasts (NF) and CAF from CRC. METHODS: DNA and RNA were extracted from cultured NF and CAF from CRC resections. Genome-wide methylation analysis from bisulfite-converted DNA was analyzed utilizing the Illumina Human Methylation27 (Illumina; San Diego). Gene expression values between NF and CAF were compared and correlated with methylation patterns. Unsupervised cluster analysis was used to visualize and characterize broad methylation and gene expression patterns in the data. All tests were 2-tailed and P<0.05 was considered significant. RESULTS: Paired methylation and gene expression analyses on 12 NF and 11 CAF colon samples were identified. One-hundred different genes: 46 were hypermethylated and 54 were hypomethylated in CAFs. An example of a gene upregulated in CAF is chemokine CXCL13 and downregulated is tumor suppressor TP73. CONCLUSIONS: Previous studies in such epigentic changes have focused on the cancer cells. We have identified genes not previously associated with CRC-specific hypo- or hyper-methylation that are CAF-specific. Elucidating the methylation status of CAF may help to identify potential biomarkers for risk and prognosis to therapy. The RON receptor tyrosine kinase is alternatively spliced in pancreatic ductal adenocarcinoma to create active protein isoforms Jeffery Chakedis, BS, MD, Randall French, BS, PhD, Michele Babicky, MD, Megan Harper, BS, Haleigh Howard, BS, Dawn Jaquish, BS, Evangeline Mose, BS, Andrew M Lowy, MD, FACS University of California-San Diego, San Diego, CA
S134
Surgical Forum Abstracts
J Am Coll Surg
INTRODUCTION: The RON tyrosine kinase receptor is overexpressed in the majority of pancreatic ductal adenocarcinomas (PDAC) which promotes pancreatic cancer cell migration, invasion and apoptotic resistance. Previous work has found the RON gene is alternatively spliced to produce protein isoforms which can be both oncogenic and tumor suppressing. Here we report the novel identification of isoforms in PDAC. METHODS: Patient-derived PDAC xenografts (PDXS) were established in the pancreata of NSG mice and low passage tumors were analyzed by Reverse Transcription PCR, western blot, and mass spectrometry. Primers specific for exons 3-8 and 10-18 were created to assess alternative splicing. DNA sequencing was used to analyze the transcripts. RESULTS: 19 PDX’s and 7 PDAC cell lines were analyzed and alternative transcription of the RON gene was identified as noted in the Table. Deletions of exons 5, 6, and 11 were common in all tumors that over expressed wild type RON, and the exon 11 deletion isoform was as common as the wild-type transcript. Two novel isoforms were discovered containing intron 13 and partial deletions of exon 5 and 6. Protein analysis by western blot and mass spectrometry confirmed protein expression and activation. Overexpression of a truncated form of RON (short-form RON) in the HPDE6/E7 cell line caused oncogenic transformation which led to increased invasion.
RON Isoform
Wild type RON Exon 5+6 deletion (ROND160, ROND155) Exon 6 deletion (ROND90) Exon 11 deletion (ROND165, ROND155) Partial exon 10, Partial exon 11 deletion (ROND100) short-form RON (ROND55) c-terminal deletion (ROND170) Intron 13 inclusion
Number of PDX which express isoform, n (%)
Number of cell lines which express isoform, n (%)
18/19 (95)
7/7 (100)
13/19 (68)
6/7 (86)
17/19 (89)
6/7 (86)
17/19 (89)
7/7 (100)
12/19 (63)
6/7 (86)
15/19 (79)
6/7 (86)
17/19 (89) 16/19 (84)
6/7 (86) 5/7 (71)
CONCLUSIONS: In human PDAC the RON gene is alternatively transcribed and spliced to produce variants which are active and can impart a more invasive phenotype. Overexpression of these isoforms has biologic implications and could impact response to therapeutics.
Identification of a regulatory T cell subset that correlates with in vivo and in vitro immune responses in breast cancer patients receiving a CD4-eliciting, HER2 peptide vaccine (AE37) Alfred Francois Trappey, MD, John S Berry, MD, Mohamed Mursal, BS, Yusuf Jama, MS, Athina Zacharia, MS, Rebecca Pham, BS, Eric Margulies, BA, Sathibalan Ponniah, PhD, Elizabeth A Mittendorf, MD, FACS, George E Peoples, MD, FACS San Antonio Military Medical Center, San Antonio, TX INTRODUCTION: CD4+ regulatory T-cells (Treg) prevent autoimmunity, and may be a potential obstacle to immunotherapy. Identifying Treg population/s that correlate with in vivo and in vitro immune responses would be useful for monitoring cancer immunotherapy. We analyzed several Treg phenotypes and compared their levels to in vivo and in vitro assays in a Phase II trial of breast cancer (BCa) patients (pts) receiving a HER2 peptide vaccine (AE37). METHODS: Disease-free, high-risk BCa pts enrolled after completion of standard therapy were randomized to 6 monthly inoculations (R0-R6) of AE37+GM-CSF (PA) or GM-CSF (AA). Delayed-type hypersensitivity (DTH) and blood draws were performed at R0 and R6. Tregs were detected by staining with CD4, CD25 and CD127 antibodies and flow cytometry. In vitro assay was proliferation (prolif) (3H-thymidine incorporation). RESULTS: CD4+ cells with all combinations of CD25 and CD127 expression were analyzed for reduction from R0-R6 in 73 pts (44AA, 29PA). Comparing PAvAA, the only significant difference was in CD4+CD25hi++CD127 low population (AAeR0 1.70.2%vR6 1.60.2%, p¼0.5; PAeR0 1.70.1% vR6 1.40.1%, p¼0.05; R6 PAvR6 AA, p¼0.04). The decrease in this Treg population from R0-R6 also correlated with increased AE37-specific DTH responses (2.9v37.2mm, p<0.01) and CD4 cell prolif (0v3915Dcpm, p<0.01). PA pts without disease recurrence (n¼27) showed a decrease in this population R0-R6 (1.70.2%v1.30.1%, p¼0.05) while those with recurrence (n¼2) did not (2.30.6%v1.91.1%, p¼0.4). CONCLUSIONS: Immune response to the AE37 vaccine monitored by the Treg phenotype CD4+CD25hi++CD127low correlates well with in vivo and in vitro tests. Decrease in this population post-vaccination demonstrates stronger vaccine responses and may confer survival benefit. Influence of thyroid shape determined by Fourier analysis on intra- and postoperative complications in patients operated on for Graves disease Franck Georges Billmann, MD, PhD, FACS, Therezia Bokor-Billmann, MD, Joachim Voigt, MD, Erhard Kiffner, MD Universita¨tsklinikum Freiburg, Freiburg, Germany INTRODUCTION: Total thyroidectomy appears superior to less radical resections in the treatment of Graves disease (GD). The disadvantages of this surgery include intra- and postoperative complications