- Email: [email protected]
541 The Tumor Suppressor MicroRNA-122 Is Suppressed in Liver Cancer Cells and Can Be Reactivated by DNA Methylation Inhibitors
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IPMC on final diagnosis. There were no differences in age, sex, tumor location, tumor distribution, average cystic size, average main duct diameter, the presence of mural nodule, and surgical procedure between the patients with IPMN and those with IPMC. 4-year disease free survival and overall survival rates of patients with IPMN were significantly better than IPMC (P=0.0002 and P=0.0086, respectively, log-rank test). The correlation between the grade of atypism and DNA methylation patterns demonstrated that there were significantly more methylated genes in patients with IPMC than those with IPMN (mean number of methylated genes 1.625±1.345 compared with 0.826±0.887 of genes; P=0.0376, log-rank test). Not only the number of methylated genes, but also the extent of methylation in different promoter regions of various genes was specifically more predominant in IPMC. Conclusion: Our study shows that IPMCs have poorer prognosis than IPMNs, and DNA methylation patterns can act as stage-dependent biomarkers of the tumorigenesis in IPMNs. It is suggested that the DNA methylation profiling provides a possible means to noninvasively screen for IPMC.
AGA Abstracts
Nuclear Translocation of FGFR1 and FGF2 in Pancreatic Stellate Cells Is Necessary for Pancreatic Cancer Cell Invasion Stacey Coleman, Athina-Myrto Chioni, Mohammed Ghallab, Nicholas Lemoine, Richard P. Grose, Hemant Kocher Background: It is now appreciated that, in pancreatic cancer, transformed cells interact with stromal cells, extracellular matrix proteins, and neighboring normal epithelial cells to exploit feedback mechanisms and facilitate tumour progression. Studies have shown that overexpression of FGF2 in pancreatic cancer correlates with poor patient survival and nuclear FGF2 has been observed in pancreatic cancer patient tissue but not in normal patient samples (Yamanka et al 1993). A number of mechanisms have been proposed for the nuclear accumulation of FGFR1 and its ligand; however, the role of intracrine FGF2 signalling in tumour invasion remains poorly understood. We investigated whether nuclear translocation of FGFR1 with FGF2 plays a role in pancreatc stellate cell (PSC) behaviour. Design: FGF2 and FGFR1 RNAi, together with an FGFR inhibitor (PD173074) were used on pancreatic cancer cell lines PSCs and in organotypic cultures to assess their effect on cell behaviour and to assess the effects of blocking FGF signalling on cancer cell behaviour and invasion. Nuclear FGFR1 and FGF2 expression were also studied in vivo. Results: FGF2 and FGFR1 localised to the nucleus in stromal fibroblasts at the invasive front of human pancreatic cancer tissue. In vitro, FGFR1 and FGF2 co-localised to the nucleus in PSCs but not in cancer or normal ductal epithelial cell lines. PSCs but not cancer cells secrete both HMW and LMW FGF2 isoforms. Abolishing nuclear FGFR1 and FGF2 in PSCs using either RNAi or FGFR inhibitor resulted in a significant reduction in cell proliferation which correlated with a G1 cell-cycle block and reduction in the G1 cyclin, cyclin D1. In an organotypic model, nuclear FGFR1 and FGF2 were significantly greater in PSCs invading into the matrix. When treated with FGFR inhibitor, PSCs were unable to invade into the extra-cellular matrix and FGFR1 and FGF2 remained cytoplasmic. Effective blockade of nuclear FGFR1 signalling in PSCs abolished cancer cell invasion. Conclusion: These studies show for the first time that nuclear FGFR1 and FGF2 plays a role in driving pancreatic stellate cell proliferation. Preventing nuclear FGF/FGFR mediated proliferation in PSCs leads to disruption of the tumour microenvironment, thus preventing pancreatic cancer cell invasion.
541 The Tumor Suppressor MicroRNA-122 Is Suppressed in Liver Cancer Cells and Can Be Reactivated by DNA Methylation Inhibitors Misa Matsuura, Yoshimasa Saito, Yoshiyuki Doke, Tomomi Yoshida, Asako Genka, Toshihide Muramatsu, Masaki Kimura, Eri Arai, Yae Kanai, Hidetsugu Saito Introduction DNA methylation and histone modification collaborate to cause silencing of tumor suppressor genes during carcinogenesis. Epigenetic therapy with DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors holds clinical promise for the treatment of human malignancies. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of various target genes and play critical roles in cancer. We have proposed that epigenetic activation of tumor suppressor miRNAs can be a novel therapeutic approach for human cancers (Cancer Cell 9: 435, 2006). To investigate potential targets for epigenetic therapy of liver cancer, we analyzed miRNA expression profiles of liver cancer cells treated with DNA methylation inhibitors. Materials and Methods The human liver cancer cell lines HepG2, PLC/PRF/5, HuH7, and OR6, and tissue specimens from 36 primary liver cancers together with samples of non-tumorous liver tissue from the same patients were analyzed. HepG2 cells were treated with the DNA methylation inhibitors 5-aza-2'deoxycytidine (5-Aza-CdR) and zebularine. MiRNA expression profiles were analyzed using microarrays and TaqMan quantitative RT-PCR. Cell proliferation was analyzed by cell counting. The target genes of miRNAs were analyzed by Western blotting, and the levels of DNA methylation were evaluated by bisulfite pyrosequencing. Results The results of microarray analyses revealed that the liver-specific microRNA-122 (miR-122) was significantly upregulated by treatment of HepG2 cells with 5-Aza-CdR. Expression levels of miR-122 were down-regulated in HepG2 cells, and in 25 of the 36 (69%) clinical samples of primary liver cancers. Treatment of HepG2 cells with the DNA methylation inhibitors 5-Aza-CdR and zebularine reduced cell proliferation activity and reactivated miR-122 expression with promoter demethylation. In addition, Cyclin G1, one of the targets of miR-122, was markedly down-regulated by treatment with the DNA methylation inhibitors. Conclusions These findings indicate that miR-122 acts as a tumor suppressor via inhibition of its target Cyclin G1. MiR-122 is suppressed in liver cancer cells and can be reactivated by DNA methylation inhibitors. DNA methylation inhibitors may have clinical promise for treatment of patients with liver cancers lacking miR-122 expression.
539 Activation-Induced Cytidine Deaminase (AID) Contributes to Pancreatic Cancer Initiation by Induction of Tumor Related Gene Mutations Yugo Sawai, Yuzo Kodama, Yuji Ota, Masahiro Shiokawa, Akira Kurita, Norimitsu Uza, Hiroyuki Marusawa, Tsutomu Chiba Background and Aims: Pancreatic ductal adenocarcinoma (PDA) is considered to develop via an accumulation of various genetic mutations. However, the mechanism underlying the induction of mutations in PDA is not fully understood. We investigated whether activationinduced cytidine deaminase (AID), a member of DNA/RNA editing enzyme, is related to the pancreatic carcinogenesis. Methods: First, we analyzed the AID expression in human PDA tissue samples by RT-PCR and immunohistochemistry. Second, the phenotypes of pancreas in AID transgenic (AID Tg) mice were investigated. Third, mutations of tumor related genes were investigated in the pancreas of AID Tg mice by using next generation sequencing (NGS). Finally, mutations of tumor related genes were separately assessed in acinar cells of wild type (WT), acinar cells of AID Tg, and precancerous lesions of AID Tg mice that were isolated by laser capture microdissection. Results: AID mRNA was expressed in both human pancreatic cancer cell lines and human PDA tissue samples. Immunohistochemistry also revealed strong AID protein expression in cancer cells in almost all the examined PDA tissue samples. The frequencies and intensity of AID protein expression were increased along with the progression of precancerous to cancerous lesion (acinar-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN), and invasive cancer) in human PDA tissue samples. AID Tg mice developed ADM and PanIN in the pancreas at 6 and 11 months of age, respectively. NGS revealed higher mutational frequencies in K-ras, Smad4, Myc, Pten, and Ctnnb1 genes in the pancreas of AID Tg than those in WT mice. Mutations of K-ras, Smad4, and Myc were found exclusively in the precancerous lesions (ADM and PanIN) of the AID Tg mice, whereas none of those mutations were found in acinar cells of AID Tg and WT mice. Conclusions: Our findings indicate that AID could induce multiple mutations in various tumor related genes including K-ras in pancreatic cells and contribute to the formation of pancreatic precancerous lesions.
542 Direct In Vivo Visualization of Esophageal Neuronal Network During Endoscopy Using Needle- Based Confocal Laser Endomicroscopic Probe. Validation Using Neuronal Probes and Correlation With Expression of CGRP, Neuronal NOS & Enolase and NGF Jason B. Samarasena, Kenneth J. Chang, Amrita Ahluwalia, Susumu Shinoura, Kee Don Choi, John G. Lee, Andrzej S. Tarnawski Background/Aims: The esophagus is innervated by the autonomous enteric neural system (AENS) mainly composed of submucosal Meissner's plexus and myenteric Auerbach's plexus. This neural network is composed of neuronal cells, interneurons, and nerve bundles and fibers. In vivo visualization of esophageal neuronal structures during endoscopy was not possible without mucosal resection. Recent introduction of the state-of-the-art needle-based confocal laser induced endomicroscopy probe (nCLE - Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France) has changed this paradigm. EUS guided nCLE now allows for minimally invasive imaging of deeper submucosal and myenteric structures. This study was aimed to perform in-vivo visualization of the submucosal and myenteric neural network in porcine esophagus using EUS guided nCLE with injection of a fluorescent neuronal probe Neurotrace®; and to determine in esophageal AENS the expression level and localization of several neuronal peptides and nerve growth factor (NGF). The expression and roles of NGF in esophageal AENS are not known. Methods: In anesthetized pigs during endoscopy NeuroTrace was injected into the submucosa of mid and distal (LES) esophagus using a 19 gauge needle introduced under EUS guidance. The nCLE imaging was then performed; pigs were euthanized and transmural esophageal specimens from areas injected with NeuroTrace were obtained. Studies: 1) quantitative analysis of nCLE images, 2) histologic evaluation, 3) expression of: a) CGRP- sensory neuronal marker, b) neuronal NOS (nNOS - marker of nitriergic inhibitory neurons, c) neuronal enolase (NES) and d) NGF and its Trk A receptor, using immunostaining and quantification of signal distribution and intensity with a Metamorph imaging system. Results: The nCLE imaging clearly visualized neuronal cells, nerve bundles and fibers providing their distinctive image patterns. Histology demonstrated normal esophageal structures; fluorescence microscopy showed that in vivo injected NeuroTrace was well retained in neuronal cells and nerves. Immunostaining for CGRP, nNOS and NES showed their strong and differential expression and localization in the neuronal network. NGF was strongly expressed in the majority of neurons and nerves, distribution of TrkA was complementary; its signal was 1.5-fold weaker p ,0.001 than NGF). Quantitatively the signal intensity was: CGRP. nNOS.NGF . NES.TrkA. In addition to neural structures, nNOS, NGF and TrkA were expressed in keratinocyte progenitor cells (KPC) and in endothelial cells. Conclusions: 1) nCLE and NeuroTrace provide reliable in vivo imaging of esophageal
540 Accumulation of Epigenetic Alteration Could Predict Malignant Formation in Intraductal Papillary Mucinous Neoplasm (IPMN) Kazuhiro Yoshida, Takeshi Nagasaka, Yuzo Umeda, Naosuke Yokomichi, Yoshiko Mori, Nobuhito Kubota, Tatsuya Morikawa, Yuko Takehara, Kiyoto Takehara, Kunitoshi Shigeyasu, Akihiro Nyuya, Rikiya Shiwaku, Manabu Suno, Naoshi Nishida, Toshiyoshi Fujiwara, Ajay Goel Introduction: Intraductal Papillary Mucinous Neoplasm (IPMN) is a pancreatic cystic neoplasm and arises in main and side branch of pancreatic duct. Accumulation of genetic and epigenetic alterations is believed to accelerate the malignant formation of IPMNs. However, the molecular events underlying the normal-adenoma-carcinoma sequence in the tumorigenesis in IPMN remain unclear. The aim of the present study is to identify DNA methylation patterns in IPMN, and evaluate their utility as potential predictive biomarkers in this malignancy. Patients and Methods: Tissue specimens were obtained from 50 patients with IPMN or pancreatic ductal carcinoma derived from IPMN (IPMC) who underwent surgery at the Okayama University Hospital from 2001 to 2012. We analyzed methylation patterns of 8 gene promoters in the DNA extracted from the formalin-fixed paraffin-embedded tissues using a single-step bisulfite modification and fluorescence polymerase chain reaction, alsocalled High Sensitive Assay for bisulfite modified DNA (Hi-SA). Subsequently, we determined whether the identified methylation patterns could act as stage-dependent biomarkers of IPMN tumorigenesis. Results: In our patient cohort, 24 patients had IPMNs and 26 had
AGA Abstracts
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IPMC on final diagnosis. There were no differences in age, sex, tumor location, tumor distribution, average cystic size, average main duct diameter, the presence of mural nodule, and surgical procedure between the patients with IPMN and those with IPMC. 4-year disease free survival and overall survival rates of patients with IPMN were significantly better than IPMC (P=0.0002 and P=0.0086, respectively, log-rank test). The correlation between the grade of atypism and DNA methylation patterns demonstrated that there were significantly more methylated genes in patients with IPMC than those with IPMN (mean number of methylated genes 1.625±1.345 compared with 0.826±0.887 of genes; P=0.0376, log-rank test). Not only the number of methylated genes, but also the extent of methylation in different promoter regions of various genes was specifically more predominant in IPMC. Conclusion: Our study shows that IPMCs have poorer prognosis than IPMNs, and DNA methylation patterns can act as stage-dependent biomarkers of the tumorigenesis in IPMNs. It is suggested that the DNA methylation profiling provides a possible means to noninvasively screen for IPMC.
AGA Abstracts
Nuclear Translocation of FGFR1 and FGF2 in Pancreatic Stellate Cells Is Necessary for Pancreatic Cancer Cell Invasion Stacey Coleman, Athina-Myrto Chioni, Mohammed Ghallab, Nicholas Lemoine, Richard P. Grose, Hemant Kocher Background: It is now appreciated that, in pancreatic cancer, transformed cells interact with stromal cells, extracellular matrix proteins, and neighboring normal epithelial cells to exploit feedback mechanisms and facilitate tumour progression. Studies have shown that overexpression of FGF2 in pancreatic cancer correlates with poor patient survival and nuclear FGF2 has been observed in pancreatic cancer patient tissue but not in normal patient samples (Yamanka et al 1993). A number of mechanisms have been proposed for the nuclear accumulation of FGFR1 and its ligand; however, the role of intracrine FGF2 signalling in tumour invasion remains poorly understood. We investigated whether nuclear translocation of FGFR1 with FGF2 plays a role in pancreatc stellate cell (PSC) behaviour. Design: FGF2 and FGFR1 RNAi, together with an FGFR inhibitor (PD173074) were used on pancreatic cancer cell lines PSCs and in organotypic cultures to assess their effect on cell behaviour and to assess the effects of blocking FGF signalling on cancer cell behaviour and invasion. Nuclear FGFR1 and FGF2 expression were also studied in vivo. Results: FGF2 and FGFR1 localised to the nucleus in stromal fibroblasts at the invasive front of human pancreatic cancer tissue. In vitro, FGFR1 and FGF2 co-localised to the nucleus in PSCs but not in cancer or normal ductal epithelial cell lines. PSCs but not cancer cells secrete both HMW and LMW FGF2 isoforms. Abolishing nuclear FGFR1 and FGF2 in PSCs using either RNAi or FGFR inhibitor resulted in a significant reduction in cell proliferation which correlated with a G1 cell-cycle block and reduction in the G1 cyclin, cyclin D1. In an organotypic model, nuclear FGFR1 and FGF2 were significantly greater in PSCs invading into the matrix. When treated with FGFR inhibitor, PSCs were unable to invade into the extra-cellular matrix and FGFR1 and FGF2 remained cytoplasmic. Effective blockade of nuclear FGFR1 signalling in PSCs abolished cancer cell invasion. Conclusion: These studies show for the first time that nuclear FGFR1 and FGF2 plays a role in driving pancreatic stellate cell proliferation. Preventing nuclear FGF/FGFR mediated proliferation in PSCs leads to disruption of the tumour microenvironment, thus preventing pancreatic cancer cell invasion.
541 The Tumor Suppressor MicroRNA-122 Is Suppressed in Liver Cancer Cells and Can Be Reactivated by DNA Methylation Inhibitors Misa Matsuura, Yoshimasa Saito, Yoshiyuki Doke, Tomomi Yoshida, Asako Genka, Toshihide Muramatsu, Masaki Kimura, Eri Arai, Yae Kanai, Hidetsugu Saito Introduction DNA methylation and histone modification collaborate to cause silencing of tumor suppressor genes during carcinogenesis. Epigenetic therapy with DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors holds clinical promise for the treatment of human malignancies. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of various target genes and play critical roles in cancer. We have proposed that epigenetic activation of tumor suppressor miRNAs can be a novel therapeutic approach for human cancers (Cancer Cell 9: 435, 2006). To investigate potential targets for epigenetic therapy of liver cancer, we analyzed miRNA expression profiles of liver cancer cells treated with DNA methylation inhibitors. Materials and Methods The human liver cancer cell lines HepG2, PLC/PRF/5, HuH7, and OR6, and tissue specimens from 36 primary liver cancers together with samples of non-tumorous liver tissue from the same patients were analyzed. HepG2 cells were treated with the DNA methylation inhibitors 5-aza-2'deoxycytidine (5-Aza-CdR) and zebularine. MiRNA expression profiles were analyzed using microarrays and TaqMan quantitative RT-PCR. Cell proliferation was analyzed by cell counting. The target genes of miRNAs were analyzed by Western blotting, and the levels of DNA methylation were evaluated by bisulfite pyrosequencing. Results The results of microarray analyses revealed that the liver-specific microRNA-122 (miR-122) was significantly upregulated by treatment of HepG2 cells with 5-Aza-CdR. Expression levels of miR-122 were down-regulated in HepG2 cells, and in 25 of the 36 (69%) clinical samples of primary liver cancers. Treatment of HepG2 cells with the DNA methylation inhibitors 5-Aza-CdR and zebularine reduced cell proliferation activity and reactivated miR-122 expression with promoter demethylation. In addition, Cyclin G1, one of the targets of miR-122, was markedly down-regulated by treatment with the DNA methylation inhibitors. Conclusions These findings indicate that miR-122 acts as a tumor suppressor via inhibition of its target Cyclin G1. MiR-122 is suppressed in liver cancer cells and can be reactivated by DNA methylation inhibitors. DNA methylation inhibitors may have clinical promise for treatment of patients with liver cancers lacking miR-122 expression.
539 Activation-Induced Cytidine Deaminase (AID) Contributes to Pancreatic Cancer Initiation by Induction of Tumor Related Gene Mutations Yugo Sawai, Yuzo Kodama, Yuji Ota, Masahiro Shiokawa, Akira Kurita, Norimitsu Uza, Hiroyuki Marusawa, Tsutomu Chiba Background and Aims: Pancreatic ductal adenocarcinoma (PDA) is considered to develop via an accumulation of various genetic mutations. However, the mechanism underlying the induction of mutations in PDA is not fully understood. We investigated whether activationinduced cytidine deaminase (AID), a member of DNA/RNA editing enzyme, is related to the pancreatic carcinogenesis. Methods: First, we analyzed the AID expression in human PDA tissue samples by RT-PCR and immunohistochemistry. Second, the phenotypes of pancreas in AID transgenic (AID Tg) mice were investigated. Third, mutations of tumor related genes were investigated in the pancreas of AID Tg mice by using next generation sequencing (NGS). Finally, mutations of tumor related genes were separately assessed in acinar cells of wild type (WT), acinar cells of AID Tg, and precancerous lesions of AID Tg mice that were isolated by laser capture microdissection. Results: AID mRNA was expressed in both human pancreatic cancer cell lines and human PDA tissue samples. Immunohistochemistry also revealed strong AID protein expression in cancer cells in almost all the examined PDA tissue samples. The frequencies and intensity of AID protein expression were increased along with the progression of precancerous to cancerous lesion (acinar-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN), and invasive cancer) in human PDA tissue samples. AID Tg mice developed ADM and PanIN in the pancreas at 6 and 11 months of age, respectively. NGS revealed higher mutational frequencies in K-ras, Smad4, Myc, Pten, and Ctnnb1 genes in the pancreas of AID Tg than those in WT mice. Mutations of K-ras, Smad4, and Myc were found exclusively in the precancerous lesions (ADM and PanIN) of the AID Tg mice, whereas none of those mutations were found in acinar cells of AID Tg and WT mice. Conclusions: Our findings indicate that AID could induce multiple mutations in various tumor related genes including K-ras in pancreatic cells and contribute to the formation of pancreatic precancerous lesions.
542 Direct In Vivo Visualization of Esophageal Neuronal Network During Endoscopy Using Needle- Based Confocal Laser Endomicroscopic Probe. Validation Using Neuronal Probes and Correlation With Expression of CGRP, Neuronal NOS & Enolase and NGF Jason B. Samarasena, Kenneth J. Chang, Amrita Ahluwalia, Susumu Shinoura, Kee Don Choi, John G. Lee, Andrzej S. Tarnawski Background/Aims: The esophagus is innervated by the autonomous enteric neural system (AENS) mainly composed of submucosal Meissner's plexus and myenteric Auerbach's plexus. This neural network is composed of neuronal cells, interneurons, and nerve bundles and fibers. In vivo visualization of esophageal neuronal structures during endoscopy was not possible without mucosal resection. Recent introduction of the state-of-the-art needle-based confocal laser induced endomicroscopy probe (nCLE - Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France) has changed this paradigm. EUS guided nCLE now allows for minimally invasive imaging of deeper submucosal and myenteric structures. This study was aimed to perform in-vivo visualization of the submucosal and myenteric neural network in porcine esophagus using EUS guided nCLE with injection of a fluorescent neuronal probe Neurotrace®; and to determine in esophageal AENS the expression level and localization of several neuronal peptides and nerve growth factor (NGF). The expression and roles of NGF in esophageal AENS are not known. Methods: In anesthetized pigs during endoscopy NeuroTrace was injected into the submucosa of mid and distal (LES) esophagus using a 19 gauge needle introduced under EUS guidance. The nCLE imaging was then performed; pigs were euthanized and transmural esophageal specimens from areas injected with NeuroTrace were obtained. Studies: 1) quantitative analysis of nCLE images, 2) histologic evaluation, 3) expression of: a) CGRP- sensory neuronal marker, b) neuronal NOS (nNOS - marker of nitriergic inhibitory neurons, c) neuronal enolase (NES) and d) NGF and its Trk A receptor, using immunostaining and quantification of signal distribution and intensity with a Metamorph imaging system. Results: The nCLE imaging clearly visualized neuronal cells, nerve bundles and fibers providing their distinctive image patterns. Histology demonstrated normal esophageal structures; fluorescence microscopy showed that in vivo injected NeuroTrace was well retained in neuronal cells and nerves. Immunostaining for CGRP, nNOS and NES showed their strong and differential expression and localization in the neuronal network. NGF was strongly expressed in the majority of neurons and nerves, distribution of TrkA was complementary; its signal was 1.5-fold weaker p ,0.001 than NGF). Quantitatively the signal intensity was: CGRP. nNOS.NGF . NES.TrkA. In addition to neural structures, nNOS, NGF and TrkA were expressed in keratinocyte progenitor cells (KPC) and in endothelial cells. Conclusions: 1) nCLE and NeuroTrace provide reliable in vivo imaging of esophageal
540 Accumulation of Epigenetic Alteration Could Predict Malignant Formation in Intraductal Papillary Mucinous Neoplasm (IPMN) Kazuhiro Yoshida, Takeshi Nagasaka, Yuzo Umeda, Naosuke Yokomichi, Yoshiko Mori, Nobuhito Kubota, Tatsuya Morikawa, Yuko Takehara, Kiyoto Takehara, Kunitoshi Shigeyasu, Akihiro Nyuya, Rikiya Shiwaku, Manabu Suno, Naoshi Nishida, Toshiyoshi Fujiwara, Ajay Goel Introduction: Intraductal Papillary Mucinous Neoplasm (IPMN) is a pancreatic cystic neoplasm and arises in main and side branch of pancreatic duct. Accumulation of genetic and epigenetic alterations is believed to accelerate the malignant formation of IPMNs. However, the molecular events underlying the normal-adenoma-carcinoma sequence in the tumorigenesis in IPMN remain unclear. The aim of the present study is to identify DNA methylation patterns in IPMN, and evaluate their utility as potential predictive biomarkers in this malignancy. Patients and Methods: Tissue specimens were obtained from 50 patients with IPMN or pancreatic ductal carcinoma derived from IPMN (IPMC) who underwent surgery at the Okayama University Hospital from 2001 to 2012. We analyzed methylation patterns of 8 gene promoters in the DNA extracted from the formalin-fixed paraffin-embedded tissues using a single-step bisulfite modification and fluorescence polymerase chain reaction, alsocalled High Sensitive Assay for bisulfite modified DNA (Hi-SA). Subsequently, we determined whether the identified methylation patterns could act as stage-dependent biomarkers of IPMN tumorigenesis. Results: In our patient cohort, 24 patients had IPMNs and 26 had
AGA Abstracts
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