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T2010 Effect of Diet on APC Mutation Spectra and Gene Promoter Methylation in Colorectal Cancer
T1943
8q24 and 9p24 (rs6983267, rs10505477, rs1447295 and rs719725) have been shown to increase risk of various solid tumours including prostate and colorectal cancer. To date, these SNP's have not been studied in the context of upper GI cancer. Aim: To assess these SNPs and risk of upper GI carcinoma. Subjects and Methods: Two independent casecontrol studies were used to assess SNP frequency: Study 1 - a gastric cancer case-control study derived from a Caucasian population in Warsaw, Poland with DNA available from 312 non-cardia gastric adenocarcinoma patients and 419 controls, Study 2 - a multi-center esophageal and gastric cancer study from the United States with DNA available from 307 subjects with gastric adenocarcinoma, 159 subjects with esophageal cancer and 211 population controls. DNA was genotyped by 5' nuclease PCR assay. Hardy-Weinberg equilibrium of alleles at individual loci was assessed using χ2 test. Odd ratio (OR) with Cornfield 95% confidence intervals (CIs) were computed by logistic regression using STATA version 7.0 software. Results: Genotyping and statistical analysis of the 4 SNPs was completed on 765 cases and 583 controls. Individuals carrying the C/C genotype of rs1447295 had an elevated risk for non-cardia gastric cancer (OR = 1.57; 95% CI 1.03 - 2.45). Individuals carrying the A/A genotype of rs1447295 had an increased risk of squamous cell esophageal cancer (OR = 7.4; 95% CI 1.4 - 49). None of the other three SNP's tested in this study showed any statistically significant associations. Conclusions: The association between the rs1447295 SNP and risks of gastric and esophageal cancer are novel and offer an opportunity to explore the genetic basis of these malignancies. Our study has extended the genetic link between the 8q24 region and risk of yet another group of solid tumours. Extensive efforts should focus on evaluating the functional significance of this region in the context of the molecular pathogenesis of these malignancies.
AGA Abstracts
Multiple Functions of Glycoprotein 2 (GP2) in the Gastrointestinal Systems; A Link to Sorting, Packaging and Recycling of the Digestive Enzymes in the Exocrine Pancreas Yu Tokuhira, Ryuta Suzuki, Rei Kawashima, Shin-Ichi Fukuoka BACKGROUND & AIMS: GP2 is expressed in the pancreatic acinar cells and the other tissues of gastrointestinal systems. GP2 was identified as GPI-anchored membrane proteins associated with apical secretory membranes; however, its biological function is poorly understood. Recently, our group reported that GP2, which is expressed specifically to M cells in the intestinal epithelial cells, serves as a transcytotic receptor for mucosal antigens (Hase et al. Nature. 2009 Nov 12;462(7270):226-30.). This result has prompted us to investigate role of GP2 in the pancreatic function. GP2 knockout (KO) mice were utilized to clarify mechanisms of the granule formation and exocrine secretion. METHODS: messenger RNA (mRNA) levels of LC3, the most commonly examined autophagy marker in mammalian cells, were quantified by real-time PCR, and its protein expression was analyzed by western blotting. The levels of enzymes, amylase in the pancreatic tissue were measured and the expression of amylase was assayed by immunohistochemistry. The mRNA levels of SNAREs, which drive membrane fusion in exocytosis, were quantified by real-time PCR. RESULTS: Up-regulation of mRNA and protein levels of LC3 in the pancreatic tissue were suppressed in 18 hours fasted GP2 KO mice compared with wild type (WT) mice. Starvation induced the reduction of pancreatic amylase activity in WT mice. In contrast, the induction was failed in GP2 KO mice. In histlogical analysis, amylase expression was significantly elevated in the GP2 deficient acinar cells at the same time points. Further, mRNA expressions of SNAREs were attenuated in the GP2 deficient pancreatic tissues when compared with those of WT mice. CONCLUSIONS: Our results suggest that zymogen granules accumulation is associated with lysosomal dysfunction and autophagic stress in the GP2 deficient pancreas under fasted condition. GP2 seems required for sorting, packaging and recycling of the digestive enzymes in the secretory pathway. GP2 is likely to play multiple roles in the gastrointestinal systems.
T2010 Effect of Diet on APC Mutation Spectra and Gene Promoter Methylation in Colorectal Cancer Laura J. Gay, Mark J. Arends, Panagiota N. Mitrou, Janusz A. Jankowski, Alison McTaggart, Robert Luben, Kay-Tee Khaw, Richard Y. Ball, Sheila A. Rodwell Epidemiological evidence suggests that the large worldwide variation in rates of colorectal cancer incidence can be attributed to differences in exposure to certain environmental factors, in particular the ‘westernised' diet. In colorectal carcinogenesis, genetic aberrations to the tumour suppressor gene Adenomatous Polyposis Coli (APC) are considered to be key to the initiation of colorectal neoplasia. Analysis of APC mutations in the extended mutation cluster region (codons 1276-1556) and promoter 1A methylation was performed on 185 archival colorectal tumour samples collected from participants of the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC), with the aim of relating these results to seven-day dietary information collected for each individual at the start of the study. Overall, 43% and 23% of tumours analysed had truncating APC mutations and promoter methylation, respectively. Comparison by clinico-pathological features revealed that proximal tumours were significantly more likely to harbour an APC mutation or promoter methylation (P=0.04). Case-case analysis by diet revealed that tumour cases with an APC mutation consumed more alcohol than their counterparts (P=0.01) and those with APC promoter methylation consumed lower levels of folate and fibre (P=0.01 and 0.004, respectively). Tumour cases with either genetic aberration consumed higher levels of processed meat compared to those without (P=0.007). However, analysis by APC mutation spectra revealed that it was specifically those tumour cases harbouring GC to AT transitions that consumed higher levels of processed meat (35 versus 24g/day, P=0.03). In conclusion, the results from this study indicate that APC mutation spectra and promoter methylation may be influenced by diet. In particular, APC mutation spectra were associated with processed meat consumption, suggesting a mechanistic link between dietary alkylating agents, such as N-Nitroso compounds, and GC to AT transitions. Analysis of the combined mutation spectra from several other genes associated with colorectal cancer (TP53, KRAS, BRAF, PTEN and PIK3CA) in the same 185 cases will be conducted to further explore this observation.
T1944 High-Resolution, 3-Dimensional Confocal Imaging of Mouse Pancreatic Microstructure and Vasculature Ya-Yuan Fu, Shiue-Cheng Tang Background & Aims: The opacity of pancreata limits optical accessibility for high-resolution light microscopy. Thus, pancreata are usually sectioned to expose their interior domain for microscopic examination. We sought to integrate optical clearing with confocal microscopy to develop a microtome-free method for in-depth, 3-dimensional (3D) visualization of mouse pancreatic microstructure and vasculature. Methods: Pancreatic tissue specimens were treated with an optical-clearing solution, FocusClear (US Patent 6472216, Fu et al., Gastroenterology, 137:p453-463, 2009), to improve photon penetration. After fluorophore labeling, such as vessel painting (lipophilic dye labeling of blood vessels), the pancreatic structures underwent confocal microscopy with efficient fluorescence excitation/emission in the optical-cleared specimen. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3D visualization. Results: The improved photon penetration resulting from treatment with the optical-clearing solution led to image acquisition with subcellular-level resolution up to 400 μm. We achieved an integral, 3D visualization of pancreatic microstructure and vasculature. Notably, the microvessels with a diameter of ~5 μm were clearly visualized in the islet and acini. Conclusion: This new optical method will change our conventional planar view of the pancreatic tissue structures into a 3D panorama for better understanding the pancreatic physiology and diseases.
T2011 Identification of the Gnas1 T393c Polymorphism as a New Independent Prognostic Marker in Oesophageal Cancer Hakan Alakus, Elfriede Bollschweiler, Ute Warnecke-Eberz, Stefan Monig, Winfried Siffert, Arnulf H. Hölscher, Ralf Metzger Introduction Recent studies have shown an association between the GNAS1 T393C polymorphism and clinical outcome for various solid tumors. Until now, nothing is known about the impact of this SNP on histopathology and on prognosis of oesophageal cancer. Methods Genomic DNA was extracted from paraffin-embedded tissues of 301 patients (248 men, 53 female, median age 62 years, range 19-86 years), who were initially treated surgically for oesophageal carcinoma [125 (41.5%) squamous cell carcinoma and 176 (58.5%) adenocarcinoma] between 1996 and 2008. Additionally, DNA was extracted from blood of 820 healthy white individuals as a reference group. Allelic discrimination was performed by quantitative real-time PCR. Genotyping was correlated with histopathologic parameters and with overall survival according to the Kaplan-Meier approach and with multivariate analysis by multiple stepwise reggression. Results 30% patients displayed a CC genotype, 47% a CT genotype and 23% a TT genotype. The genotype distribution of the patient group was not different from that of healthy blood donors and it was compatible with the Hardy-Weinberg equilibrium. Analysis of clinicopathological parameters did not show any significant correlation of the T393C genotype with pT (p=0.5), pN (p=0.6), pM (p=0.5), gender (p=0.6), histology (p=0.2) and tumor localisation (p=0.5). The 5-year-survival rate for patients with a TT-genotype was 47% and 42% for patients with a CT-genotype. Homozygous C allele carrier (CC) had a 5-year-survival rate of 59% which was significantly higher compared to T allele carriers (CT+TT) (p=0.015). In multivariate analysis beside age (p<0.001), pT (<0.001), pN (<0.001) und pM (p<0.001), the T393C polymorphism also remained as an independent prognostic factor (p=0.048). Conclusions This study is the first to demonstrate that the GNAS1 T393C polymorphism is an independent prognostic marker in oesophageal cancer. This SNP could be used in future treatment protocols of oesophageal cancer to identify high-risk patients.
Penetrative 3D microscopy of mouse pancreas. T2009 Assessment of Novel Genetic Polymorphisms and Risk of Upper Gastrointestinal Carcinoma Mike T. Ng, Charles S. Rabkin, Paul Lochhead, Jolanta Lissowska, Thomas L. Vaughan, Marilie Gammon, Harvey Risch, Wong-Ho Chow, Georgina L. Hold, Emad El-Omar Background: Upper gastrointestinal malignancies comprising gastric and esophageal cancers remain a major global health problem. Recently genome wide association studies have been used to identify risk factors for several diseases. Four SNPs identified at chromosome regions
AGA Abstracts
S-612
8q24 and 9p24 (rs6983267, rs10505477, rs1447295 and rs719725) have been shown to increase risk of various solid tumours including prostate and colorectal cancer. To date, these SNP's have not been studied in the context of upper GI cancer. Aim: To assess these SNPs and risk of upper GI carcinoma. Subjects and Methods: Two independent casecontrol studies were used to assess SNP frequency: Study 1 - a gastric cancer case-control study derived from a Caucasian population in Warsaw, Poland with DNA available from 312 non-cardia gastric adenocarcinoma patients and 419 controls, Study 2 - a multi-center esophageal and gastric cancer study from the United States with DNA available from 307 subjects with gastric adenocarcinoma, 159 subjects with esophageal cancer and 211 population controls. DNA was genotyped by 5' nuclease PCR assay. Hardy-Weinberg equilibrium of alleles at individual loci was assessed using χ2 test. Odd ratio (OR) with Cornfield 95% confidence intervals (CIs) were computed by logistic regression using STATA version 7.0 software. Results: Genotyping and statistical analysis of the 4 SNPs was completed on 765 cases and 583 controls. Individuals carrying the C/C genotype of rs1447295 had an elevated risk for non-cardia gastric cancer (OR = 1.57; 95% CI 1.03 - 2.45). Individuals carrying the A/A genotype of rs1447295 had an increased risk of squamous cell esophageal cancer (OR = 7.4; 95% CI 1.4 - 49). None of the other three SNP's tested in this study showed any statistically significant associations. Conclusions: The association between the rs1447295 SNP and risks of gastric and esophageal cancer are novel and offer an opportunity to explore the genetic basis of these malignancies. Our study has extended the genetic link between the 8q24 region and risk of yet another group of solid tumours. Extensive efforts should focus on evaluating the functional significance of this region in the context of the molecular pathogenesis of these malignancies.
AGA Abstracts
Multiple Functions of Glycoprotein 2 (GP2) in the Gastrointestinal Systems; A Link to Sorting, Packaging and Recycling of the Digestive Enzymes in the Exocrine Pancreas Yu Tokuhira, Ryuta Suzuki, Rei Kawashima, Shin-Ichi Fukuoka BACKGROUND & AIMS: GP2 is expressed in the pancreatic acinar cells and the other tissues of gastrointestinal systems. GP2 was identified as GPI-anchored membrane proteins associated with apical secretory membranes; however, its biological function is poorly understood. Recently, our group reported that GP2, which is expressed specifically to M cells in the intestinal epithelial cells, serves as a transcytotic receptor for mucosal antigens (Hase et al. Nature. 2009 Nov 12;462(7270):226-30.). This result has prompted us to investigate role of GP2 in the pancreatic function. GP2 knockout (KO) mice were utilized to clarify mechanisms of the granule formation and exocrine secretion. METHODS: messenger RNA (mRNA) levels of LC3, the most commonly examined autophagy marker in mammalian cells, were quantified by real-time PCR, and its protein expression was analyzed by western blotting. The levels of enzymes, amylase in the pancreatic tissue were measured and the expression of amylase was assayed by immunohistochemistry. The mRNA levels of SNAREs, which drive membrane fusion in exocytosis, were quantified by real-time PCR. RESULTS: Up-regulation of mRNA and protein levels of LC3 in the pancreatic tissue were suppressed in 18 hours fasted GP2 KO mice compared with wild type (WT) mice. Starvation induced the reduction of pancreatic amylase activity in WT mice. In contrast, the induction was failed in GP2 KO mice. In histlogical analysis, amylase expression was significantly elevated in the GP2 deficient acinar cells at the same time points. Further, mRNA expressions of SNAREs were attenuated in the GP2 deficient pancreatic tissues when compared with those of WT mice. CONCLUSIONS: Our results suggest that zymogen granules accumulation is associated with lysosomal dysfunction and autophagic stress in the GP2 deficient pancreas under fasted condition. GP2 seems required for sorting, packaging and recycling of the digestive enzymes in the secretory pathway. GP2 is likely to play multiple roles in the gastrointestinal systems.
T2010 Effect of Diet on APC Mutation Spectra and Gene Promoter Methylation in Colorectal Cancer Laura J. Gay, Mark J. Arends, Panagiota N. Mitrou, Janusz A. Jankowski, Alison McTaggart, Robert Luben, Kay-Tee Khaw, Richard Y. Ball, Sheila A. Rodwell Epidemiological evidence suggests that the large worldwide variation in rates of colorectal cancer incidence can be attributed to differences in exposure to certain environmental factors, in particular the ‘westernised' diet. In colorectal carcinogenesis, genetic aberrations to the tumour suppressor gene Adenomatous Polyposis Coli (APC) are considered to be key to the initiation of colorectal neoplasia. Analysis of APC mutations in the extended mutation cluster region (codons 1276-1556) and promoter 1A methylation was performed on 185 archival colorectal tumour samples collected from participants of the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC), with the aim of relating these results to seven-day dietary information collected for each individual at the start of the study. Overall, 43% and 23% of tumours analysed had truncating APC mutations and promoter methylation, respectively. Comparison by clinico-pathological features revealed that proximal tumours were significantly more likely to harbour an APC mutation or promoter methylation (P=0.04). Case-case analysis by diet revealed that tumour cases with an APC mutation consumed more alcohol than their counterparts (P=0.01) and those with APC promoter methylation consumed lower levels of folate and fibre (P=0.01 and 0.004, respectively). Tumour cases with either genetic aberration consumed higher levels of processed meat compared to those without (P=0.007). However, analysis by APC mutation spectra revealed that it was specifically those tumour cases harbouring GC to AT transitions that consumed higher levels of processed meat (35 versus 24g/day, P=0.03). In conclusion, the results from this study indicate that APC mutation spectra and promoter methylation may be influenced by diet. In particular, APC mutation spectra were associated with processed meat consumption, suggesting a mechanistic link between dietary alkylating agents, such as N-Nitroso compounds, and GC to AT transitions. Analysis of the combined mutation spectra from several other genes associated with colorectal cancer (TP53, KRAS, BRAF, PTEN and PIK3CA) in the same 185 cases will be conducted to further explore this observation.
T1944 High-Resolution, 3-Dimensional Confocal Imaging of Mouse Pancreatic Microstructure and Vasculature Ya-Yuan Fu, Shiue-Cheng Tang Background & Aims: The opacity of pancreata limits optical accessibility for high-resolution light microscopy. Thus, pancreata are usually sectioned to expose their interior domain for microscopic examination. We sought to integrate optical clearing with confocal microscopy to develop a microtome-free method for in-depth, 3-dimensional (3D) visualization of mouse pancreatic microstructure and vasculature. Methods: Pancreatic tissue specimens were treated with an optical-clearing solution, FocusClear (US Patent 6472216, Fu et al., Gastroenterology, 137:p453-463, 2009), to improve photon penetration. After fluorophore labeling, such as vessel painting (lipophilic dye labeling of blood vessels), the pancreatic structures underwent confocal microscopy with efficient fluorescence excitation/emission in the optical-cleared specimen. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3D visualization. Results: The improved photon penetration resulting from treatment with the optical-clearing solution led to image acquisition with subcellular-level resolution up to 400 μm. We achieved an integral, 3D visualization of pancreatic microstructure and vasculature. Notably, the microvessels with a diameter of ~5 μm were clearly visualized in the islet and acini. Conclusion: This new optical method will change our conventional planar view of the pancreatic tissue structures into a 3D panorama for better understanding the pancreatic physiology and diseases.
T2011 Identification of the Gnas1 T393c Polymorphism as a New Independent Prognostic Marker in Oesophageal Cancer Hakan Alakus, Elfriede Bollschweiler, Ute Warnecke-Eberz, Stefan Monig, Winfried Siffert, Arnulf H. Hölscher, Ralf Metzger Introduction Recent studies have shown an association between the GNAS1 T393C polymorphism and clinical outcome for various solid tumors. Until now, nothing is known about the impact of this SNP on histopathology and on prognosis of oesophageal cancer. Methods Genomic DNA was extracted from paraffin-embedded tissues of 301 patients (248 men, 53 female, median age 62 years, range 19-86 years), who were initially treated surgically for oesophageal carcinoma [125 (41.5%) squamous cell carcinoma and 176 (58.5%) adenocarcinoma] between 1996 and 2008. Additionally, DNA was extracted from blood of 820 healthy white individuals as a reference group. Allelic discrimination was performed by quantitative real-time PCR. Genotyping was correlated with histopathologic parameters and with overall survival according to the Kaplan-Meier approach and with multivariate analysis by multiple stepwise reggression. Results 30% patients displayed a CC genotype, 47% a CT genotype and 23% a TT genotype. The genotype distribution of the patient group was not different from that of healthy blood donors and it was compatible with the Hardy-Weinberg equilibrium. Analysis of clinicopathological parameters did not show any significant correlation of the T393C genotype with pT (p=0.5), pN (p=0.6), pM (p=0.5), gender (p=0.6), histology (p=0.2) and tumor localisation (p=0.5). The 5-year-survival rate for patients with a TT-genotype was 47% and 42% for patients with a CT-genotype. Homozygous C allele carrier (CC) had a 5-year-survival rate of 59% which was significantly higher compared to T allele carriers (CT+TT) (p=0.015). In multivariate analysis beside age (p<0.001), pT (<0.001), pN (<0.001) und pM (p<0.001), the T393C polymorphism also remained as an independent prognostic factor (p=0.048). Conclusions This study is the first to demonstrate that the GNAS1 T393C polymorphism is an independent prognostic marker in oesophageal cancer. This SNP could be used in future treatment protocols of oesophageal cancer to identify high-risk patients.
Penetrative 3D microscopy of mouse pancreas. T2009 Assessment of Novel Genetic Polymorphisms and Risk of Upper Gastrointestinal Carcinoma Mike T. Ng, Charles S. Rabkin, Paul Lochhead, Jolanta Lissowska, Thomas L. Vaughan, Marilie Gammon, Harvey Risch, Wong-Ho Chow, Georgina L. Hold, Emad El-Omar Background: Upper gastrointestinal malignancies comprising gastric and esophageal cancers remain a major global health problem. Recently genome wide association studies have been used to identify risk factors for several diseases. Four SNPs identified at chromosome regions
AGA Abstracts
S-612