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QS296. Differential Expression of Palladin in Breast Cancer and Its Potential Role in Tumor Metastasis
384 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS (gamma) and ␦ (delta), the purpose of this study is to determine which isoforms of p38 MAPK is involved in the cross-talk between TGF- receptors and GRP-R. Rat intestinal epithelial cell line stably transfected with GRP-R (RIE/GRP-R) were treated with TGF- (40 pM) and BBS (10 ⫺8 M), the activation of p38 MAPK was determined by phospho-p38 MAPK antibody. Activation of p38 MAPK began 15 min after BBS treatment and 30 min after TGF- treatment. Treatment with TGF- and BBS together induced activation of p38 MAPK greater than either treatment alone. Treatment of RIE-1/GRP-R cells with p38 MAPK␣ siRNAs inhibited p38 MAPK␣ expression and blocked TGF- and BBS induced p38 MAPK activation suggesting that the ␣ isoform of p38 MAPK is the main isoform. Furthermore, p38 MAPK␣ siRNAs attenuated the synergistic effect of TGF- and BBS on the expression of COX-2. These results suggest that p38 MAPK␣ is the isoform involved in the cross-talk between TGF- receptors and GRP-R. Selective targeting of p38 MAPK␣ may provide a novel chemoprevention strategy in the management of colorectal cancer. QS294. A GENE SIGNATURE FOR THE PREDICTION OF HEPATIC METASTASIS FROM COLORECTAL CARCINOMA. Kenneth L. Meredith, Erin Siegel, James Mcloughlin, James Lewis, Steve Escherich, Eric Jensen, Quan Ly, Michael Alvarado, Tim Yeatman, David Shibata; H. Lee Moffitt Cancer Center, Tampa, FL Purpose: Approximately 20% of patients with colorectal carcinoma (CRC) present with hepatic metastases. Genome-wide alterations that occur during the metastatic progression of CRC remain poorly understood. We have previously used a clinicogenomic CRC database to compare clinical variables as well as microarray gene expression profiles between metastastic and non-metastatic primary CRCs. We have sought to utilize this dataset to establish a gene signature to predict the hepatic metastastic potential of primary CRCs. Methods: From 1993 to 2003, primary CRC specimens were collected from patients consenting to our tissue bank protocol. RNA was extracted from tissue collected at surgery. All specimens underwent gene profiling using the Affymetrix 133 Plus 2.0 Gene chip. The data was normalized using RMA normalization. A c4.5 decision tree was implemented in a 5-fold cross-validation setup. The input to the decision tree within each fold was a set of 20 significant genes ranked by p-values. Each fold trained on 90% of the samples, and tested the signature on 10% held-out samples. The gene signature was then internally validated on our own data set. Clinical data were collected by chart review, and statistical comparisons performed using the Fisher Exact Test. Results: We identified a total of 96 analyzable cases of which there were 66 patients without evidence of nodal or distant metastases. An additional 30 patients were noted to have either synchronous or metachronous hepatic metastases. The study population was comprised of 58 males and 38 females with a median age of 72 years (range 39-90). At a false discovery rate of 1%, metastatic tumors displayed a total 234 upregulated genes (0 downregulated) as compared to non-metastatic CRCs. A 20-gene signature created from this list demonstrated an overall accuracy of 74 % at prediction of hepatic metastasis on validation testing. The signature included genes associated with cytoskeletal integrity (dystonin, actin), ubiquitin proteolysis (EDD1), cellular growth via insulin like growth factor binding gene (CYR61), and cellular signal/ transduction via TGF receptor binding protein (LTBP1). Conclusions: Patients with colorectal carcinoma who develop hepatic metastasis display a dramatically altered panel of differentially expressed genes in the primary tumor. We have developed a gene signature that identifies these patients at risk for developing hepatic metastasis with significant accuracy. Our data may allow for the development of means to more accurately determine prognosis in early stage primary CRCs. This may subsequently assist with the personalized selection of patients for adjuvant therapy in the absence of conventional indications such as nodal positivity and poor histologic features.
QS295. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) OVER EXPRESSION IN MICROSATELLITE UNSTABLE (MSI) COLORECTAL CANCER (CRC) IS CHARACTERIZED BY A DELETION MUTATION IN THE 3’ UTR POLY (A) 13 TRACT AND IS NOT ASSOCIATED WITH INTRON 1 CA REPEAT LENGTH VARIATION. Joongho Shin1, Ziqian Yuan1, Prashanth Sreeramoju1, Kenneth Fordyce2, Victoria Lai1, Noam White5, John Mariadason7, Thomas K. Weber1; 1Albert Einstein College of Medicine, New York, NY; 2IBM USA, Burlington, VT; 5Salanter Akiva Riverdale High School, New York, NY; 7Montefiore Medical Center, New York, NY Introduction: Last year we presented to this forum our finding of significantly increased EGFR expression in MSI over microsatellite stable (MSS) human CRC cell lines (p⫽ 0.009). Epidermal Growth Factor Receptor (EGFR) is over-expressed in multiple malignancies including colorectal cancer, and multiple EGFR targeted molecular therapies have demonstrated important but modest effectiveness against human CRC. The underlying cellular mechanism of EGFR over-expression has not been elucidated, however, multiple reports suggest an inverse correlation between CA repeat length within the first intron of the EGFR gene and its level of expression in head and neck, lung, and breast carcinomas in vitro. The aim of our present study was to; (1) test the hypothesis that EGFR over expression in MSI CRC is inversely related with CA repeat length as suggested by published studies on other solid tumors, noted above. (2) expand our analysis of EGFR over expression in MSI CRC to human ex-vivo colorectal cancers in order to further test the hypothesis that MSI CRC is characterized by the novel 3’UTR deletion A mutation in the poly (A) 13 tract first identified by our laboratory. Methods: 30 human CRC cell lines (19 MSS and 11MSI) and 33 human ex-vivo colorectal cancers (16 MSS and 17 MSI) were used for our study. In the cell lines, EGFR DNA regions containing the intron 1 CA repeats and the 3’ UTR poly (A) 13 tract were PCR amplified separately and analyzed using direct sequencing and rigorous nucleotide fragment analysis. Using the ex-vivo tumors, DNA regions containing the poly (A) 13 tract were PCR amplified, directly sequenced and submitted for fragment analysis. Results: EGFR intron 1 CA repeat lengths ranged from 16 to 28 nucleotides with 18 being the most frequently observed length. An inverse relationship was not observed between EGFR over expression and intron 1 CA repeat length (p ⬍ 0.01). The deletion A mutation was highly prevalent in both the MSI cell lines and ex-vivo tumors tested; 7 of 11 MSI CRC cell lines and 11 of 17 MSI ex-vivo human colorectal cancers contained the mutation. In contrast, 0 of 19 MSS cell lines and 0 of 16 MSS tumors demonstrated the deletion A mutation in our analysis (p ⬍ 0.001). Conclusions: These results clearly indicate the relationship between EGFR intron 1 CA repeat length and EGFR over expression observed in multiple other solid tumors does not apply in MSI human colorectal cancer supporting our original hypothesis that the 3’UTR poly A (13) del mutation contributes to EGFR over expression in MSI colorectal cancer. Importantly our ex-vivo tumor results expand and confirm our original finding of a strong association between the 3’ UTR del A mutation and EGFR over expression in human MSI colorectal cancer cell lines. The results of this study, combined with published reports from our laboratory focused on CDK2-AP1 expression in MSI CRC, strongly support our broader hypothesis that 3’UTR simple repeat sequence variation contributes to malignant transformation and progression in human MSI colorectal cancer. QS296. DIFFERENTIAL EXPRESSION OF PALLADIN IN BREAST CANCER AND ITS POTENTIAL ROLE IN TUMOR METASTASIS. Brian K. Bednarski, Silvia Goicoechea, Xiaoyu Ding, Carol Otey, Hong J. Kim; University of North Carolina, Chapel Hill, NC
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 385 Introduction: Palladin is a cytoskeletal protein involved in actin organization and cell motility. Recent research showed that a mutation in palladin is linked to a form of familial pancreatic cancer, and that palladin may be overexpressed in sporadic pancreatic cancers. The role of palladin in breast cancer has not been well elucidated. Previous work suggests that palladin expression is increased in phenotypically invasive breast cancer cells, while others report a decrease in palladin mRNA levels in metastatic lesions. Research in palladin is complicated in that multiple isoforms are generated from a single gene with nested promoters, so that different probes may detect different isoforms. Our purpose was to determine the role of palladin in human breast cancer by examining protein levels in both in vitro models and human breast cancer specimens using multiple well-characterized palladin antibodies. Materials and Methods: Three breast cancer cell lines (MCF-7, MDA-MB-231, and SUM159) were selected based on their different metastatic potentials. The presence of palladin was assessed in whole cell extracts using western blot analysis with a commercial antibody (Proteintech Group, Inc., Chicago, IL). Three primary human breast cancer specimens, three metastatic breast cancer specimens and three samples of benign breast tissue were obtained from the UNC Tissue Procurement Facility, and extracts were prepared and analyzed using western blots with two additional palladin polyclonal antibodies (4IG and 622). Results: Examination of palladin protein levels in the in vitro models demonstrated increases correlating directly with increasing metastatic potential. Although palladin was expressed in all cell lines, the highest expression was noted in the cells characterized to have the highest metastatic potential (SUM159) and the lowest expression was in the ER(⫹) MCF-7 cells. In the human breast cancer tissue specimens, the commercial antibody illustrated an increase in palladin levels in 2/3 primary tumors and 2/3 metastatic lesions when compared to normals. Evaluation with the 622 antibody demonstrated a similar pattern of palladin expression (4/6 tumors) but different isoforms were noted. Finally, the 4IG antibody revealed that all (6/6) primary and metastatic specimens had more palladin than normal breast tissue (see Figure). Conclusion: Palladin protein levels in cultured cells are directly correlated to their metastatic potential. Importantly, the expression of palladin is increased in human breast cancer specimens when compared to benign breast tissue. Further investigations are ongoing to determine the role of palladin and its isoforms in breast cancer, and whether palladin expression can be linked to metastatic potential, severity of disease, tumor type, or clinical outcome.
QS297. DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE DETECTION OF GERMLINE MUTATIONS IN PHAEOCHROMOCYTOMA. Goswin Y. Meyer-Rochow1, Janine M. Smith2, Debbie J. Marsh3, Bruce G. Robinson1, Stanley B. Sidhu1, Diana E. Benn1; 1Cancer Genetics Laboratory, Hormone and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia; 2Department of Clinical Genetics, The Children’s Hospital at Westmead, Westmead, Australia; 3Functional
Genomics, Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, Sydney, Australia Introduction: Pheochromocytomas are neuroendocrine tumours of chromaffin cell origin, arising from the adrenal medulla and less commonly from extra-adrenal sympathetic paraganglia within the abdomen, thorax and neck. For some time it has been recognised that pheochromocytomas are component tumours in the following familial syndromes: Multiple Endocrine Neoplasia type 2, von Hippel Lindau disease and Neurofibromatosis type 1 caused by mutations in RET, VHL and NF1 respectively. More recently germline mutations in the genes encoding succinate dehydrogenase subunit B (SDHB) and subunit D (SDHD) have been identified as high risk factors for the development of Pheochromocytoma/Paraganglioma syndromes. Approximately 11-24% of pheochromocytomas with an apparently sporadic presentation have been shown to carry a germline mutation indicating familial disease. Finding a germline mutation in a patient is an indication for the screening of tumours associated with the identified syndrome, thereby allowing earlier detection of associated disease. The aim of this study was to perform mutation analysis for the VHL, SDHB, and SDHD genes in a cohort of 82 patients diagnosed with a pheochromocytoma, using denaturing high performance liquid chromatography (dHPLC). Methods: A polymerase chain reaction (PCR) of all exons of the three genes was performed on leukocyte DNA extracted from stored blood samples of 82 patients treated for pheochromocytoma. Institutional ethics was obtained to use samples stored in the Kolling Institue of Medical Research Neuroendocrine tumour bank. There were 40 males with a median age of 51 (11-89) years and 42 females with a median age of 48 (11-77) years in the sample population. dHPLC was undertaken on a WAVE TM DNA fragment analysis system (Transgenomic, Omaha, NE, USA) with analysis performed using the WAVEMaker TM software. Samples demonstrating variance when compared to normal controls were selected for sequencing. Results: Using this method a total of five mutations and 13 polymorphisms were detected in SDHB and seven mutations and one polymorphism were identified in VHL. All mutations were confirmed by re-extraction of DNA from the original blood sample, repeat PCR and dHPLC, followed by sequencing. Of 52 apparently sporadic pheochromocytoma, three SDHB (6%) and one VHL (2%) mutations were identified which is consistent with previously published literature. SDHD mutation analysis is in progress. With PCR and dHPLC (followed by sequencing of variants only), the total amount of DNA sequencing required was able to be reduced by approximately 90% when compared to screening by sequence analysis alone. Conclusions: dHPLC appears to be a cost effective screening tool for the detection of germ-line mutations in SDHB and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients. The detection of germline mutations in a patient with pheochromocytoma identifies familial disease which demonstrates the clinical importance of mutation analysis in the management of pheochromocytoma. QS298. ROLE OF CPG ISLAND METHYLATOR PHENOTYPE (CIMP) IN ULCERATIVE COLITIS ONCOGENESIS. Julian A. Sanchez, Kathryn L. DeJulius, Craig A. Messick, Mary P. Bronner, Graham Casey, James M. Church, Matthew F. Kalady; Cleveland Clinic Foundation, Cleveland, OH Introduction: Patients with ulcerative colitis (UC) harbor increased colorectal cancer risk which is believed to be secondary to repetitive colonic mucosal injury. However, underlying causes for malignant transformation remain incompletely defined. Epigenetic methylation of DNA has been implicated as a mechanism for oncogenesis in a variety of inflammation-associated cancers including gastric MALT lymphoma, Barrett’s-associated esophageal cancer, and hepatocellular carcinoma. This study evaluates the relative contribution of epigenetic methylation as well as the more classical
QS295. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) OVER EXPRESSION IN MICROSATELLITE UNSTABLE (MSI) COLORECTAL CANCER (CRC) IS CHARACTERIZED BY A DELETION MUTATION IN THE 3’ UTR POLY (A) 13 TRACT AND IS NOT ASSOCIATED WITH INTRON 1 CA REPEAT LENGTH VARIATION. Joongho Shin1, Ziqian Yuan1, Prashanth Sreeramoju1, Kenneth Fordyce2, Victoria Lai1, Noam White5, John Mariadason7, Thomas K. Weber1; 1Albert Einstein College of Medicine, New York, NY; 2IBM USA, Burlington, VT; 5Salanter Akiva Riverdale High School, New York, NY; 7Montefiore Medical Center, New York, NY Introduction: Last year we presented to this forum our finding of significantly increased EGFR expression in MSI over microsatellite stable (MSS) human CRC cell lines (p⫽ 0.009). Epidermal Growth Factor Receptor (EGFR) is over-expressed in multiple malignancies including colorectal cancer, and multiple EGFR targeted molecular therapies have demonstrated important but modest effectiveness against human CRC. The underlying cellular mechanism of EGFR over-expression has not been elucidated, however, multiple reports suggest an inverse correlation between CA repeat length within the first intron of the EGFR gene and its level of expression in head and neck, lung, and breast carcinomas in vitro. The aim of our present study was to; (1) test the hypothesis that EGFR over expression in MSI CRC is inversely related with CA repeat length as suggested by published studies on other solid tumors, noted above. (2) expand our analysis of EGFR over expression in MSI CRC to human ex-vivo colorectal cancers in order to further test the hypothesis that MSI CRC is characterized by the novel 3’UTR deletion A mutation in the poly (A) 13 tract first identified by our laboratory. Methods: 30 human CRC cell lines (19 MSS and 11MSI) and 33 human ex-vivo colorectal cancers (16 MSS and 17 MSI) were used for our study. In the cell lines, EGFR DNA regions containing the intron 1 CA repeats and the 3’ UTR poly (A) 13 tract were PCR amplified separately and analyzed using direct sequencing and rigorous nucleotide fragment analysis. Using the ex-vivo tumors, DNA regions containing the poly (A) 13 tract were PCR amplified, directly sequenced and submitted for fragment analysis. Results: EGFR intron 1 CA repeat lengths ranged from 16 to 28 nucleotides with 18 being the most frequently observed length. An inverse relationship was not observed between EGFR over expression and intron 1 CA repeat length (p ⬍ 0.01). The deletion A mutation was highly prevalent in both the MSI cell lines and ex-vivo tumors tested; 7 of 11 MSI CRC cell lines and 11 of 17 MSI ex-vivo human colorectal cancers contained the mutation. In contrast, 0 of 19 MSS cell lines and 0 of 16 MSS tumors demonstrated the deletion A mutation in our analysis (p ⬍ 0.001). Conclusions: These results clearly indicate the relationship between EGFR intron 1 CA repeat length and EGFR over expression observed in multiple other solid tumors does not apply in MSI human colorectal cancer supporting our original hypothesis that the 3’UTR poly A (13) del mutation contributes to EGFR over expression in MSI colorectal cancer. Importantly our ex-vivo tumor results expand and confirm our original finding of a strong association between the 3’ UTR del A mutation and EGFR over expression in human MSI colorectal cancer cell lines. The results of this study, combined with published reports from our laboratory focused on CDK2-AP1 expression in MSI CRC, strongly support our broader hypothesis that 3’UTR simple repeat sequence variation contributes to malignant transformation and progression in human MSI colorectal cancer. QS296. DIFFERENTIAL EXPRESSION OF PALLADIN IN BREAST CANCER AND ITS POTENTIAL ROLE IN TUMOR METASTASIS. Brian K. Bednarski, Silvia Goicoechea, Xiaoyu Ding, Carol Otey, Hong J. Kim; University of North Carolina, Chapel Hill, NC
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 385 Introduction: Palladin is a cytoskeletal protein involved in actin organization and cell motility. Recent research showed that a mutation in palladin is linked to a form of familial pancreatic cancer, and that palladin may be overexpressed in sporadic pancreatic cancers. The role of palladin in breast cancer has not been well elucidated. Previous work suggests that palladin expression is increased in phenotypically invasive breast cancer cells, while others report a decrease in palladin mRNA levels in metastatic lesions. Research in palladin is complicated in that multiple isoforms are generated from a single gene with nested promoters, so that different probes may detect different isoforms. Our purpose was to determine the role of palladin in human breast cancer by examining protein levels in both in vitro models and human breast cancer specimens using multiple well-characterized palladin antibodies. Materials and Methods: Three breast cancer cell lines (MCF-7, MDA-MB-231, and SUM159) were selected based on their different metastatic potentials. The presence of palladin was assessed in whole cell extracts using western blot analysis with a commercial antibody (Proteintech Group, Inc., Chicago, IL). Three primary human breast cancer specimens, three metastatic breast cancer specimens and three samples of benign breast tissue were obtained from the UNC Tissue Procurement Facility, and extracts were prepared and analyzed using western blots with two additional palladin polyclonal antibodies (4IG and 622). Results: Examination of palladin protein levels in the in vitro models demonstrated increases correlating directly with increasing metastatic potential. Although palladin was expressed in all cell lines, the highest expression was noted in the cells characterized to have the highest metastatic potential (SUM159) and the lowest expression was in the ER(⫹) MCF-7 cells. In the human breast cancer tissue specimens, the commercial antibody illustrated an increase in palladin levels in 2/3 primary tumors and 2/3 metastatic lesions when compared to normals. Evaluation with the 622 antibody demonstrated a similar pattern of palladin expression (4/6 tumors) but different isoforms were noted. Finally, the 4IG antibody revealed that all (6/6) primary and metastatic specimens had more palladin than normal breast tissue (see Figure). Conclusion: Palladin protein levels in cultured cells are directly correlated to their metastatic potential. Importantly, the expression of palladin is increased in human breast cancer specimens when compared to benign breast tissue. Further investigations are ongoing to determine the role of palladin and its isoforms in breast cancer, and whether palladin expression can be linked to metastatic potential, severity of disease, tumor type, or clinical outcome.
QS297. DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE DETECTION OF GERMLINE MUTATIONS IN PHAEOCHROMOCYTOMA. Goswin Y. Meyer-Rochow1, Janine M. Smith2, Debbie J. Marsh3, Bruce G. Robinson1, Stanley B. Sidhu1, Diana E. Benn1; 1Cancer Genetics Laboratory, Hormone and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia; 2Department of Clinical Genetics, The Children’s Hospital at Westmead, Westmead, Australia; 3Functional
Genomics, Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, Sydney, Australia Introduction: Pheochromocytomas are neuroendocrine tumours of chromaffin cell origin, arising from the adrenal medulla and less commonly from extra-adrenal sympathetic paraganglia within the abdomen, thorax and neck. For some time it has been recognised that pheochromocytomas are component tumours in the following familial syndromes: Multiple Endocrine Neoplasia type 2, von Hippel Lindau disease and Neurofibromatosis type 1 caused by mutations in RET, VHL and NF1 respectively. More recently germline mutations in the genes encoding succinate dehydrogenase subunit B (SDHB) and subunit D (SDHD) have been identified as high risk factors for the development of Pheochromocytoma/Paraganglioma syndromes. Approximately 11-24% of pheochromocytomas with an apparently sporadic presentation have been shown to carry a germline mutation indicating familial disease. Finding a germline mutation in a patient is an indication for the screening of tumours associated with the identified syndrome, thereby allowing earlier detection of associated disease. The aim of this study was to perform mutation analysis for the VHL, SDHB, and SDHD genes in a cohort of 82 patients diagnosed with a pheochromocytoma, using denaturing high performance liquid chromatography (dHPLC). Methods: A polymerase chain reaction (PCR) of all exons of the three genes was performed on leukocyte DNA extracted from stored blood samples of 82 patients treated for pheochromocytoma. Institutional ethics was obtained to use samples stored in the Kolling Institue of Medical Research Neuroendocrine tumour bank. There were 40 males with a median age of 51 (11-89) years and 42 females with a median age of 48 (11-77) years in the sample population. dHPLC was undertaken on a WAVE TM DNA fragment analysis system (Transgenomic, Omaha, NE, USA) with analysis performed using the WAVEMaker TM software. Samples demonstrating variance when compared to normal controls were selected for sequencing. Results: Using this method a total of five mutations and 13 polymorphisms were detected in SDHB and seven mutations and one polymorphism were identified in VHL. All mutations were confirmed by re-extraction of DNA from the original blood sample, repeat PCR and dHPLC, followed by sequencing. Of 52 apparently sporadic pheochromocytoma, three SDHB (6%) and one VHL (2%) mutations were identified which is consistent with previously published literature. SDHD mutation analysis is in progress. With PCR and dHPLC (followed by sequencing of variants only), the total amount of DNA sequencing required was able to be reduced by approximately 90% when compared to screening by sequence analysis alone. Conclusions: dHPLC appears to be a cost effective screening tool for the detection of germ-line mutations in SDHB and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients. The detection of germline mutations in a patient with pheochromocytoma identifies familial disease which demonstrates the clinical importance of mutation analysis in the management of pheochromocytoma. QS298. ROLE OF CPG ISLAND METHYLATOR PHENOTYPE (CIMP) IN ULCERATIVE COLITIS ONCOGENESIS. Julian A. Sanchez, Kathryn L. DeJulius, Craig A. Messick, Mary P. Bronner, Graham Casey, James M. Church, Matthew F. Kalady; Cleveland Clinic Foundation, Cleveland, OH Introduction: Patients with ulcerative colitis (UC) harbor increased colorectal cancer risk which is believed to be secondary to repetitive colonic mucosal injury. However, underlying causes for malignant transformation remain incompletely defined. Epigenetic methylation of DNA has been implicated as a mechanism for oncogenesis in a variety of inflammation-associated cancers including gastric MALT lymphoma, Barrett’s-associated esophageal cancer, and hepatocellular carcinoma. This study evaluates the relative contribution of epigenetic methylation as well as the more classical