- Email: [email protected]
Sa1922 miRNA Expression Signatures of Colorectal Adenoma-Carcinoma Sequence in Tissue and Matched Plasma Specimens
also discriminate CRN in Lynch Syndrome. Aims: In a blinded, prospective tissue study, to (1) determine if molecular markers in MT-sDNA are similarly discriminant for sporadic and Lynch-related CRN, and (2) assess the potential yield in Lynch Syndrome of methylated DNA markers optimized for detection of sporadic CRN. Methods: We studied 283 paraffinembedded specimens, including 184 from sporadic patients (62 normal mucosa, 20 adenoma >1cm (AD), 32 sessile serrated polyp >1cm (SSP), and 70 adenocarcinoma (ACA) and 99 from Lynch patients (36 normal-appearing mucosa in resected specimens with synchronous CRN, 30 AD, and 33 ACA). Assays on extracted DNA were performed using quantitative allele specific real-time target and signal amplification. MT-sDNA markers (without hemoglobin) included 7 KRAS mutations and aberrantly methylated BMP3 and NDRG4 normalized to βactin (total human DNA); a composite MT-sDNA score was created using logistic regression. We also assayed 8 novel markers from a whole methylome discovery (Gastroenterology 2014;146,S-30) including SFMBT2 (Regions 895, 896, and 897), CHST2 (Regions 7889 and 7890), PDGFD, VAV3, and DTX1. Results: Median age in sporadic patients was 68 (range 22-99) and in Lynch patients 56 (27-96); 46% and 52%, respectively, were women. Based on a composite score of MT-sDNA markers, the distributions with normal tissues were comparable between groups, similar between groups with ACA, and were significantly lower in Lynch with AD (Fig.1). At 95% specificity, sensitivity for ACA was 91% in sporadic and 83% in Lynch (p=0.311) but for AD was 88% and 26%, respectively (p=0.0001). One of the best 8 novel markers (SFMB2-896) was comparably discriminant for CRN in sporadic and Lynch tissues based on marker distributions (Fig.2). However, at 95% specificity, sensitivity of SFMB2-896 for ACA was 96% in sporadic and 88% in Lynch (p=0.207) and for AD was 100% and 70%, respectively (p=0.002). At 95% specificity, a panel of 3 methylated markers (SFMB2-896, PDGFD, CHST2-7899) detected 99% of ACA in sporadic and 94% in Lynch (p=0.240) and 100% and 73%, respectively, in AD (p=0.015). Conclusions: Based on our study, acquired DNA changes selected as markers for optimal discrimination of sporadic CRN should detect Lynch ACAs well but will likely miss a proportion of Lynch ADs. These findings may reflect fundamental differences in the molecular biology of neoplasm progression between groups.
Sa1920 Clonal Diversity Based on Single-Cell Analysis Predicts Progression in Barrett's Esophagus Margriet R. Timmer, Pierre Martinez, Chiu T. Lau, Paul Fockens, Jacques J. Bergman, Carlo C. Maley, Trevor A. Graham, Kausilia K. Krishnadath Background: Barrett's esophagus (BE) is associated with a low annual risk of developing esophageal adenocarcinoma (EAC). Predictive biomarkers would be of great clinical value in facilitating more cost-effective surveillance. Here we attempted to evaluate the predictive value of clonal diversity in a large prospective cohort of Barrett's patients without dysplasia at baseline. Methods: We have performed single-cell genetic analysis on brush cytology specimens. Genetic abnormalities were detected by fluorescence in situ hybridization using two probe sets including probes for CEP17, Her-2, P53 and P16 (Set 1) and 20q, MYC and the chromosomal centromeric probes 7 and 17 (Set 2). For each individual cell, a ‘per cell' signal pattern was recorded for each set to provide detailed information on genetically different clones. Clones were defined as the collection of cells with the same genotype. We then aimed to risk stratify patients according to measures of clonal diversity (e.g. richness, Simpson diversity, Shannon diversity and average pairwise distance) using Cox proportionalhazard models. Endpoints were progression to high-grade dysplasia (HGD) or EAC. Thresholds for each variable (e.g. a threshold to distinguish normal from abnormal) were determined by a bootstrap-resampling procedure. Results: A total of 320 patients (81% men; mean age 59 years ±12; median BE length 2 cm) were followed for a median of 43 (95% CI 40-46) months during which 20 patients progressed to HGD (n=8) or EAC (n=12). Only 30 patients displayed a single genotype throughout their respective cell population in set 1, and 107 in set 2 (9.4 % and 33.4%, respectively). The diversity obtained from set 1 was significantly higher than the one obtained from set 2 (p=0.0001, paired t-test), owing to the prominence of P16 and P53 abnormalities which were assayed in set 1. All measures of clonal diversity, except for the Simpson diversity (set 1), were significantly associated with progression (see Table 1). Conclusion: Quantification of genetic diversity can be considered a proxy measure of the evolutionary process that underpins carcinogenesis in Barrett's Esophagus. In our cohort, measures of clonal diversity based on single-cell genetics predict progression to highgrade dysplasia or adenocarcinoma. Our data support the hypothesis that Barrett's segments with higher levels of genetic diversity likely indicate more evolvable lesions. Table 1. Hazard ratios for the risk of progression among patients with non-dysplastic Barrett's oesophagus.
Figure 1: Composite Score Distribution of MT-sDNA Test Markers
Figure 2: SFMBT-2896 Levels
Sa1921 Molecular Detection of Colorectal Neoplasia: Do Markers That Target Acquired DNA Alterations in Sporadic Cases Also Discriminate Lynch Syndrome Cases? Veroushka Ballester, Maria Giakoumopoulos, Tracy C. Yab, William R. Taylor, Patrick Foote, Mary E. Devens, Douglas W. Mahoney, Lisa A. Boardman, John B. Kisiel, Hatim T. Allawi, Graham P. Lidgard, Marcia R. Cruz-Correa, David A. Ahlquist
Sa1922 miRNA Expression Signatures of Colorectal Adenoma-Carcinoma Sequence in Tissue and Matched Plasma Specimens Zsofia B. Nagy, Alexandra Kalmár, Barnabás Wichmann, Barbara Bartak, Béla Molnár, Zsolt Tulassay
Background: A multi-target stool DNA test (MT-sDNA) has recently been validated in a multicenter study (N Engl J Med 2014;370:1287), approved by the U.S. Food & Drug Administration for average risk colorectal cancer screening, and become clinically available (CologuardTM, Exact Sciences, Madison WI). It is not known if molecular markers in MTsDNA or other markers optimized for detection of sporadic colorectal neoplasia (CRN) will
BACKGROUND: MicroRNA has been found to play critical role in the colorectal cancer (CRC) development. Tissue specific miRNA changes could be recovered in plasma samples in CRC. However, relevant microRNA expression alterations have not been identified in the adenoma stages, yet. AIMS: The purpose of this study was to identify miRNA expression
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AGA Abstracts
AGA Abstracts
study the role of IRS-4 on proliferation and the cell cycle, an expression vector of pcDNA3IRS-4 was constructed. RKO cells were transfected i) with the recombinant plasmid pcDNA3IRS-4 expressing the protein, or ii) with the empty plasmid pcDNA3 as a control. The impact of IRS-4 overexpression was analyzed on cell cycle and IGF-1 signaling. Sixteen colon tumor samples and paired tumor-adjacent normal tissue were collected during surgery and processed after obtaining informed consent. We evaluated the relationship between IRS-4 level and the proteins expression involved in G1/S checkpoint in the tumor and paired normal of all the reported cases by western blot. RESULTS: As we expected, RKO cells overexpressing IRS-4 showed higher levels of this protein than the control cells. The high levels of IRS-4 caused an increase of pRb, Rb, E2F, Cyclin E and Cyclin D1 expression in RKO cells. IRS-4, pRb, Rb and E2F were highly expressed in the cancerous tissue, in comparison with the normal epithelium. We evaluated the correlations between IRS-4 expression and the level of proteins involved in the control of cell cycle. The levels of IRS4 correlated positively with pRb (rpearson=0.745, P value <0.0001), Rb (rpearson=0.810, P value <0.0001) and E2F (rpearson=0.856, P value <0.0001). Regarding to the IGF-1 pathway, the levels of IRS-4 showed a positive correlation with pIGF-1R (rpearson=0.657, P value <0.0001), pAKT (rpearson=0.537, P value <0.01), AKT (rpearson=0.520, P value <0.01) and GSK-3 (rpearson=0.911, P value <0.0001) expression. Our general finding showed that IRS4 stimulates the expression of the proteins that unlock the G1/S restriction point in the cell cycle in RKO cells. Interestingly, the proteins involved in the cell cycle increased especially in the cancerous tissue of the patients overexpressing IRS-4. CONCLUSION: Our results suggest that IRS-4 is an important protein involved in the cell cycle regulation in colon cancer cells and it could be a key protein in the CRC development.
AGA Abstracts
patterns between normal colonic tissue (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and colorectal cancer (CRC) biopsy tissue samples. Furthermore, our aim was to analyze and confirm the expression level of adenoma and CRC related miRNAs in matched plasma samples. METHODS: Sixty fresh frozen colorectal biopsy (n=20 N, n=11 ADT, n= 9 ADTV, n=20 CRC) and 16 matched plasma samples (n=4 N, n=4 ADT, n=4 ADTV, n=4 CRC) were collected and total RNA including miRNA was isolated (High Pure miRNA Isolation Kit, Roche,Germany). MiRNA expression analysis was performed on GeneChip® miRNA 3.0 Arrays (Affymetrix,USA). Then, the results of microarray analysis were confirmed on RT-qPCR arrays (microRNA Ready-to-use PCR Human Panel I+II V2.R; Exiqon, Denmark). The matched plasma samples were also evaluated by the same miRNA expression and RTqPCR arrays. The obtained results were analyzed by Expression Console (Affymetrix,USA). RESULTS: Out of the 1733 detectable number of miRNAs on the array, in normal biopsies 442, in adenomas 460, and in CRC 441 miRNA could be identified. In plasma these were 306, 334, and 321 miRNA, respectively. Interestingly, 12 miRNAs were upregulated (e.g. miR-31 logFC=3, p<0,001) and 11 miRNA were downregulated only (e.g. miR-10b, logFC=1.7 p<0.001) in neoplastic lesions (AD+CRC) compared to N tissue samples. 11 miRNAs showed altered expression between ADT and ADTV (e.g. miR-183 LogFC=1.5 p<0.007). Expression levels of 9 miRNAs were found to be changed between ADT,TV and CRC groups based on tissue biopsy microarray data (e.g. miR-196a logFC=-1.8 p<0.001). Only three miRNA(-31;-4506;-452*) have been found to be differentially expressed in adenoma compared to normal. Significant positive overexpression was observed in tissue (logFC=5 p= 0.003) and in plasma (logFC=0.3 p=0.02) in case of miR-31 in the adenoma cases. Moreover, increased expressions were detected in CRC compared to healthy controls in tissue and plasma levels of miR-187;-675;-3591-3p (p<0.05). The observed miRNA expression changes could be confirmed by RT-qPCR arrays in both plasma and tissue samples. CONCLUSION: A small number (n=23) of miRNA showed characteristic alterations during neoplastic development in biopsy tissue. Our observations suggest that miRNA are also present in the plasma fraction and their expression is positively correlated with matched tissue expression levels. The identified miRNA expression changes are consistent by different methods through the adenoma-dysplasia-carcinoma sequence.
Figure: ROC for the performance of ML in predicting progression at index time points for each patient. AUC = 0.95 (95% CI: 0.89-0.99). ML = mutational load. Sa1924 Androgen Receptor Pathway As a Prognostic Indicator in Esophageal Adenocarcinoma Eric Smith, Helen M. Palethorpe, Andrew Ruszkiewicz, Damien Leach, Eleanor Need, Paul Drew Background: Esophageal adenocarcinoma (EAC) is a male dominant disease. The role of male sex steroid hormones (androgens) in the biology of EAC is unknown. Androgens activate the androgen receptor (AR), thereby altering the expression of AR-responsive genes, eg. FK506 binding protein 5 (FKBP5), cyclin B1 (CCNB1) and vascular endothelial growth factor A (VEGFA). The role of androgen signalling mediated by AR is unknown in this cancer. Methods: Immunohistochemistry for AR and FKBP5 was performed on 77 cases of EAC. Expression of AR and FKBP5 in cell lines was determined by Western blot, and functional AR by transactivation assays. The AR-negative EAC cell line OE33 was stably transduced with AR (OE33-AR). Expression of FKBP5, CCNB1, VEGFA, E2F transcription factor 1 (E2F1) and cyclin D1 (CCND1) was measured by qRT-PCR in cell lines treated with 0 nM or 10 nM of the androgen dihydrotestosterone (DHT). Results: AR staining was observed in 75 of 77 cases of EAC (97%). Staining was both nuclear and cytoplasmic in 63 (82%) cases, nuclear only in seven (9%), and cytoplasmic only in five (6%). FKBP5 staining was observed in 49 cases (64%), and all of these also had nuclear localisation of AR. Of the 28 cases that did not express FKBP5, 21 had nuclear localisation of AR and 7 did not. There was a significant association between FKBP5 expression and AR nuclear localisation (p=0.0005). Clinicopathological data were available for 76 cases. FKBP5 expression was associated with decreased median overall survival (451 vs 1338 days) and 5-year survival (32 vs 44%). By multivariable Cox Proportional Hazard Models analysis, FKBP5 expression (HR 3.043, 95% CI 1.417-6.531), T- and N-stage, but not patient age nor the presence of Barrett's esophagus were associated with decreased survival. Functional AR expression was not detected in the EAC cell lines OE33, OE19, JH-EsoAd1 or FLO-1. DHT induced a time-dependent increase in FKBP5 expression in OE33-AR, but not in the ARnegative EAC cell lines. DHT induced a dose-dependent inhibition of cell proliferation of OE33-AR, but not OE33. This inhibition of cell proliferation was associated with an increased number of cells in the G0/G1 phase of the cell cycle, reduced expression of CCNB1 (p<0.0001) and E2F1 (p<0.0001), and increased expression of cyclin D1 (p=0.0006). There was no significant difference in p16 expression. DHT inhibited cell migration of OE33-AR. Expression of VEGFA, a potent angiogenic factor which enhances metastasis, was increased 3-fold in response to DHT in OE33-AR (p<0.0001), but not in OE33. Conclusions: AR was expressed frequently in EAC, and expression of the AR-responsive gene FKBP5 was associated with decreased patient survival. These data suggest that androgen receptor mediated signalling may play a significant role in the biology of EAC, and have implications for new therapeutic interventions.
Sa1923 Genetic Mutations at Key Loci Predict Progression to High-grade Dysplasia or Esophageal Adenocarcinoma in Barrett's Esophagus Swathi Eluri, William R. Brugge, Ebubekir S. Daglilar, Sara A. Jackson, Mindi A. Styn, Keith M. Callenberg, Derek C. Welch, Todd M. Barr, Lucas C. Duits, Jacques J. Bergman, Nicholas J. Shaheen Background: Dysplasia surveillance in Barrett's esophagus (BE) is a challenging and costly problem. Current methods based on histological classification are inaccurate. We assessed a panel of genetic markers to determine its value in risk stratification for the development of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). Methods: We conducted a case-control study to measure genetic instability, assessed by the mutational load (ML), in pre-progression BE tissue as a marker to predict progression to HGD or EAC. Patients with BE were from 3 sites (Massachusetts General Hospital, Allegheny General Hospital, and PathGroup). Cases were BE patients with no dysplasia or low-grade dysplasia (LGD) at baseline, and who had HGD or EAC on a follow-up biopsy done at least 1 year later. Controls were matched 2:1, and had non-dysplastic BE or LGD at baseline and no progression of BE at follow-up. Formalin-fixed, paraffin-embedded tissue was microdissected for epithelium. The presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) was investigated using polymerase chain reaction and quantitative capillary electrophoresis of DNA extracted from each microdissected target. ML was calculated from genetic derangements in 10 genomic loci: 1p (CMM1, L-myc), 3p (VHL, HoGG1), 5q (MCC, APC), 9p (CDKN2A), 10q (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q (TFF1, PSEN2) and 22q (NF2). High clonality LOH mutations were assigned a value of 1, low clonality mutations assigned a value of 0.5, and MSI assigned a value of 0.75 for the first loci and 0.5 for all additional loci. These values were summed to constitute the ML. Receiver operator characteristic (ROC) curves were created to test the ability of ML at different thresholds to predict progression. Results: Of the 69 patients, there were 46 non-progressors and 23 progressors. The groups were similar in age, follow-up time, and mean number of microdissected targets (Table). Both groups had a similar proportion of nondysplastic and LGD histology at index biopsy. The mean per-patient ML in the pre-progression biopsies was significantly higher in progressors (mean ML=2.21) compared to non-progressors (mean ML = 0.42, p<0.0001). The test was 100% sensitive in discriminating cases from controls at an ML cutoff ≥ 0.5 and 96% specific at ML cutoff ≥ 1.5. Accuracy of the test was highest at 89.9% (95% CI: 80.2-95.8) at ML ≥ 1. ROC curves were constructed with varying ML thresholds (Figure), and the corresponding AUC was 0.946 (95% CI 0.894-0.999). Conclusion: The mutational load in pre-progression tissue of BE patients with no dysplasia or LGD, predicts progression to HGD or EAC. ML may have utility as a clinical biomarker in endoscopic surveillance of BE patients to predict progression to HGD and EAC approximately 3-4 years prior to the histological onset of HGD and EAC. Table: Description of Barrett's esophagus non-progressor and progressor patients included in the study
AGA Abstracts
Sa1925 Cross-Talk Between Gamma-Aminobutyric Acid and Prostaglandin E2 in Colon Carcinogenesis Grace O'Callaghan, Philana Fernandes, Fergus Shanahan, Niall P. Hyland, Aileen Houston Introduction: Colorectal cancer is the third leading cause of cancer-related death worldwide. The role of cyclooxygenase-2 (COX-2) and its major metabolite prostaglandin E2 (PGE2) in colon carcinogenesis is well established. Recent studies suggest that gamma-aminobutyric acid (GABA) may have potential tumor suppressive effects. Whether GABA exerts a direct anti-tumorigenic effect on colonic tumors is unclear. Aim: To investigate the role of GABA in colon carcinogenesis, and to determine if it alters the tumor-promoting properties of PGE2. Methods: SW480 and CT26 colon cancer cells were treated with GABA (500nM 100μM), Cell migration and invasion towards 10% serum were investigated using transwell assays (8μM pores). SW480, HCT116 and HT29 colon cancer cells were treated with GABA(100mM), PGE2 (1μM) or were co-treated with both GABA and PGE2. 24 hrs later, cell proliferation was measured by resazurin reduction. Changes in IL-6 expression were assessed by real-time RT-PCR. Results: GABA reduced the proliferation of both the SW480 and CT26 colon tumor cells in a concentration-dependant manner (p < 0.001), and significantly reduced the proliferation of HT29 and HCT116 cells (p < 0.01). GABA also significantly reduced tumor cell migration by 50% and reduced invasion by 20%. PGE2 induced cell proliferation, migration and invasion (p<0.01), as well as increasing IL-6 and IL-8 production
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Sa1920 Clonal Diversity Based on Single-Cell Analysis Predicts Progression in Barrett's Esophagus Margriet R. Timmer, Pierre Martinez, Chiu T. Lau, Paul Fockens, Jacques J. Bergman, Carlo C. Maley, Trevor A. Graham, Kausilia K. Krishnadath Background: Barrett's esophagus (BE) is associated with a low annual risk of developing esophageal adenocarcinoma (EAC). Predictive biomarkers would be of great clinical value in facilitating more cost-effective surveillance. Here we attempted to evaluate the predictive value of clonal diversity in a large prospective cohort of Barrett's patients without dysplasia at baseline. Methods: We have performed single-cell genetic analysis on brush cytology specimens. Genetic abnormalities were detected by fluorescence in situ hybridization using two probe sets including probes for CEP17, Her-2, P53 and P16 (Set 1) and 20q, MYC and the chromosomal centromeric probes 7 and 17 (Set 2). For each individual cell, a ‘per cell' signal pattern was recorded for each set to provide detailed information on genetically different clones. Clones were defined as the collection of cells with the same genotype. We then aimed to risk stratify patients according to measures of clonal diversity (e.g. richness, Simpson diversity, Shannon diversity and average pairwise distance) using Cox proportionalhazard models. Endpoints were progression to high-grade dysplasia (HGD) or EAC. Thresholds for each variable (e.g. a threshold to distinguish normal from abnormal) were determined by a bootstrap-resampling procedure. Results: A total of 320 patients (81% men; mean age 59 years ±12; median BE length 2 cm) were followed for a median of 43 (95% CI 40-46) months during which 20 patients progressed to HGD (n=8) or EAC (n=12). Only 30 patients displayed a single genotype throughout their respective cell population in set 1, and 107 in set 2 (9.4 % and 33.4%, respectively). The diversity obtained from set 1 was significantly higher than the one obtained from set 2 (p=0.0001, paired t-test), owing to the prominence of P16 and P53 abnormalities which were assayed in set 1. All measures of clonal diversity, except for the Simpson diversity (set 1), were significantly associated with progression (see Table 1). Conclusion: Quantification of genetic diversity can be considered a proxy measure of the evolutionary process that underpins carcinogenesis in Barrett's Esophagus. In our cohort, measures of clonal diversity based on single-cell genetics predict progression to highgrade dysplasia or adenocarcinoma. Our data support the hypothesis that Barrett's segments with higher levels of genetic diversity likely indicate more evolvable lesions. Table 1. Hazard ratios for the risk of progression among patients with non-dysplastic Barrett's oesophagus.
Figure 1: Composite Score Distribution of MT-sDNA Test Markers
Figure 2: SFMBT-2896 Levels
Sa1921 Molecular Detection of Colorectal Neoplasia: Do Markers That Target Acquired DNA Alterations in Sporadic Cases Also Discriminate Lynch Syndrome Cases? Veroushka Ballester, Maria Giakoumopoulos, Tracy C. Yab, William R. Taylor, Patrick Foote, Mary E. Devens, Douglas W. Mahoney, Lisa A. Boardman, John B. Kisiel, Hatim T. Allawi, Graham P. Lidgard, Marcia R. Cruz-Correa, David A. Ahlquist
Sa1922 miRNA Expression Signatures of Colorectal Adenoma-Carcinoma Sequence in Tissue and Matched Plasma Specimens Zsofia B. Nagy, Alexandra Kalmár, Barnabás Wichmann, Barbara Bartak, Béla Molnár, Zsolt Tulassay
Background: A multi-target stool DNA test (MT-sDNA) has recently been validated in a multicenter study (N Engl J Med 2014;370:1287), approved by the U.S. Food & Drug Administration for average risk colorectal cancer screening, and become clinically available (CologuardTM, Exact Sciences, Madison WI). It is not known if molecular markers in MTsDNA or other markers optimized for detection of sporadic colorectal neoplasia (CRN) will
BACKGROUND: MicroRNA has been found to play critical role in the colorectal cancer (CRC) development. Tissue specific miRNA changes could be recovered in plasma samples in CRC. However, relevant microRNA expression alterations have not been identified in the adenoma stages, yet. AIMS: The purpose of this study was to identify miRNA expression
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AGA Abstracts
AGA Abstracts
study the role of IRS-4 on proliferation and the cell cycle, an expression vector of pcDNA3IRS-4 was constructed. RKO cells were transfected i) with the recombinant plasmid pcDNA3IRS-4 expressing the protein, or ii) with the empty plasmid pcDNA3 as a control. The impact of IRS-4 overexpression was analyzed on cell cycle and IGF-1 signaling. Sixteen colon tumor samples and paired tumor-adjacent normal tissue were collected during surgery and processed after obtaining informed consent. We evaluated the relationship between IRS-4 level and the proteins expression involved in G1/S checkpoint in the tumor and paired normal of all the reported cases by western blot. RESULTS: As we expected, RKO cells overexpressing IRS-4 showed higher levels of this protein than the control cells. The high levels of IRS-4 caused an increase of pRb, Rb, E2F, Cyclin E and Cyclin D1 expression in RKO cells. IRS-4, pRb, Rb and E2F were highly expressed in the cancerous tissue, in comparison with the normal epithelium. We evaluated the correlations between IRS-4 expression and the level of proteins involved in the control of cell cycle. The levels of IRS4 correlated positively with pRb (rpearson=0.745, P value <0.0001), Rb (rpearson=0.810, P value <0.0001) and E2F (rpearson=0.856, P value <0.0001). Regarding to the IGF-1 pathway, the levels of IRS-4 showed a positive correlation with pIGF-1R (rpearson=0.657, P value <0.0001), pAKT (rpearson=0.537, P value <0.01), AKT (rpearson=0.520, P value <0.01) and GSK-3 (rpearson=0.911, P value <0.0001) expression. Our general finding showed that IRS4 stimulates the expression of the proteins that unlock the G1/S restriction point in the cell cycle in RKO cells. Interestingly, the proteins involved in the cell cycle increased especially in the cancerous tissue of the patients overexpressing IRS-4. CONCLUSION: Our results suggest that IRS-4 is an important protein involved in the cell cycle regulation in colon cancer cells and it could be a key protein in the CRC development.
AGA Abstracts
patterns between normal colonic tissue (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and colorectal cancer (CRC) biopsy tissue samples. Furthermore, our aim was to analyze and confirm the expression level of adenoma and CRC related miRNAs in matched plasma samples. METHODS: Sixty fresh frozen colorectal biopsy (n=20 N, n=11 ADT, n= 9 ADTV, n=20 CRC) and 16 matched plasma samples (n=4 N, n=4 ADT, n=4 ADTV, n=4 CRC) were collected and total RNA including miRNA was isolated (High Pure miRNA Isolation Kit, Roche,Germany). MiRNA expression analysis was performed on GeneChip® miRNA 3.0 Arrays (Affymetrix,USA). Then, the results of microarray analysis were confirmed on RT-qPCR arrays (microRNA Ready-to-use PCR Human Panel I+II V2.R; Exiqon, Denmark). The matched plasma samples were also evaluated by the same miRNA expression and RTqPCR arrays. The obtained results were analyzed by Expression Console (Affymetrix,USA). RESULTS: Out of the 1733 detectable number of miRNAs on the array, in normal biopsies 442, in adenomas 460, and in CRC 441 miRNA could be identified. In plasma these were 306, 334, and 321 miRNA, respectively. Interestingly, 12 miRNAs were upregulated (e.g. miR-31 logFC=3, p<0,001) and 11 miRNA were downregulated only (e.g. miR-10b, logFC=1.7 p<0.001) in neoplastic lesions (AD+CRC) compared to N tissue samples. 11 miRNAs showed altered expression between ADT and ADTV (e.g. miR-183 LogFC=1.5 p<0.007). Expression levels of 9 miRNAs were found to be changed between ADT,TV and CRC groups based on tissue biopsy microarray data (e.g. miR-196a logFC=-1.8 p<0.001). Only three miRNA(-31;-4506;-452*) have been found to be differentially expressed in adenoma compared to normal. Significant positive overexpression was observed in tissue (logFC=5 p= 0.003) and in plasma (logFC=0.3 p=0.02) in case of miR-31 in the adenoma cases. Moreover, increased expressions were detected in CRC compared to healthy controls in tissue and plasma levels of miR-187;-675;-3591-3p (p<0.05). The observed miRNA expression changes could be confirmed by RT-qPCR arrays in both plasma and tissue samples. CONCLUSION: A small number (n=23) of miRNA showed characteristic alterations during neoplastic development in biopsy tissue. Our observations suggest that miRNA are also present in the plasma fraction and their expression is positively correlated with matched tissue expression levels. The identified miRNA expression changes are consistent by different methods through the adenoma-dysplasia-carcinoma sequence.
Figure: ROC for the performance of ML in predicting progression at index time points for each patient. AUC = 0.95 (95% CI: 0.89-0.99). ML = mutational load. Sa1924 Androgen Receptor Pathway As a Prognostic Indicator in Esophageal Adenocarcinoma Eric Smith, Helen M. Palethorpe, Andrew Ruszkiewicz, Damien Leach, Eleanor Need, Paul Drew Background: Esophageal adenocarcinoma (EAC) is a male dominant disease. The role of male sex steroid hormones (androgens) in the biology of EAC is unknown. Androgens activate the androgen receptor (AR), thereby altering the expression of AR-responsive genes, eg. FK506 binding protein 5 (FKBP5), cyclin B1 (CCNB1) and vascular endothelial growth factor A (VEGFA). The role of androgen signalling mediated by AR is unknown in this cancer. Methods: Immunohistochemistry for AR and FKBP5 was performed on 77 cases of EAC. Expression of AR and FKBP5 in cell lines was determined by Western blot, and functional AR by transactivation assays. The AR-negative EAC cell line OE33 was stably transduced with AR (OE33-AR). Expression of FKBP5, CCNB1, VEGFA, E2F transcription factor 1 (E2F1) and cyclin D1 (CCND1) was measured by qRT-PCR in cell lines treated with 0 nM or 10 nM of the androgen dihydrotestosterone (DHT). Results: AR staining was observed in 75 of 77 cases of EAC (97%). Staining was both nuclear and cytoplasmic in 63 (82%) cases, nuclear only in seven (9%), and cytoplasmic only in five (6%). FKBP5 staining was observed in 49 cases (64%), and all of these also had nuclear localisation of AR. Of the 28 cases that did not express FKBP5, 21 had nuclear localisation of AR and 7 did not. There was a significant association between FKBP5 expression and AR nuclear localisation (p=0.0005). Clinicopathological data were available for 76 cases. FKBP5 expression was associated with decreased median overall survival (451 vs 1338 days) and 5-year survival (32 vs 44%). By multivariable Cox Proportional Hazard Models analysis, FKBP5 expression (HR 3.043, 95% CI 1.417-6.531), T- and N-stage, but not patient age nor the presence of Barrett's esophagus were associated with decreased survival. Functional AR expression was not detected in the EAC cell lines OE33, OE19, JH-EsoAd1 or FLO-1. DHT induced a time-dependent increase in FKBP5 expression in OE33-AR, but not in the ARnegative EAC cell lines. DHT induced a dose-dependent inhibition of cell proliferation of OE33-AR, but not OE33. This inhibition of cell proliferation was associated with an increased number of cells in the G0/G1 phase of the cell cycle, reduced expression of CCNB1 (p<0.0001) and E2F1 (p<0.0001), and increased expression of cyclin D1 (p=0.0006). There was no significant difference in p16 expression. DHT inhibited cell migration of OE33-AR. Expression of VEGFA, a potent angiogenic factor which enhances metastasis, was increased 3-fold in response to DHT in OE33-AR (p<0.0001), but not in OE33. Conclusions: AR was expressed frequently in EAC, and expression of the AR-responsive gene FKBP5 was associated with decreased patient survival. These data suggest that androgen receptor mediated signalling may play a significant role in the biology of EAC, and have implications for new therapeutic interventions.
Sa1923 Genetic Mutations at Key Loci Predict Progression to High-grade Dysplasia or Esophageal Adenocarcinoma in Barrett's Esophagus Swathi Eluri, William R. Brugge, Ebubekir S. Daglilar, Sara A. Jackson, Mindi A. Styn, Keith M. Callenberg, Derek C. Welch, Todd M. Barr, Lucas C. Duits, Jacques J. Bergman, Nicholas J. Shaheen Background: Dysplasia surveillance in Barrett's esophagus (BE) is a challenging and costly problem. Current methods based on histological classification are inaccurate. We assessed a panel of genetic markers to determine its value in risk stratification for the development of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). Methods: We conducted a case-control study to measure genetic instability, assessed by the mutational load (ML), in pre-progression BE tissue as a marker to predict progression to HGD or EAC. Patients with BE were from 3 sites (Massachusetts General Hospital, Allegheny General Hospital, and PathGroup). Cases were BE patients with no dysplasia or low-grade dysplasia (LGD) at baseline, and who had HGD or EAC on a follow-up biopsy done at least 1 year later. Controls were matched 2:1, and had non-dysplastic BE or LGD at baseline and no progression of BE at follow-up. Formalin-fixed, paraffin-embedded tissue was microdissected for epithelium. The presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) was investigated using polymerase chain reaction and quantitative capillary electrophoresis of DNA extracted from each microdissected target. ML was calculated from genetic derangements in 10 genomic loci: 1p (CMM1, L-myc), 3p (VHL, HoGG1), 5q (MCC, APC), 9p (CDKN2A), 10q (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q (TFF1, PSEN2) and 22q (NF2). High clonality LOH mutations were assigned a value of 1, low clonality mutations assigned a value of 0.5, and MSI assigned a value of 0.75 for the first loci and 0.5 for all additional loci. These values were summed to constitute the ML. Receiver operator characteristic (ROC) curves were created to test the ability of ML at different thresholds to predict progression. Results: Of the 69 patients, there were 46 non-progressors and 23 progressors. The groups were similar in age, follow-up time, and mean number of microdissected targets (Table). Both groups had a similar proportion of nondysplastic and LGD histology at index biopsy. The mean per-patient ML in the pre-progression biopsies was significantly higher in progressors (mean ML=2.21) compared to non-progressors (mean ML = 0.42, p<0.0001). The test was 100% sensitive in discriminating cases from controls at an ML cutoff ≥ 0.5 and 96% specific at ML cutoff ≥ 1.5. Accuracy of the test was highest at 89.9% (95% CI: 80.2-95.8) at ML ≥ 1. ROC curves were constructed with varying ML thresholds (Figure), and the corresponding AUC was 0.946 (95% CI 0.894-0.999). Conclusion: The mutational load in pre-progression tissue of BE patients with no dysplasia or LGD, predicts progression to HGD or EAC. ML may have utility as a clinical biomarker in endoscopic surveillance of BE patients to predict progression to HGD and EAC approximately 3-4 years prior to the histological onset of HGD and EAC. Table: Description of Barrett's esophagus non-progressor and progressor patients included in the study
AGA Abstracts
Sa1925 Cross-Talk Between Gamma-Aminobutyric Acid and Prostaglandin E2 in Colon Carcinogenesis Grace O'Callaghan, Philana Fernandes, Fergus Shanahan, Niall P. Hyland, Aileen Houston Introduction: Colorectal cancer is the third leading cause of cancer-related death worldwide. The role of cyclooxygenase-2 (COX-2) and its major metabolite prostaglandin E2 (PGE2) in colon carcinogenesis is well established. Recent studies suggest that gamma-aminobutyric acid (GABA) may have potential tumor suppressive effects. Whether GABA exerts a direct anti-tumorigenic effect on colonic tumors is unclear. Aim: To investigate the role of GABA in colon carcinogenesis, and to determine if it alters the tumor-promoting properties of PGE2. Methods: SW480 and CT26 colon cancer cells were treated with GABA (500nM 100μM), Cell migration and invasion towards 10% serum were investigated using transwell assays (8μM pores). SW480, HCT116 and HT29 colon cancer cells were treated with GABA(100mM), PGE2 (1μM) or were co-treated with both GABA and PGE2. 24 hrs later, cell proliferation was measured by resazurin reduction. Changes in IL-6 expression were assessed by real-time RT-PCR. Results: GABA reduced the proliferation of both the SW480 and CT26 colon tumor cells in a concentration-dependant manner (p < 0.001), and significantly reduced the proliferation of HT29 and HCT116 cells (p < 0.01). GABA also significantly reduced tumor cell migration by 50% and reduced invasion by 20%. PGE2 induced cell proliferation, migration and invasion (p<0.01), as well as increasing IL-6 and IL-8 production
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