- Email: [email protected]
S1958 Expression and Localization of Calreticulin in Human Colorectal Polyps
at colonoscopy were examined. Tissues were first investigated biochemically with 2D electrophoresis and mass spectrometry. They were also investigated histologically and ultrastructurally. Calreticulin expression was finally analysed immunohistochemically, using peroxidase anti-peroxidase (PAP) method and immunogold labelling method, for light and electron microscope respectively. All polyp tissues examined were adenoma of low, mild and highgrade dysplasia as showed the histopathological reports. Results: Using 2D-electrophoresis and mass spectrometry analysis, we detected calreticulin in cytoplasmic fraction which was differentially expressed in the analysed samples. The statistical analysis showed a significant correlation (non-paired Student t-test) between calreticulin expression and colon lesions progression. The investigation of the respective nuclear matrix fraction showed that calreticulin was absent in the majority of the samples. The examination of the above specimens with electron microscope revealed that a proportion of adenoma tissues of mild dysplasia had more prominent subcellular changes. Calreticulin immunoreactivity was noted in all adenomas connected of the degree of dysplastic change with the lowest relative levels occurring in low-grade adenoma and the highest levels found in high-grade adenoma. Calreticulin was located not only in rough endoplasmic reticulum of adenoma cells but also in other membranes of cytoplasmic organelles. In low grade dysplasia and mild grade dysplasia whithout significant subcellular changes calreticulin was also localized in nucleus. Conclusion: Calreticulin overexpression in high grade dysplasia samples and in mild grade dysplasia samples with significant ultrastructural changes as well as its specific cellular localization could provide useful information for the preneoplastic transformation in adenoma tissues.
Expression of S100 Proteins May Help Screen for the Transition of Chronic Inflammation to Dysplasia Jose A. Serrano, Edelmarie Rivera, Angel A. Isidro, Beatriz Pagan, Richard J. Noel, Virgilio Salvo, Caroline B. Appleyard Patients with Inflammatory Bowel Disease (IBD) are at increased risk of developing colorectal cancer. The use of biomarkers to aid in the early detection and diagnosis of colorectal cancer is of interest due to their potential to improve treatment preventing further progression. Calprotectin (S100A8/S100A9) has been identified previously as a potential marker for colorectal cancer, but few studies have looked at its possible role in secondary prevention. Aim: To evaluate the potential for calprotectin in screening for, and early detection of, the transition from inflammation to dysplasia. Methods: Chronic colitis was induced in male Sprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for three days. To induce colitis-associated dysplasia the rats then received TNBS (iv) twice a week for ten weeks. A group of age-matched normal untreated animals were also included as controls. After sacrifice the colons were removed and analyzed for damage, pathology, and expression of S100A8 and S100A9 were measured by real time RT-PCR. Tissue distribution of the S100 proteins was measured by immunohistochemistry. Results: Administration of TNBS caused ulceration and damage as expected, which correlated with the pathological diagnosis. Animals diagnosed with dysplasia had the highest macroscopic damage (p<0.05 vs. normal). Using the ΔΔCt method the fold expression of S100A8 was found to be upregulated and similar in both IBD and dysplasia compared to non specific inflammation (NSI) or untreated, whereas expression of S100A9 was more than 2-fold higher in dysplasia than in IBD. The IHC score for S100A8 showed the highest intensity of staining among the samples diagnosed with IBD and low grade dysplasia. In each case this was significantly higher than the staining intensity found in areas of NSI or high grade dysplasia. When the protein expression for the heterodimer S100A8 & S100A9 was examined the intensity of staining in areas of low grade dysplasia was high, with no difference in the intensity of staining between the other diagnoses. These all showed comparable but lower intensity values compared to low grade dysplasia. In each case there appeared to be a shift from weaker staining in NSI to increased staining during the transition to dysplasia. Interestingly, as the pathology worsens the expression once again decreases. Conclusion:There is a potential role for S100A8 and S100A9 in secondary prevention via early disease detection of low grade dysplasia or active IBD. Supported in by 1U56 CA126379, RR003050 & 1F31DK077584
S1959 Identification and Validation of New Biomarkers for Colorectal Cancer Using Isolated Epithelial Stem Cells David Harrison, S. Suchitha, Ansamma Joseph, Asit Panja BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States (US) and is the most common malignancy in the US. Although, it has been known for many decades that the transformation of the epithelial cells lining the colon and/or rectum are the cause of CRC, the actual causative molecular mechanism of CRC remains unknown. Furthermore, no specific biomarker currently exists to aid in the early diagnosis or therapeutic treatment of CRC. AIM: The aim of this study was to identify and validate cell and disease state specific biomarkers for CRC. METHODS: Adult human gastrointestinal stem cells were isolated and cultured concurrently from both the actual cancerous and normal/unaffected colonic tissue of the same individual suffering from CRC. Isolated total RNA from these stem cells were subjected to gene expression microarray analysis. Potential molecular biomarkers/targets associated with cancerous transformation of the epithelial stem cells in the cancerous tissues compared to its normal counterpart were identified. Gene array data was validated at the mRNA expression level by RT-PCR analysis and at the protein expression level by immunocytochemical, flow cytometric, and Western blot analysis. The corresponding identified molecules were further validated by immunohistochemical analysis of paired (normal and cancer) paraffin block tissue sections. RESULTS: Our data from comparative gene array analysis of normal vs. CRC stem cells (positive for Nanog, Oct4, SOX2, LIN28, and Bmi-1) identified 160 dysregulated genes. Many of the identified gene products were shown for the first time to be potential biomarkers for CRC. Several of these identified potential biomarkers were further analyzed, including: proprotein convertase subtilisin/kexin type 5 (PCSK5) and chemokine ligand 20 (BST2 or CCL20) which were shown to be dramatically upregulated in CRC, as well as, superoxide dismutase 3 (SOD3) which demonstrated a 482 fold decrease in expression in CRC derived stem cells. CONCLUSION: Our results demonstrated that paired stem cells isolated concurrently from both the cancerous/affected and normal/unaffected colonic tissue of an individual suffering CRC and the differentiation and culturing of epithelial cell lines from these stem cells provides a unique and superior system for the identification of specific biomarkers and potential therapeutic targets for CRC.
S1957 Potential Role of Biological Ageing in Colorectal Cancer: How Many Miles on the Clock? Fraser Maxwell, Liane McGlynn, Campbell S. Roxburgh, Donald C. McMillan, Paul G. Horgan, Paul Shiels Introduction Ageing is a complex multi-factorial process of which the fundamental determinants remain to be fully elucidated. A key manifestation of biological ageing at the cellular level is telomere attrition. Telomeres are nucleo-protein complexes found at the ends of eukaryotic chromosomes and play a critical role in the maintenance of genomic integrity. They undergo progressive shortening with each round of cell division and, therefore act as a mitotic clock. Telomere attrition and hence dysfunction can induce genomic instability and cellular senescence, which can in turn cause cancer predisposition. Factors which accelerate telomere attrition and thus put ‘miles on the clock' include inflammation and oxidative stress. An association between telomere length and cancer however has resulted in many equivocal results in the field. We have investigated whether patients with colorectal cancer display evidence of accelerated telomere attrition. Methods After full ethical approval and informed consent, DNA was extracted from peripheral blood leucocytes of patients with a diagnosis of colorectal cancer using routine methods. Telomere lengths were determined by quantitative-PCR using the method of Cawthon (2002) and correlated with common clinic-pathological characteristics, markers of inflammation and antioxidant status. Statistical analysis was performed using SPSS version 15.0. Results Data were available for 64 patients in the CRC and 59 in the control group. Mean T/S ratio (telomere length) was 0.64 (±0.17) in the CRC group and 0.82 (±0.22) in the control group (p=0.000). There was a significant negative correlation between age and telomere length (rho=-.279, p<0.05) in patients with CRC. Patients in the quartile with the shortest telomere length had a significantly higher neutrophil to lymphocyte ratio (NLR) (p<0.05). Furthermore, there was a significant association between short telomere length and high risk pathological features (Peterson Index). Conclusion We have demonstrated that patients with CRC display clear evidence of accelerated biological ageing. The observed association between telomere length and NLR may implicate inflammation in this process. In addition, telomere length may prove a useful marker of disease severity.
S1960 Clinical Significance of HER2/Neu Overexpression in Gastric Cancer Paola Figueroa Barojas, Zarate Osorno Alejandra, Enoe E. Quiñonez-Urrego, Angelica Hernandez-Guerrero, Sergio R. Sobrino-Cossio, Juan Octavio Alonso-Larraga Gastric cancer is the 2nd most common worldwide, causing 3-10% of cancer related deaths. 95% of gastric tumors are adenocarcinoma, 65% present at an advanced stage, and up to 85% have lymph node metastasis at diagnosis. Studies have pointed to an association between gene/protein expression and higher rates of invasion, lymph node metastasis and/or advanced stage. The amplification of the C-erb2 gene and the overexpression of its protein (HER2/ neu) has been implicated as a poor prognostic factor, both have been extensively studied in breast cancer. In gastric cancer, immunohistochemical studies show a frequency of overexpression of HER2/neu of 8-91%, but there is no consensus regarding its clinical significance. OBJECTIVE: Determine the frequency of overexpression and clinical significance of HER2/neu protein in patients with advanced gastric adenocarcinoma. MATERIAL AND METHODS: Prospective, observational study of a series of consecutive cases sent to the Endoscopy Department at Instituto Nacional de Cancerologia in Mexico City between March and June 2009 for diagnostic confirmation of gastric adenocarcinoma; clinical characteristics were recorded and tissue samples obtained during gastroscopy. Once adenocarcinoma was confirmed, the samples were immunohistochemically stained for HER-2/neu. Protein overexpression was measured according to the standardized scale, which has been validated for gastric cancer: 0 and 1+ (negative); 2+ and 3+ (positive). RESULTS: 45 patients were recruited. 5 patients were excluded due to lack of histological confirmation of malignancy. 40 patients, 23 male and 17 female (ratio 1.3:1)were included. The mean age was 52.77 ± 13.7 years. 35% (14/40)overexpressed HER2/neu. There was no association between HER2/ neu overexpression and hemoglobin, albumin, platelet count, weight loss, histological type, degree of differentiation and the presence of metastasis; however there was a positive correlation in regards to sex (p=0.036), age (≥60 yo p=0.0146) and lymph node metastasis (p=0.035). The different histological types were compared and a statically significant difference with regards to age and degree of differentiation was found. Diffuse type gastric adenocarcinoma was seen more frequently among younger patients and showed a lesser
S1958 Expression and Localization of Calreticulin in Human Colorectal Polyps Eleni Seretis, Eleni Gaitanarou, Ageliki Toumpanaki, Kostas Vougas, Dimitrios Xinopoulos, Niki Arnogiannaki, Stefanos P. Bassioukas, Dimitrios Kypreos, Klisthenis Tsamakidis, Emmanouil Paraskevas, Irene Voloudakis -Baltatzis Introduction: Calreticulin is an abundant, high-capacity Ca2+ binding protein. It has been shown to be implicated in a large number of cellular functions as regulation of intracellular Ca2+ homeostasis, chaperone activity, gene expression and modulation of cell adhesion.The fact that calreticulin affects many cellular functions, reinforce the possibility that it is involved in many pathologic conditions, especially in cancers. The up or down-regulation of calreticulin expression in colorectal cancers remain a controversial issue among the investigators. Methods: The aim of this study was to investigate the distribution of calreticulin in colon polyps in order to evaluate its role in precancerous conditions. 16 polyp tissues removed
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AGA Abstracts
AGA Abstracts
S1956
Expression of S100 Proteins May Help Screen for the Transition of Chronic Inflammation to Dysplasia Jose A. Serrano, Edelmarie Rivera, Angel A. Isidro, Beatriz Pagan, Richard J. Noel, Virgilio Salvo, Caroline B. Appleyard Patients with Inflammatory Bowel Disease (IBD) are at increased risk of developing colorectal cancer. The use of biomarkers to aid in the early detection and diagnosis of colorectal cancer is of interest due to their potential to improve treatment preventing further progression. Calprotectin (S100A8/S100A9) has been identified previously as a potential marker for colorectal cancer, but few studies have looked at its possible role in secondary prevention. Aim: To evaluate the potential for calprotectin in screening for, and early detection of, the transition from inflammation to dysplasia. Methods: Chronic colitis was induced in male Sprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for three days. To induce colitis-associated dysplasia the rats then received TNBS (iv) twice a week for ten weeks. A group of age-matched normal untreated animals were also included as controls. After sacrifice the colons were removed and analyzed for damage, pathology, and expression of S100A8 and S100A9 were measured by real time RT-PCR. Tissue distribution of the S100 proteins was measured by immunohistochemistry. Results: Administration of TNBS caused ulceration and damage as expected, which correlated with the pathological diagnosis. Animals diagnosed with dysplasia had the highest macroscopic damage (p<0.05 vs. normal). Using the ΔΔCt method the fold expression of S100A8 was found to be upregulated and similar in both IBD and dysplasia compared to non specific inflammation (NSI) or untreated, whereas expression of S100A9 was more than 2-fold higher in dysplasia than in IBD. The IHC score for S100A8 showed the highest intensity of staining among the samples diagnosed with IBD and low grade dysplasia. In each case this was significantly higher than the staining intensity found in areas of NSI or high grade dysplasia. When the protein expression for the heterodimer S100A8 & S100A9 was examined the intensity of staining in areas of low grade dysplasia was high, with no difference in the intensity of staining between the other diagnoses. These all showed comparable but lower intensity values compared to low grade dysplasia. In each case there appeared to be a shift from weaker staining in NSI to increased staining during the transition to dysplasia. Interestingly, as the pathology worsens the expression once again decreases. Conclusion:There is a potential role for S100A8 and S100A9 in secondary prevention via early disease detection of low grade dysplasia or active IBD. Supported in by 1U56 CA126379, RR003050 & 1F31DK077584
S1959 Identification and Validation of New Biomarkers for Colorectal Cancer Using Isolated Epithelial Stem Cells David Harrison, S. Suchitha, Ansamma Joseph, Asit Panja BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States (US) and is the most common malignancy in the US. Although, it has been known for many decades that the transformation of the epithelial cells lining the colon and/or rectum are the cause of CRC, the actual causative molecular mechanism of CRC remains unknown. Furthermore, no specific biomarker currently exists to aid in the early diagnosis or therapeutic treatment of CRC. AIM: The aim of this study was to identify and validate cell and disease state specific biomarkers for CRC. METHODS: Adult human gastrointestinal stem cells were isolated and cultured concurrently from both the actual cancerous and normal/unaffected colonic tissue of the same individual suffering from CRC. Isolated total RNA from these stem cells were subjected to gene expression microarray analysis. Potential molecular biomarkers/targets associated with cancerous transformation of the epithelial stem cells in the cancerous tissues compared to its normal counterpart were identified. Gene array data was validated at the mRNA expression level by RT-PCR analysis and at the protein expression level by immunocytochemical, flow cytometric, and Western blot analysis. The corresponding identified molecules were further validated by immunohistochemical analysis of paired (normal and cancer) paraffin block tissue sections. RESULTS: Our data from comparative gene array analysis of normal vs. CRC stem cells (positive for Nanog, Oct4, SOX2, LIN28, and Bmi-1) identified 160 dysregulated genes. Many of the identified gene products were shown for the first time to be potential biomarkers for CRC. Several of these identified potential biomarkers were further analyzed, including: proprotein convertase subtilisin/kexin type 5 (PCSK5) and chemokine ligand 20 (BST2 or CCL20) which were shown to be dramatically upregulated in CRC, as well as, superoxide dismutase 3 (SOD3) which demonstrated a 482 fold decrease in expression in CRC derived stem cells. CONCLUSION: Our results demonstrated that paired stem cells isolated concurrently from both the cancerous/affected and normal/unaffected colonic tissue of an individual suffering CRC and the differentiation and culturing of epithelial cell lines from these stem cells provides a unique and superior system for the identification of specific biomarkers and potential therapeutic targets for CRC.
S1957 Potential Role of Biological Ageing in Colorectal Cancer: How Many Miles on the Clock? Fraser Maxwell, Liane McGlynn, Campbell S. Roxburgh, Donald C. McMillan, Paul G. Horgan, Paul Shiels Introduction Ageing is a complex multi-factorial process of which the fundamental determinants remain to be fully elucidated. A key manifestation of biological ageing at the cellular level is telomere attrition. Telomeres are nucleo-protein complexes found at the ends of eukaryotic chromosomes and play a critical role in the maintenance of genomic integrity. They undergo progressive shortening with each round of cell division and, therefore act as a mitotic clock. Telomere attrition and hence dysfunction can induce genomic instability and cellular senescence, which can in turn cause cancer predisposition. Factors which accelerate telomere attrition and thus put ‘miles on the clock' include inflammation and oxidative stress. An association between telomere length and cancer however has resulted in many equivocal results in the field. We have investigated whether patients with colorectal cancer display evidence of accelerated telomere attrition. Methods After full ethical approval and informed consent, DNA was extracted from peripheral blood leucocytes of patients with a diagnosis of colorectal cancer using routine methods. Telomere lengths were determined by quantitative-PCR using the method of Cawthon (2002) and correlated with common clinic-pathological characteristics, markers of inflammation and antioxidant status. Statistical analysis was performed using SPSS version 15.0. Results Data were available for 64 patients in the CRC and 59 in the control group. Mean T/S ratio (telomere length) was 0.64 (±0.17) in the CRC group and 0.82 (±0.22) in the control group (p=0.000). There was a significant negative correlation between age and telomere length (rho=-.279, p<0.05) in patients with CRC. Patients in the quartile with the shortest telomere length had a significantly higher neutrophil to lymphocyte ratio (NLR) (p<0.05). Furthermore, there was a significant association between short telomere length and high risk pathological features (Peterson Index). Conclusion We have demonstrated that patients with CRC display clear evidence of accelerated biological ageing. The observed association between telomere length and NLR may implicate inflammation in this process. In addition, telomere length may prove a useful marker of disease severity.
S1960 Clinical Significance of HER2/Neu Overexpression in Gastric Cancer Paola Figueroa Barojas, Zarate Osorno Alejandra, Enoe E. Quiñonez-Urrego, Angelica Hernandez-Guerrero, Sergio R. Sobrino-Cossio, Juan Octavio Alonso-Larraga Gastric cancer is the 2nd most common worldwide, causing 3-10% of cancer related deaths. 95% of gastric tumors are adenocarcinoma, 65% present at an advanced stage, and up to 85% have lymph node metastasis at diagnosis. Studies have pointed to an association between gene/protein expression and higher rates of invasion, lymph node metastasis and/or advanced stage. The amplification of the C-erb2 gene and the overexpression of its protein (HER2/ neu) has been implicated as a poor prognostic factor, both have been extensively studied in breast cancer. In gastric cancer, immunohistochemical studies show a frequency of overexpression of HER2/neu of 8-91%, but there is no consensus regarding its clinical significance. OBJECTIVE: Determine the frequency of overexpression and clinical significance of HER2/neu protein in patients with advanced gastric adenocarcinoma. MATERIAL AND METHODS: Prospective, observational study of a series of consecutive cases sent to the Endoscopy Department at Instituto Nacional de Cancerologia in Mexico City between March and June 2009 for diagnostic confirmation of gastric adenocarcinoma; clinical characteristics were recorded and tissue samples obtained during gastroscopy. Once adenocarcinoma was confirmed, the samples were immunohistochemically stained for HER-2/neu. Protein overexpression was measured according to the standardized scale, which has been validated for gastric cancer: 0 and 1+ (negative); 2+ and 3+ (positive). RESULTS: 45 patients were recruited. 5 patients were excluded due to lack of histological confirmation of malignancy. 40 patients, 23 male and 17 female (ratio 1.3:1)were included. The mean age was 52.77 ± 13.7 years. 35% (14/40)overexpressed HER2/neu. There was no association between HER2/ neu overexpression and hemoglobin, albumin, platelet count, weight loss, histological type, degree of differentiation and the presence of metastasis; however there was a positive correlation in regards to sex (p=0.036), age (≥60 yo p=0.0146) and lymph node metastasis (p=0.035). The different histological types were compared and a statically significant difference with regards to age and degree of differentiation was found. Diffuse type gastric adenocarcinoma was seen more frequently among younger patients and showed a lesser
S1958 Expression and Localization of Calreticulin in Human Colorectal Polyps Eleni Seretis, Eleni Gaitanarou, Ageliki Toumpanaki, Kostas Vougas, Dimitrios Xinopoulos, Niki Arnogiannaki, Stefanos P. Bassioukas, Dimitrios Kypreos, Klisthenis Tsamakidis, Emmanouil Paraskevas, Irene Voloudakis -Baltatzis Introduction: Calreticulin is an abundant, high-capacity Ca2+ binding protein. It has been shown to be implicated in a large number of cellular functions as regulation of intracellular Ca2+ homeostasis, chaperone activity, gene expression and modulation of cell adhesion.The fact that calreticulin affects many cellular functions, reinforce the possibility that it is involved in many pathologic conditions, especially in cancers. The up or down-regulation of calreticulin expression in colorectal cancers remain a controversial issue among the investigators. Methods: The aim of this study was to investigate the distribution of calreticulin in colon polyps in order to evaluate its role in precancerous conditions. 16 polyp tissues removed
S-289
AGA Abstracts
AGA Abstracts
S1956