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S1931 Colon Cancers With Defective DNA Mismatch Repair Show Reduced Recurrence, Delayed Time-to-Recurrence and Fewer Distant Metastases
interrogated in single cell preparation (prepared using the standard Weiser technique which employs divalent cation chelation ). LEBS markers were calculated by observer blinded to treatment group. Crypt morphology studies were also performed using standard H&E staining as well as confocal microscopy imaging using techniques. Results: In the tissue LEBS analysis, there were profound alterations in several spectral markers. Indeed, spectral slope in the tissue sections was altered by 58% (p<0.01), consistent with our clinical data. On the other hand, when the colonocytes were assessed without the cryptal context, the effect size decreased to 11.3% (p=n.s.). Despite being microscopically normal, when we rigorously measured crypt length, we noted a marked increase in the AOM-treated rats (207 vs 273 microns respectively, p<0.0001) with a corresponding increase in crypt diameter (p<0.0001). Conclusions: We provide the first evidence that spectral markers originate, at least partly (i.e. spectral slope), from the overall cryptal organization. We also make the novel observation that in the visually normal mucosa of premalignant colonic mucosa the crypt length and diameter are markedly altered. The crypt alterations may reflect the overall alterations in proliferation/apoptosis ratio that is characteristic of field carcinogenesis. This work may serve as a platform to understand and develop novel biomarkers of field carcinogenesis enabling accurate risk stratification through biophotonic rectal interrogation.
Reprimo: A Novel Non-Invasive Biomarker for Gastric Cancer Detection Alejandro H. Corvalan, Catterina Ferreccio, Cynthia Villarroel, Francisco R. Aguayo, Eudocia Santibañez, Carlos Barrientos, Edmundo Aravena, Alfonso Calvo, Maria J. Maturana Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fisher's exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer. Supported by FONDECYT grant 1080563 from Government of Chile
S1931 Colon Cancers With Defective DNA Mismatch Repair Show Reduced Recurrence, Delayed Time-to-Recurrence and Fewer Distant Metastases Frank A. Sinicrope, Nathan R. Foster, Daniel J. Sargent, Stephen N. Thibodeau, Silvia Marsoni, Genevieve M. Monges, Roberto Labianca, Steven Gallinger Background. Colon cancers with deficient DNA mismatch repair (MMR) appear to have a more favorable stage-adjusted survival rate than do cases with proficient MMR. To gain further insights into their clinical behavior, we analyzed time-to-recurrence and site of recurrence data in relation to MMR status. Materials & Methods. Curatively resected TNM stage II and III colon carcinoma patients (N= 1,606) were treated in nine 5-fluorouracilbased, randomized adjuvant therapy trials. Tumors were analyzed for microsatellite instability (MSI) and/or MMR protein expression. Deficient MMR was defined as MSI-H or loss of an MMR protein by immunohistochemistry. Association of MMR status with time-to-recurrence and survival data were analyzed using a Cox regression model, and with site of recurrence using Chi-square or Fisher's Exact tests. Results. Colon cancers with deficient versus proficient MMR showed recurrence rates of 21% and 34%, respectively, within 5 years (p <0.0001). Tumors with deficient MMR showed a significantly longer time-to-recurrence (TTR) compared to tumors with proficient MMR [HR = 0.59 (95% CI: 0.44, 0.78); p=0.0003]. Divergence in TTR curves occurred between 1 to 2 years post surgical resection. Patients whose tumors showed deficient MMR had significantly improved DFS [5 yr rate: 75% vs. 62%; HR = 0.65 (95% CI: 0.50, 0.84); p=0.0011] and OS [5 yr rate: 79% vs. 69%; HR = 0.68 (95% CI: 0.53, 0.88); p=0.0027]. Patient tumors with deficient MMR had fewer hepatic and pulmonary metastases compared to cases with proficient MMR (38% vs 54%; p=0.0226). For patients that recurred within 2 yr post resection, hepatic metastases were detected in 17% of MMR deficient versus 38% of MMR proficient tumors (p=0.0152). Intra-abdominal recurrence was significantly more common in MMR deficient vs proficient tumors (43% vs 22%, p=0.0068). For recurrences more than 2 years post resection, no significant differences were found based upon MMR status. MMR status had a similar impact on survival and TTR in stage II vs. III colon cancer patients. Conclusion. Colon cancers with deficient DNA MMR show significantly reduced recurrence rates and delayed time-to-recurrence. Within 2 yr of resection, tumors with deficient MMR show higher rates of intra-abdominal recurrence and fewer hepatic metastases contributing to their better prognosis.
S1929 Comparison of the Prognostic Value of Selected Markers of the Systemic Inflammatory Response in Patients With Gastro-Oesophageal Cancer Sumanta Dutta, Andrew B. Crumley, Grant Fullarton, Paul G. Horgan, Donald C. McMillan Background: There is increasing evidence that the presence of an ongoing systemic inflammatory response is associated with poor outcome in patients with gastro-oesophageal cancer. However, it is not clear what components of systemic inflammatory response best predict cancer survival. The aim of the present study was to compare the prognostic value of selected markers of the systemic inflammatory response in gastro-oesophageal cancer. Patient and Methods: Two patient groups were studied: 169 patients who underwent potentially curative resection and 252 patients with inoperable cancer. All patients had measurement of white cell, lymphocyte and platelet counts, albumin and C-reactive protein at the time of diagnosis and the Glasgow Prognostic Score (mGPS)1, Neutrophil Lymphocyte Ratio (NLR) and Platelet Lymphocyte Ratio (PLR) were calculated. In the patients who underwent surgery, the positive to total lymph node ratio (LNR) were calculated. Clinical information was obtained from a gastro-oesophageal cancer database and analysis of the case notes. Results: In the operable group median follow up for survivors was 48 months. 61 patients died of cancer and median survival was 17 months (range 2 to 88 months). On multivariate analysis only LNR (HR 2.44, 95% CI 1.58-3.76, P<0.001) and preoperative mGPS (HR 2.06, 95% CI 1.2-3.52, P= 0.009) were independently associated with cancer-specific survival. In the inoperable group median follow up was 7 months. 235 patients died of cancer and median survival was 7 months (range 1 to 76 months). On multivariate analysis, only tumour stage (HR 1.49, 95% CI 1.3-1.71, P<0.001), treatment (HR 2.24, 95% CI 1.63-3.08, P<0.001) and mGPS (HR 1.48, 95% CI 1.22-1.80, P<0.001) were independently associated with cancer-specific survival. mGPS was directly associated with age (p<0.05), stage (p<0.001), white cell count(p<0.001), neutrophil count (p<0.001), lymphocyte count (p<0.05), platelet count (p<0.001), NLR (p<0.001) and PLR (p<0.001). Conclusion: The present study indicates that, in patients with gastro-oesophageal cancer, an acute-phase protein-based prognostic score, the mGPS, is a superior predictor of cancer survival compared with the cellular components of the systemic inflammatory response. References: 1. McMillan DC. Systemic inflammation, nutritional status and survival in patients with cancer. Curr Opin Clin Nutr Metab Care 2009;12:223-226.
S1932 Identification of Potential Novel Risk Stratification Markers for Ulcerative Colitis-Associated Dysplasia Using Methylation Arrays Andrew Kaz, Mary P. Bronner, Ru Chen, David Crispin, Teresa Brentnall, William M. Grady INTRODUCTION: Individuals with long-standing ulcerative colitis (UC) are at increased risk for colorectal cancer (CRC) because of chronic inflammation of the colon. To prevent CRC in UC patients, they undergo annual colonoscopy with endoscopic biopsies to assess for pre-cancerous changes in the colonic mucosa. Currently, the best marker for cancer risk is dysplasia in biopsies of normal colon mucosa, but this method is suboptimal due to its costs and variability. Thus, there has been intense investigation for other risk markers, including molecular alterations in the normal colon mucosa of UC patients (e.g. mutant TP53; methylated genes) to determine if they are more accurate predictive markers for dysplasia/cancer than histology. The data on methylated genes as risk markers is conflicting to date, likely because of differences in the genes studied. HYPOTHESIS: Aberrantly methylated genes occur in the normal mucosa of UC patients, reflect a field cancerization process, and can be used to identify those patients with high-grade dysplasia/cancer. METHODS: Epithelial DNA isolated from normal-appearing colonic mucosa from four UC patients without cancer (UC-N) and from three UC patients with dysplasia/cancer in the colon (UCD/C) was bisulfite treated and analyzed using the Illumina GoldenGate Cancer Panel I microarray. The array results were validated by bisulfite sequencing DNA isolated from one UC-N and one UC-D/C patient. RESULTS: After filtering the array data, we identified 23 genes for further analysis, including: extracellular matrix genes (4), proliferation regulator genes (5), growth factors (5), transcription factors (4), tyrosine kinase pathway members (3), DNA repair genes (2), and an IGF pathway gene (1). We validated six genes (TFPI2, GSTM2, HIN1, NTRK3, TUSC3, and TWIST1) by bisulfite sequencing, confirming relatively high methylation in an UC-D/C patient and low methylation in an UC-N patient. DISCUSSION: Using a methylation array-based approach, we identified novel aberrantly methylated genes in histologically normal colonic mucosa from UC patients who had cancer/dysplasia located elsewhere in the colon. Bisulfite sequencing of the original DNA confirmed the methylation differences between UC-N and UC-D/C in 6/6 genes tested. Thus, even histologically normal-appearing colonic mucosa in UC patients with dysplasia/cancer contains aberrantly methylated DNA, suggesting that epigenetic alterations are part of a field cancerization process in the colon. Equally as important, we have identified a set of genes that show
S1930 Biophotonic Detection of Colonic Field Carcinogenesis Spectral Slope Represents Fundamental Alterations in Crypt Architecture Yolanda Stypula, Nikhil Mutyal, Vladimir Turzhitsky, Mona L. Cornwell, Ramesh K. Wali, Ashish K. Tiwari, Susan Crawford, Vadim Backman, Hemant K. Roy Background: Our group has employed a variety of optical technologies to identify field carcinogenesis as a means for risk stratification (Gastro, 2009; Cancer Res, 2009; PNAS, 2008). One powerful approach has been using a technology low coherence enhanced backscattering spectroscopy (LEBS) which probes tissue organization in microscopically normal mucosa (Clin Can Res, 2006). While the clinical value is unequivocal with rectal LEBS AUROC for advanced adenomas was 0.90 (Cancer Res, 2009), the biological determinants for these spectral markers remain unclear. Fundamentally, these markers could represent alterations in submicron cellular structure or more cryptal architectural changes. We, therefore, investigated this using the azoxymethane (AOM)-treated rat model and focused on the marker spectral slope which seemed to be very clinically diagnostic(Dis Colon Rectum, 2008). Methods: Thirty Fisher 344 rats were randomized to two weekly treatments of AOM or saline. Animals were sacrificed at a premalignant time point (12 weeks post injection) and underwent LEBS analysis either when tissue architecture intact (standard fresh tissue sections) or colonocytes
S-283
AGA Abstracts
AGA Abstracts
S1928
Reprimo: A Novel Non-Invasive Biomarker for Gastric Cancer Detection Alejandro H. Corvalan, Catterina Ferreccio, Cynthia Villarroel, Francisco R. Aguayo, Eudocia Santibañez, Carlos Barrientos, Edmundo Aravena, Alfonso Calvo, Maria J. Maturana Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fisher's exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer. Supported by FONDECYT grant 1080563 from Government of Chile
S1931 Colon Cancers With Defective DNA Mismatch Repair Show Reduced Recurrence, Delayed Time-to-Recurrence and Fewer Distant Metastases Frank A. Sinicrope, Nathan R. Foster, Daniel J. Sargent, Stephen N. Thibodeau, Silvia Marsoni, Genevieve M. Monges, Roberto Labianca, Steven Gallinger Background. Colon cancers with deficient DNA mismatch repair (MMR) appear to have a more favorable stage-adjusted survival rate than do cases with proficient MMR. To gain further insights into their clinical behavior, we analyzed time-to-recurrence and site of recurrence data in relation to MMR status. Materials & Methods. Curatively resected TNM stage II and III colon carcinoma patients (N= 1,606) were treated in nine 5-fluorouracilbased, randomized adjuvant therapy trials. Tumors were analyzed for microsatellite instability (MSI) and/or MMR protein expression. Deficient MMR was defined as MSI-H or loss of an MMR protein by immunohistochemistry. Association of MMR status with time-to-recurrence and survival data were analyzed using a Cox regression model, and with site of recurrence using Chi-square or Fisher's Exact tests. Results. Colon cancers with deficient versus proficient MMR showed recurrence rates of 21% and 34%, respectively, within 5 years (p <0.0001). Tumors with deficient MMR showed a significantly longer time-to-recurrence (TTR) compared to tumors with proficient MMR [HR = 0.59 (95% CI: 0.44, 0.78); p=0.0003]. Divergence in TTR curves occurred between 1 to 2 years post surgical resection. Patients whose tumors showed deficient MMR had significantly improved DFS [5 yr rate: 75% vs. 62%; HR = 0.65 (95% CI: 0.50, 0.84); p=0.0011] and OS [5 yr rate: 79% vs. 69%; HR = 0.68 (95% CI: 0.53, 0.88); p=0.0027]. Patient tumors with deficient MMR had fewer hepatic and pulmonary metastases compared to cases with proficient MMR (38% vs 54%; p=0.0226). For patients that recurred within 2 yr post resection, hepatic metastases were detected in 17% of MMR deficient versus 38% of MMR proficient tumors (p=0.0152). Intra-abdominal recurrence was significantly more common in MMR deficient vs proficient tumors (43% vs 22%, p=0.0068). For recurrences more than 2 years post resection, no significant differences were found based upon MMR status. MMR status had a similar impact on survival and TTR in stage II vs. III colon cancer patients. Conclusion. Colon cancers with deficient DNA MMR show significantly reduced recurrence rates and delayed time-to-recurrence. Within 2 yr of resection, tumors with deficient MMR show higher rates of intra-abdominal recurrence and fewer hepatic metastases contributing to their better prognosis.
S1929 Comparison of the Prognostic Value of Selected Markers of the Systemic Inflammatory Response in Patients With Gastro-Oesophageal Cancer Sumanta Dutta, Andrew B. Crumley, Grant Fullarton, Paul G. Horgan, Donald C. McMillan Background: There is increasing evidence that the presence of an ongoing systemic inflammatory response is associated with poor outcome in patients with gastro-oesophageal cancer. However, it is not clear what components of systemic inflammatory response best predict cancer survival. The aim of the present study was to compare the prognostic value of selected markers of the systemic inflammatory response in gastro-oesophageal cancer. Patient and Methods: Two patient groups were studied: 169 patients who underwent potentially curative resection and 252 patients with inoperable cancer. All patients had measurement of white cell, lymphocyte and platelet counts, albumin and C-reactive protein at the time of diagnosis and the Glasgow Prognostic Score (mGPS)1, Neutrophil Lymphocyte Ratio (NLR) and Platelet Lymphocyte Ratio (PLR) were calculated. In the patients who underwent surgery, the positive to total lymph node ratio (LNR) were calculated. Clinical information was obtained from a gastro-oesophageal cancer database and analysis of the case notes. Results: In the operable group median follow up for survivors was 48 months. 61 patients died of cancer and median survival was 17 months (range 2 to 88 months). On multivariate analysis only LNR (HR 2.44, 95% CI 1.58-3.76, P<0.001) and preoperative mGPS (HR 2.06, 95% CI 1.2-3.52, P= 0.009) were independently associated with cancer-specific survival. In the inoperable group median follow up was 7 months. 235 patients died of cancer and median survival was 7 months (range 1 to 76 months). On multivariate analysis, only tumour stage (HR 1.49, 95% CI 1.3-1.71, P<0.001), treatment (HR 2.24, 95% CI 1.63-3.08, P<0.001) and mGPS (HR 1.48, 95% CI 1.22-1.80, P<0.001) were independently associated with cancer-specific survival. mGPS was directly associated with age (p<0.05), stage (p<0.001), white cell count(p<0.001), neutrophil count (p<0.001), lymphocyte count (p<0.05), platelet count (p<0.001), NLR (p<0.001) and PLR (p<0.001). Conclusion: The present study indicates that, in patients with gastro-oesophageal cancer, an acute-phase protein-based prognostic score, the mGPS, is a superior predictor of cancer survival compared with the cellular components of the systemic inflammatory response. References: 1. McMillan DC. Systemic inflammation, nutritional status and survival in patients with cancer. Curr Opin Clin Nutr Metab Care 2009;12:223-226.
S1932 Identification of Potential Novel Risk Stratification Markers for Ulcerative Colitis-Associated Dysplasia Using Methylation Arrays Andrew Kaz, Mary P. Bronner, Ru Chen, David Crispin, Teresa Brentnall, William M. Grady INTRODUCTION: Individuals with long-standing ulcerative colitis (UC) are at increased risk for colorectal cancer (CRC) because of chronic inflammation of the colon. To prevent CRC in UC patients, they undergo annual colonoscopy with endoscopic biopsies to assess for pre-cancerous changes in the colonic mucosa. Currently, the best marker for cancer risk is dysplasia in biopsies of normal colon mucosa, but this method is suboptimal due to its costs and variability. Thus, there has been intense investigation for other risk markers, including molecular alterations in the normal colon mucosa of UC patients (e.g. mutant TP53; methylated genes) to determine if they are more accurate predictive markers for dysplasia/cancer than histology. The data on methylated genes as risk markers is conflicting to date, likely because of differences in the genes studied. HYPOTHESIS: Aberrantly methylated genes occur in the normal mucosa of UC patients, reflect a field cancerization process, and can be used to identify those patients with high-grade dysplasia/cancer. METHODS: Epithelial DNA isolated from normal-appearing colonic mucosa from four UC patients without cancer (UC-N) and from three UC patients with dysplasia/cancer in the colon (UCD/C) was bisulfite treated and analyzed using the Illumina GoldenGate Cancer Panel I microarray. The array results were validated by bisulfite sequencing DNA isolated from one UC-N and one UC-D/C patient. RESULTS: After filtering the array data, we identified 23 genes for further analysis, including: extracellular matrix genes (4), proliferation regulator genes (5), growth factors (5), transcription factors (4), tyrosine kinase pathway members (3), DNA repair genes (2), and an IGF pathway gene (1). We validated six genes (TFPI2, GSTM2, HIN1, NTRK3, TUSC3, and TWIST1) by bisulfite sequencing, confirming relatively high methylation in an UC-D/C patient and low methylation in an UC-N patient. DISCUSSION: Using a methylation array-based approach, we identified novel aberrantly methylated genes in histologically normal colonic mucosa from UC patients who had cancer/dysplasia located elsewhere in the colon. Bisulfite sequencing of the original DNA confirmed the methylation differences between UC-N and UC-D/C in 6/6 genes tested. Thus, even histologically normal-appearing colonic mucosa in UC patients with dysplasia/cancer contains aberrantly methylated DNA, suggesting that epigenetic alterations are part of a field cancerization process in the colon. Equally as important, we have identified a set of genes that show
S1930 Biophotonic Detection of Colonic Field Carcinogenesis Spectral Slope Represents Fundamental Alterations in Crypt Architecture Yolanda Stypula, Nikhil Mutyal, Vladimir Turzhitsky, Mona L. Cornwell, Ramesh K. Wali, Ashish K. Tiwari, Susan Crawford, Vadim Backman, Hemant K. Roy Background: Our group has employed a variety of optical technologies to identify field carcinogenesis as a means for risk stratification (Gastro, 2009; Cancer Res, 2009; PNAS, 2008). One powerful approach has been using a technology low coherence enhanced backscattering spectroscopy (LEBS) which probes tissue organization in microscopically normal mucosa (Clin Can Res, 2006). While the clinical value is unequivocal with rectal LEBS AUROC for advanced adenomas was 0.90 (Cancer Res, 2009), the biological determinants for these spectral markers remain unclear. Fundamentally, these markers could represent alterations in submicron cellular structure or more cryptal architectural changes. We, therefore, investigated this using the azoxymethane (AOM)-treated rat model and focused on the marker spectral slope which seemed to be very clinically diagnostic(Dis Colon Rectum, 2008). Methods: Thirty Fisher 344 rats were randomized to two weekly treatments of AOM or saline. Animals were sacrificed at a premalignant time point (12 weeks post injection) and underwent LEBS analysis either when tissue architecture intact (standard fresh tissue sections) or colonocytes
S-283
AGA Abstracts
AGA Abstracts
S1928