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109 Discovery of Novel DNA Methylation Markers for the Detection of Colorectal Neoplasia: Selection by Methylome-Wide Analysis
AGA Abstracts Figure 1. Percentage of positive detection of cell-free methylated DNA of Reprimo(cfmDNARPRM) among 1.076 consecutive cases. Samples were collected from three institutions and include Non-Atrophic Gastritis (NACG), Atrophic Chronic Gastritis (ACG), Intestinal Metaplasia (IM), Low Grade Dysplasia (LGD), High Grade Dysplasia (HGD), Early Gastric Cancer (EGC) and Advanced Gastric Cancer (AGC).
110 MiR-320e Is a Prognostic and Predictive Biomarker in Colorectal Cancer Lucía Pérez-Carbonell, Frank A. Sinicrope, Steven R. Alberts, Ann L. Oberg, Francesc Balaguer, Antoni Castells, C. Richard Boland, Ajay Goel
109 Discovery of Novel DNA Methylation Markers for the Detection of Colorectal Neoplasia: Selection by Methylome-Wide Analysis William R. Taylor, John B. Kisiel, Tracy C. Yab, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Hatim Allawi, Graham P. Lidgard, David A. Ahlquist
Background. Although advances in detection and treatment have improved outcomes in patients with colorectal cancer (CRC), there is still a need for better prognostic and predictive biomarkers which can help identify patients that are at risk for developing tumor recurrence following chemotherapy. Therefore, we conducted a systematic discovery and validation of potential miRNA biomarkers in two independent clinic-based cohorts of CRC patients treated with a 5FU-based chemotherapeutic regimen. Methods. Affymetrix miRNA expression arrays were used to determine miRNA expression profiles in a discovery cohort comprised of stage III CRC patients with (N=50) and without (N=50) recurrence, with 3-year follow-up. All patients received 5FU and oxaliplatin adjuvant chemotherapy. In the validation cohort, we studied miRNA expression using qRT-PCR in an independent cohort of 237 stages II-IV CRC patients treated with 5FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy individuals. Association of miRNA expression patterns with tumor recurrence and disease-free survival (DFS) was examined using Cox regression models. Results. In the initial discovery cohort, elevated miR-320e expression was found to be significantly higher in stage III CRCs from patients who experienced tumor recurrence within 3 years, compared to those with DFS during follow-up (p<0.0001). In the validation cohort, mean levels of miR-320e expression in primary CRCs were ~10-fold higher than in normal colonic mucosa (p<0.0001). More interestingly, high expression of miR-320e was associated with tumor recurrence and reduced DFS in CRC patients with stage II & III (p<0.05), and stage III (p<0.0001) alone. Furthermore, high expression of miR-320e expression was an independent predictor of poor survival among patients with stage II & III CRC (HR=1.88; 95% CI, 0.93.56, p=0.05). Conclusions. We identified a miRNA, miR-320e, which was associated with adverse outcomes in patients with stage III CRC treated with 5FU-based adjuvant chemotherapy. Our data suggest that miR-320e status could have important clinical implications in improving therapeutic strategies and personalized management of colon cancer.
Background: DNA hypermethylation of CpG-rich promoters is an epigenetic hallmark of carcinogenesis. Numerous methylation markers for colorectal cancer (CRC) have been derived from functional gene-expression studies, but fewer from unbiased sequencing. Aims: 1) Conduct a whole methylome search and develop methodology for identification of CRCspecific DNA methylation sequences. 2) Select top candidate markers for detection of CRC and pre-cancer. Methods: For discovery, we evaluated frozen tissues from 18 sporadic CRCs (9 proximal, 9 distal), 18 adenomas ≥1cm (9 proximal, 9 distal), and 18 mucosae from age-sex matched patients with normal colonoscopy plus18 buffy-coat samples from matched normal patients. Differentially methylated regions (DMRs) were identified by reduced representation bisulfite sequencing (RRBS). Reads were mapped to a reference genome and annotated. Sequence coverage and univariate χ2 filters were applied and non-informative CpG sites excluded. Informative sites were parsed into defined contiguous in-silico "islands" and were analyzed for differential %methylation density by logistic regression. Marker selection was based on receiver operating characteristic discrimination, signal to noise (S/N) ratios, and background methylation. Biological validation of top markers was by methylationspecific PCR (MSP) on unique tissue sets from age-sex matched CRC (36), adenoma (36), and normal (36) patients. The most discriminant 8 marker candidates were tested by a quantitative allele specific real-time target and signal amplification method on pooled stools from 90 CRC cases and 90 normal controls. Results: RRBS discovery yielded 2-3 million quality CpGs, which after filtering produced 1068 and 1200 highly discriminant sites for CRC and adenomas, respectively. These clustered into 185 and 244 localized DMRs, respectively, extending from 30 to >1000 bases. By literature review, only15% of these DMRs had been reported with CRC. Many remaining sites are involved in functional pathways relevant to tumorigenesis. We found no annotation and no references for 30 DMRs in CRCs and 42 in adenomas. After MSP validation, 18 candidate markers passed our filter criteria with area under the ROC curve (AUC) for both CRC and adenoma exceeding 0.88, S/N ratios >50, and background methylation < 1%, (table). Several markers achieved almost perfect discrimination for CRC and adenomas. The top 8 tissue markers were tested on stool sample pools; 7 highly discriminated CRC from controls. The top marker (SFMBT2 region 1) had an S/N ratio of 283. Conclusion: Whole methylome sequencing, stringent filtering criteria, and biological validation yield outstanding candidate markers for detection of colorectal neoplasia. In stool and other media with excess of wild-type signal, DMRs with high S/N ratios offer particular promise for non-invasive clinical testing. Most Discriminate Markers
AGA Abstracts
111 Angiopoietin-Like Protein 2 Is a Novel Serum Biomarker of Diagnosis and Prognosis in Patients With Gastrointestinal Cancer Yuji Toiyama, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Satoru Kondo, Shozo Ide, Masato Okigami, Hiromi Yasuda, Mikio Kawamura, Aya Kawamoto, Junichiro Hiro, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Masato Kusunoki Background: Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family. It has been reported to act as a causative mediator of chronic inflammation and metabolic abnormalities. ANGPTL2 increases inflammatory carcinogenesis in several cancers, and its expression in tumor cells is highly correlated with the frequency of tumor cell metastasis through increased tumor angiogenesis and tumor cell epithelial-to-mesenchymal transitions. However, to our own knowledge, clinical significance of serum ANGPTL2 remains completely unknown in patients with cancer. Aim: The aim of this study was to quantify serum ANGPTL2 level using ELISA, and to evaluate its clinical significance in patients with gastrointestinal cancer including colorectal cancer (CRC) and gastric cancer (GC). Methods: We screened serum ANGPTL2 levels from 32 CRC (16 stage I and 16 stage IV), 32 GC (16 stage I and 16 stage IV) and 23 normal controls (NC), and validated its serum level from 194 CRC, 194 GC and 45 NC to evaluate the utility as a diagnostic and prognostic biomarker by ELISA. Results: In the screening step, serum ANGPTL2 levels were significantly higher in CRC and GC than in NC respectively (CRC: p<0.01, GC: p<0.01) and gradually increased according to TNM stage progression in CRC and GC. Serum ANGPTL2 levels differentiated CRC or GC from NC with high accuracy (CRC: AUC=0.81, GC: AUC=0.86, respectively). Next, validation step indicated that serum ANGPTL2 levels in CRC patients were significantly higher compared to NC (p<0.0001), had a promising AUC (0.88) to distinguish CRC from NC. In addition, serum ANGPTL2 levels also discriminated early CRC patients (stage I)
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110 MiR-320e Is a Prognostic and Predictive Biomarker in Colorectal Cancer Lucía Pérez-Carbonell, Frank A. Sinicrope, Steven R. Alberts, Ann L. Oberg, Francesc Balaguer, Antoni Castells, C. Richard Boland, Ajay Goel
109 Discovery of Novel DNA Methylation Markers for the Detection of Colorectal Neoplasia: Selection by Methylome-Wide Analysis William R. Taylor, John B. Kisiel, Tracy C. Yab, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Hatim Allawi, Graham P. Lidgard, David A. Ahlquist
Background. Although advances in detection and treatment have improved outcomes in patients with colorectal cancer (CRC), there is still a need for better prognostic and predictive biomarkers which can help identify patients that are at risk for developing tumor recurrence following chemotherapy. Therefore, we conducted a systematic discovery and validation of potential miRNA biomarkers in two independent clinic-based cohorts of CRC patients treated with a 5FU-based chemotherapeutic regimen. Methods. Affymetrix miRNA expression arrays were used to determine miRNA expression profiles in a discovery cohort comprised of stage III CRC patients with (N=50) and without (N=50) recurrence, with 3-year follow-up. All patients received 5FU and oxaliplatin adjuvant chemotherapy. In the validation cohort, we studied miRNA expression using qRT-PCR in an independent cohort of 237 stages II-IV CRC patients treated with 5FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy individuals. Association of miRNA expression patterns with tumor recurrence and disease-free survival (DFS) was examined using Cox regression models. Results. In the initial discovery cohort, elevated miR-320e expression was found to be significantly higher in stage III CRCs from patients who experienced tumor recurrence within 3 years, compared to those with DFS during follow-up (p<0.0001). In the validation cohort, mean levels of miR-320e expression in primary CRCs were ~10-fold higher than in normal colonic mucosa (p<0.0001). More interestingly, high expression of miR-320e was associated with tumor recurrence and reduced DFS in CRC patients with stage II & III (p<0.05), and stage III (p<0.0001) alone. Furthermore, high expression of miR-320e expression was an independent predictor of poor survival among patients with stage II & III CRC (HR=1.88; 95% CI, 0.93.56, p=0.05). Conclusions. We identified a miRNA, miR-320e, which was associated with adverse outcomes in patients with stage III CRC treated with 5FU-based adjuvant chemotherapy. Our data suggest that miR-320e status could have important clinical implications in improving therapeutic strategies and personalized management of colon cancer.
Background: DNA hypermethylation of CpG-rich promoters is an epigenetic hallmark of carcinogenesis. Numerous methylation markers for colorectal cancer (CRC) have been derived from functional gene-expression studies, but fewer from unbiased sequencing. Aims: 1) Conduct a whole methylome search and develop methodology for identification of CRCspecific DNA methylation sequences. 2) Select top candidate markers for detection of CRC and pre-cancer. Methods: For discovery, we evaluated frozen tissues from 18 sporadic CRCs (9 proximal, 9 distal), 18 adenomas ≥1cm (9 proximal, 9 distal), and 18 mucosae from age-sex matched patients with normal colonoscopy plus18 buffy-coat samples from matched normal patients. Differentially methylated regions (DMRs) were identified by reduced representation bisulfite sequencing (RRBS). Reads were mapped to a reference genome and annotated. Sequence coverage and univariate χ2 filters were applied and non-informative CpG sites excluded. Informative sites were parsed into defined contiguous in-silico "islands" and were analyzed for differential %methylation density by logistic regression. Marker selection was based on receiver operating characteristic discrimination, signal to noise (S/N) ratios, and background methylation. Biological validation of top markers was by methylationspecific PCR (MSP) on unique tissue sets from age-sex matched CRC (36), adenoma (36), and normal (36) patients. The most discriminant 8 marker candidates were tested by a quantitative allele specific real-time target and signal amplification method on pooled stools from 90 CRC cases and 90 normal controls. Results: RRBS discovery yielded 2-3 million quality CpGs, which after filtering produced 1068 and 1200 highly discriminant sites for CRC and adenomas, respectively. These clustered into 185 and 244 localized DMRs, respectively, extending from 30 to >1000 bases. By literature review, only15% of these DMRs had been reported with CRC. Many remaining sites are involved in functional pathways relevant to tumorigenesis. We found no annotation and no references for 30 DMRs in CRCs and 42 in adenomas. After MSP validation, 18 candidate markers passed our filter criteria with area under the ROC curve (AUC) for both CRC and adenoma exceeding 0.88, S/N ratios >50, and background methylation < 1%, (table). Several markers achieved almost perfect discrimination for CRC and adenomas. The top 8 tissue markers were tested on stool sample pools; 7 highly discriminated CRC from controls. The top marker (SFMBT2 region 1) had an S/N ratio of 283. Conclusion: Whole methylome sequencing, stringent filtering criteria, and biological validation yield outstanding candidate markers for detection of colorectal neoplasia. In stool and other media with excess of wild-type signal, DMRs with high S/N ratios offer particular promise for non-invasive clinical testing. Most Discriminate Markers
AGA Abstracts
111 Angiopoietin-Like Protein 2 Is a Novel Serum Biomarker of Diagnosis and Prognosis in Patients With Gastrointestinal Cancer Yuji Toiyama, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Satoru Kondo, Shozo Ide, Masato Okigami, Hiromi Yasuda, Mikio Kawamura, Aya Kawamoto, Junichiro Hiro, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Masato Kusunoki Background: Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family. It has been reported to act as a causative mediator of chronic inflammation and metabolic abnormalities. ANGPTL2 increases inflammatory carcinogenesis in several cancers, and its expression in tumor cells is highly correlated with the frequency of tumor cell metastasis through increased tumor angiogenesis and tumor cell epithelial-to-mesenchymal transitions. However, to our own knowledge, clinical significance of serum ANGPTL2 remains completely unknown in patients with cancer. Aim: The aim of this study was to quantify serum ANGPTL2 level using ELISA, and to evaluate its clinical significance in patients with gastrointestinal cancer including colorectal cancer (CRC) and gastric cancer (GC). Methods: We screened serum ANGPTL2 levels from 32 CRC (16 stage I and 16 stage IV), 32 GC (16 stage I and 16 stage IV) and 23 normal controls (NC), and validated its serum level from 194 CRC, 194 GC and 45 NC to evaluate the utility as a diagnostic and prognostic biomarker by ELISA. Results: In the screening step, serum ANGPTL2 levels were significantly higher in CRC and GC than in NC respectively (CRC: p<0.01, GC: p<0.01) and gradually increased according to TNM stage progression in CRC and GC. Serum ANGPTL2 levels differentiated CRC or GC from NC with high accuracy (CRC: AUC=0.81, GC: AUC=0.86, respectively). Next, validation step indicated that serum ANGPTL2 levels in CRC patients were significantly higher compared to NC (p<0.0001), had a promising AUC (0.88) to distinguish CRC from NC. In addition, serum ANGPTL2 levels also discriminated early CRC patients (stage I)
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