ABSTRACT Colorectal cancer is the second most common cause of malignant death in industrialized c... more ABSTRACT Colorectal cancer is the second most common cause of malignant death in industrialized countries. More accurate detection markers are needed to improve the effectiveness and efficiency of both the screening and surveillance of colorectal neoplasia. Aberrant DNA methylation occurs early in oncogenesis, is stable once initiated, and can be assayed in both primary tissues and remote clinical samples. While DNA methylation markers are attractive candidates for colorectal cancer screening, robust searches are needed to discover DNA methylation sequences that optimally discriminate colorectal neoplasia from other diseased and histologically normal tissues for accurate testing in body fluid samples. Using well-characterized normal and neoplastic specimens, a candidate methylation-based gene microarray of oligonucleotides and a genome-wide methylation discovery process was utilized to identify markers for colorectal neoplasia. To identify markers highly specific for colorectal neoplasia, 12 genes selected from the candidate gene array and 30 fragments from the discovery process were validated on a second methylation-based microarray using a larger, more diverse sample set. Sequences were probed with DNA extracted from 89 colorectal adenocarcinomas, 56 colorectal polyps, 31 inflammatory bowel disease samples, 123 extracolonic cancers, and 67 endoscopically proven normal tissues. We found markers highly methylated in colorectal neoplasia. The 16 most discriminating methylated sequences comprise both known and predicted genes including EYA4, N33, BCL6, FCGR2A, SIX6, NGFR, Q96PX8, DLX5, C14orf59, PCDH17, HVIAAT, SMAD7, CSPG2, GSK3B, CD44, and TMEFF2. Normal epithelium and extracolonic cancers revealed significantly lower or no methylation. Individual markers displayed sensitivities > 80% and specificities > 85% indicating the DNA methylation markers identified in this study can correctly classify colorectal neoplasia from other normal and diseased tissues and are attractive marker candidates for clinical testing.
Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues an... more Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.
Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establ... more Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.
ABSTRACT Colorectal cancer is the second most common cause of malignant death in industrialized c... more ABSTRACT Colorectal cancer is the second most common cause of malignant death in industrialized countries. More accurate detection markers are needed to improve the effectiveness and efficiency of both the screening and surveillance of colorectal neoplasia. Aberrant DNA methylation occurs early in oncogenesis, is stable once initiated, and can be assayed in both primary tissues and remote clinical samples. While DNA methylation markers are attractive candidates for colorectal cancer screening, robust searches are needed to discover DNA methylation sequences that optimally discriminate colorectal neoplasia from other diseased and histologically normal tissues for accurate testing in body fluid samples. Using well-characterized normal and neoplastic specimens, a candidate methylation-based gene microarray of oligonucleotides and a genome-wide methylation discovery process was utilized to identify markers for colorectal neoplasia. To identify markers highly specific for colorectal neoplasia, 12 genes selected from the candidate gene array and 30 fragments from the discovery process were validated on a second methylation-based microarray using a larger, more diverse sample set. Sequences were probed with DNA extracted from 89 colorectal adenocarcinomas, 56 colorectal polyps, 31 inflammatory bowel disease samples, 123 extracolonic cancers, and 67 endoscopically proven normal tissues. We found markers highly methylated in colorectal neoplasia. The 16 most discriminating methylated sequences comprise both known and predicted genes including EYA4, N33, BCL6, FCGR2A, SIX6, NGFR, Q96PX8, DLX5, C14orf59, PCDH17, HVIAAT, SMAD7, CSPG2, GSK3B, CD44, and TMEFF2. Normal epithelium and extracolonic cancers revealed significantly lower or no methylation. Individual markers displayed sensitivities > 80% and specificities > 85% indicating the DNA methylation markers identified in this study can correctly classify colorectal neoplasia from other normal and diseased tissues and are attractive marker candidates for clinical testing.
Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues an... more Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.
Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establ... more Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.
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