Andrew Sledziewski

Circulating Methylated SEPT9 DNA in Plasma Is a Biomarker for Colorectal Cancer

BACKGROUND The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. METHODS A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. RESULTS The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. CONCLUSIONS Circulating methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

Background Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. Methodology/Principal Findings Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%. Conclusions/Significance Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome‐wide search.

Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

BackgroundThe septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa.MethodsLaser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC).ResultsRegions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues.ConclusionsHypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon®), providing further support for the clinical relevance of this biomarker.

Identification and Validation of Colorectal Neoplasia–Specific Methylation Markers for Accurate Classification of Disease

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. (Mol Cancer Res 2007;5(2):153–63)

T2026 Circulating Methylated Septin 9 DNA in Plasma Is a Biomarker for Colorectal Cancer

Introduction: The low sensitivity of the fecal occult blood tests (FOBT) based on the guaiac have made to develop new immunochemical tests with an efficiency to detect advanced neoplasia (advanced adenoma and colorectal cancer) still not well established. Aim: Determine the sensitivity and specificity of a FOBT based on the guaiac (FOBTg) with a quantitative immunochemical FOBT (FOBTi) to detect advanced neoplasia. Material and methods: Patients: all individuals with a colonoscopy programmed by any cause (screening, surveillance or symptoms) were included. Individuals with personal history of inflammatory bowel diseases or colorectal cancer were excluded. During the days previous to the exploration three high sensitivity FOBTg and two FOBTi (positive test ≥100ng/ml) were completed. The sample was calculated for demonstrating the superiority of the FOBTi of 15% of sensitivity with a final sample of 700 individuals. Results We presented the results of the first 140 individuals. The colonoscopy was performed for symptoms in 66% of cases and for screening or surveillance in 34% of cases. The colonoscopy detected 16 individuals (11%) with an advanced neoplasia (2 infiltrant carcinoma and 14 advanced adenomes). Other finds were: 20 patients with non advanced adenomes, 11 with polyps not adenomatous and 4 inflammatory colitis. The colonoscopy was normal in 88 (63%) individuals. The percentage of positive test was from 3,5% (5/140) and 16,5% (23/140) for the FOBTg and FOBTi, respectively. The sensitivity, specificity, positive predictive value and negative predictive value to detect an advanced neoplasia were 12%, 97%, 40% and 89% with the FOBTg and of 62,5%, 89%, 60,8%, 81% for the FOBTi. If considered only the first immunochemical test, the sensitivity, specificity, positive predictive value and negative predictive value were 43,7%, 92,7%, 43,7%, 92,7%, respectively. Conclusion The FOBTi is very superior to the FOBTg to detect advanced neoplasia although the high number of false positives could limit its use. The utilization of one or two FOBTi should be appraised depending on the disposable endoscopic resources.

Identifizierung und Validierung von spezifischen Methylierungsmarkern zur Klassifizierung von kolorektalen Karzinomen und normalem Kolongewebe

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.