Comparative Analysis of PCR-Based Biomarker Assay Methods for Colorectal Polyp Detection from Fecal DNA

Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Clinical Chemistry (Impact Factor: 7.91). 07/2009; 55(8):1559-63. DOI: 10.1373/clinchem.2008.122937
Source: PubMed


Aberrantly methylated genes are promising biomarkers for the detection of colon adenomas and colorectal cancers (CRCs). The optimal assay type and specific methylated genes for these assays remain to be determined.
We used genomewide microarray-based assays to identify methylated genes as candidate biomarkers for colon neoplasms. The frequency of aberrant methylation of these genes in primary tumors was assessed with methylation-specific PCR (MSP). The limits of detection and specificities for different types of PCR-based assays were then assessed with the most promising genes identified in this screen. Finally, we assessed the best-performing MSP assay as an early-detection marker using fecal DNA samples.
ITGA4 [integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)] was identified as a novel gene frequently methylated in CRC. Methylated ITGA4 is present in 75% of colon adenomas (n = 36) and 92% of colon adenocarcinomas (n = 75). Comparison of end point MSP, end point MSP with clamped primers, and quantitative fluorescent MSP (qMSP) approaches revealed that both types of end point MSP assays could routinely detect as little as 70 pg DNA, whereas the qMSP assay could routinely detect as little as 7 pg. A fecal DNA qMSP assay for methylated ITGA4 can detect 69% of individuals with colon adenomas (n = 13) with a diagnostic specificity of 79% (n = 28).
Methylated ITGA4 is a promising marker gene for the early detection of colonic neoplasms. qMSP has the lowest limit of detection of the MSP assay types tested, and a qMSP assay that detects methylated ITGA4 has potential as an early-detection assay for colon neoplasms.

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    • "Numerous studies use qualitative techniques to assess methylation status by defining a cut-off value based on the amount of methylated cytosines that are required to repress gene expression. Based on a common PCR-based method of methylation analysis using bisulfite treatment of DNA, the minimum methylation level for a gene to be considered HM is 10–20% methylation (22). In the present study, using a qPCR-based technique, a group of five HM genes with the highest percentage of methylation were identified in CRC patients, RUNX3, PCDH10, SFRP5, IGF2 and Hnf1b. "
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    ABSTRACT: Sporadic colorectal cancer (CRC) is a consequence of the accumulation of genetic and epigenetic alterations that result in the transformation of normal colonic epithelial cells to adenocarcinomas. Studies have indicated that a common event in the tumorigenesis of CRC is the association of global hypomethylation with discrete hypermethylation at the promoter regions of specific genes that are involved in cell cycle regulation, DNA repair, apoptosis, angiogenesis, adhesion and invasion. The present study aimed to investigate the epigenetic changes (DNA methylation) in 24 candidate genes in CRC. A total of 10 candidate hypermethylated (HM) and unmethylated (UM) genes were identified that may be useful epigenetic markers for non-invasive CRC screening. The five genes that had the highest average UM percentages in the control group were MLH1 (71.7%), DKK2 (69.6%), CDKN2A (68.4%), APC (67.5%) and hsa-mir-342 (67.4%). RUNX3 (58.9%), PCDH10 (55.5%), SFRP5 (52.1%), IGF2 (50.4%) and Hnf1b (50.0%) were the five genes with the highest average HM percentages in the test group. In summary, the present preliminary study identified the methylation profiles of normal and cancerous colonic epithelial tissues, and provided the groundwork for future large-scale methylation studies.
    Oncology letters 12/2013; 6(6):1687-1692. DOI:10.3892/ol.2013.1606 · 1.55 Impact Factor
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    • "(ii) ITGA4 [67] "
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    ABSTRACT: Despite multiple screening techniques, including colonoscopy, flexible sigmoidoscopy, radiological imaging, and fecal occult blood testing, colorectal cancer remains a leading cause of death. As these techniques improve, their sensitivity to detect malignant lesions is increasing; however, detection of precursor lesions remains problematic and has generated a lack of general acceptance for their widespread usage. Early detection by an accurate, noninvasive, cost-effective, simple-to-use screening technique is central to decreasing the incidence and mortality of this disease. Recent advances in the development of molecular markers in faecal specimens are encouraging for its use as a screening tool. Genetic mutations and epigenetic alterations that result from the carcinogenetic process can be detected by coprocytobiology in the colonocytes exfoliated from the lesion into the fecal matter. These markers have shown promising sensitivity and specificity in the detection of both malignant and premalignant lesions and are gaining popularity as a noninvasive technique that is representative of the entire colon. In this paper, we summarize the genetic and epigenetic fecal molecular markers that have been identified as potential targets in the screening of colorectal cancer.
    Gastroenterology Research and Practice 09/2012; 2012(3):184343. DOI:10.1155/2012/184343 · 1.75 Impact Factor
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    • "In spite of this, only few genes, including GALR2, ITGA4, NTRK2, OSMR, SFRP1, SFRP2, SLC16A12, TUBG2, MAL and NMDAR2A [8,14,21,22,36,41-43] (reviewed in [44] and [15]) have been found to harbor promoter methylation in more than 80% of colorectal cancers analyzed, and simultaneously in less than 10% of normal mucosa samples, opening up the possibility of achieving high sensitivities and specificities in a future test. Up to now, only a few of the markers mentioned above have been analyzed in non-invasive sample material [13,14,42,45-47]. Additionally, the promising biomarkers SEPT9 [48] and VIM [12] have been analyzed in blood/serum samples and stool samples, respectively and are the only markers included in currently available non-invasive tests. "
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    ABSTRACT: The presence of cancer-specific DNA methylation patterns in epithelial colorectal cells in human feces provides the prospect of a simple, non-invasive screening test for colorectal cancer and its precursor, the adenoma. This study investigates a panel of epigenetic markers for the detection of colorectal cancer and adenomas. Candidate biomarkers were subjected to quantitative methylation analysis in test sets of tissue samples from colorectal cancers, adenomas, and normal colonic mucosa. All findings were verified in independent clinical validation series. A total of 523 human samples were included in the study. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the biomarker panel. Promoter hypermethylation of the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 was frequent in both colorectal cancers (65-94%) and adenomas (35-91%), whereas normal mucosa samples were rarely (0-5%) methylated. The combined sensitivity of at least two positives among the six markers was 94% for colorectal cancers and 93% for adenoma samples, with a specificity of 98%. The resulting areas under the ROC curve were 0.984 for cancers and 0.968 for adenomas versus normal mucosa. The novel epigenetic marker panel shows very high sensitivity and specificity for both colorectal cancers and adenomas. Our findings suggest this biomarker panel to be highly suitable for early tumor detection.
    Molecular Cancer 07/2011; 10(1):85. DOI:10.1186/1476-4598-10-85 · 4.26 Impact Factor
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