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: 7.77). 07/2009; 55(8):1559-63. DOI: 10.1373/clinchem.2008.122937
Source: PubMed

ABSTRACT 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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    ABSTRACT: Aberrantly methylated genes are increasingly being established as biomarkers for the detection of colorectal cancer (CRC). In the present study, the methylation levels of the secreted frizzled-related protein gene 2 (SFRP2), GATA binding protein 4/5 (GATA4/5), N-Myc downstream-regulated gene 4 (NDRG4) and vimentin (VIM) promoters were evaluated for their use as markers in the noninvasive detection of CRC. Methylation-specific polymerase chain reaction was performed to analyze promoter CpG methylation of SFRP2, GATA4/5, NDRG4 and VIM in the fecal DNA of 56 patients with CRC and 40 individuals exhibiting normal colonoscopy results. Promoter methylation levels of SFRP2, GATA4/5, NDRG4 and VIM in CRC patients were 57.1% (32/56), 42.9% (24/56), 83.9% (47/56), 28.6% (16/56) and 41.1% (23/56), respectively. Furthermore, the specificity of the genes were 90.0% (4/40), 95.0% (2/40), 82.5% (7/40), 97.5% (4/40) and 85.0% (6/40), respectively. The overall sensitivity of detection for fecal DNA with at least one methylated gene was 96.4% (54/56) in CRC patients. By contrast, only 14 of the 40 normal individuals exhibited methylated DNA in the aforementioned promoter regions. Methylation of the SFRP2, GATA4/5, NDRG4 and VIM promoters in fecal DNA is associated with the presence of colorectal tumors. Therefore, the detection of aberrantly methylated DNA in fecal samples may present a promising, noninvasive screening method for CRC.
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    ABSTRACT: To evaluate the accuracy of methylation of genes in stool samples for diagnosing colorectal tumours.
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    ABSTRACT: To investigate GATA5, SFRP2, and ITGA4 methylation in plasma DNA as noninvasive biomarkers for colorectal cancer (CRC) or adenomas. There were 57 CRC patients, 30 adenomas patients, and 47 control patients enrolled in this study. Methylation-specific polymerase chain reaction was used to determine the promoter methylation status of GATA5, SFRP2, and ITGA4 genes in plasma DNA, and their association with clinical outcome in CRC. The predictive ability of GATA5, SFRP2, and ITGA4 methylation, individually or in combination, to detect CRC or adenomas was further analyzed. Hypermethylated GATA5 was detected in plasma in 61.4% (35/57) of CRC cases, 43.33% (13/30) of adenoma cases, and 21.28% (10/47) of control cases. The hypermethylation of SFRP2 was detected in 54.39% (31/57), 40.00% (12/30), and 27.66% (13/47) in plasma samples from CRC, adenomas, and controls, respectively. ITGA4 methylation was detected in 36.84% (21/57) of plasma samples of CRC patients and in 30.00% (9/30) of plasma samples from patients with colorectal adenomas, and the specificity of this individual biomarker was 80.85% (9/47). Moreover, GATA5 methylation in the plasma was significantly correlated with larger tumor size (P = 0.019), differentiation status (P = 0.038), TNM stage (P = 0.008), and lymph node metastasis (P = 0.008). SFRP2 and ITGA4 methylation in plasma significantly correlated with differentiation status (SFRP2, P = 0.012; ITGA4, P = 0.007), TNM stage (SFRP2, P = 0.034; ITGA4, P = 0.021), and lymph node metastasis (SFRP2, P = 0.034; ITGA4, P = 0.021). From the perspective of predictive power and cost-performance, using GATA5 and SFRP2 together as methylation markers seemed the most favorable predictor for CRC (OR = 8.06; 95%CI: 2.54-25.5; P < 0.01) and adenomas (OR = 3.35; 95%CI: 1.29-8.71; P = 0.012). A combination of GATA5 and SFRP2 methylation could be promising as a marker for the detection and diagnosis of CRC and adenomas.


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