Original articles
Analysis of promoter methylation in stool: A novel method for the detection of colorectal cancer

https://doi.org/10.1016/S1542-3565(04)00624-XGet rights and content

Background & Aims: Detection of tumor-derived DNA alterations in stool is an intriguing new approach with high potential for the noninvasive detection of colorectal cancer (CRC). Because of heterogeneity of tumors, usually multiple markers distributed throughout the human genome need to be analyzed. This is labor intensive and does not allow for high through-put screening. Therefore, markers with high sensitivity and good specificity are needed. We explored the potential of a single epigenetic marker in comparison with fecal occult blood testing (FOBT) for the discrimination of patients with CRCs and adenomas from those without. Methods: Methylation-specific polymerase chain reaction (PCR) was performed to analyze hypermethylated in cancer 1 (HIC1) promoter methylation status in a blinded fashion in stool samples from 26 patients with CRC, 13 with adenoma ≥1 cm, 9 with hyperplastic polyps, 9 with chronic inflammatory bowel disease, and 32 with endoscopically normal colon. Results: Ninety-seven percent of the stool samples contained amplifiable DNA. Forty-two percent of the samples from patients with CRC and 31% of the samples from patients with colorectal adenoma ≥1 cm were positive for HIC1 promoter methylation. No methylated HIC1 promoter DNA was detected in the fecal DNA from patients with endoscopically normal colon or hyperplastic polyps. Conclusions: The epigenetic marker HIC1 promoter methylation carries high potential for the remote detection of CRCs. We postulate that a panel of merely a few genetic and epigenetic markers will be required for the highly sensitive and specific detection of CRCs and adenomas in fecal samples from affected patients.

Section snippets

Patients and stool samples

The investigation was approved by the ethical committee of the Medical Faculty of the University of Munich. Stool samples were collected preoperatively from patients with verified CRCs (Table 1), and before endoscopy from patients with adenomas ≥1 cm (Table 2), endoscopically normal colons, hyperplastic polyps, and chronic inflammatory bowel disease (Table 3). Most patients with CRCs had been referred with a known diagnosis, all persons with adenomas and normal colons were asymptomatic and

Establishment of hypermethylated in cancer 1 promoter methylation analysis and determination of the sensitivity of the assay

DNA was extracted from microdissected tissues from primary CRCs, microdissected samples from normal colonic epithelium from individual patients, and peripheral blood leukocytes from healthy individuals. A nested MSP amplifying a CpG island within the HIC1 promoter 1b was established (Figure 1A). We found 7 of 9 cancers (78%) to contain both methylated and unmethylated and 2 cancers to contain unmethylated HIC1 DNA only (Figure 1B and data not shown). Furthermore, none of 8 samples from normal

Discussion

Highly sensitive, specific, and easily analyzable markers are required for noninvasive stool-based CRC screening. We show here that DNA methylation can be detected in stool of CRC patients with high specificity. The use of the DNA methylation marker, HIC1 promoter methylation, allowed for the highly specific detection of CRCs in 42% and of adenomas ≥1cm in 31% of cases. Interestingly, sensitivity was quite high for early cancers and higher for adenoma than FOBT. The combination of HIC1 MSP with

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    Supported by the Sturm-Stiftung and the Bohnewand-Stiftung (to F.T.K.).

    1

    K.L. and G.T.B. contributed equally to this work.

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