Abstract
BackgroundDetermination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.MethodsPlasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.ResultsThe percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.ConclusionsMethylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.
Highlights
Colorectal cancer (CRC) is the most frequently diagnosed malignant tumor after lung cancer with an incidence of 13.1% in Europe [1]
A total of 84 matching tissue and plasma samples were analysed using a commercially available real time duplex PCR assay which determines in parallel the amount of methylated Septin 9 (mSEPT9) and total amounts of DNA in the sample
Our analysis of methylated Septin 9 (SEPT9) in matching tissue and plasma samples revealed very low levels of mSEPT9 in the tissue of healthy subjects, which may suggest a physiological role of this epigenetic modification in normal colon tissue
Summary
Colorectal cancer (CRC) is the most frequently diagnosed malignant tumor after lung cancer with an incidence of 13.1% in Europe [1]. CRC screening tests currently in use can be divided into two groups: 1) noninvasive tests for primary cancer detection, such as guaiac fecal occult blood test (gFOBT), fecal immunochemical test (FIT) and stool DNA tests; 2) invasive tests that can detect cancer and advanced lesions, such as flexible sigmoidoscopy, colonoscopy, double-contrast barium enema and virtual colonoscopy [5]. The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with
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