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. 2014 Dec 19;9(12):e115415.
doi: 10.1371/journal.pone.0115415. eCollection 2014.

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA

Kinga Tóth  1 , Reinhold Wasserkort  2 , Ferenc Sipos  1 , Alexandra Kalmár  3 , Barnabás Wichmann  4 , Katalin Leiszter  1 , Gábor Valcz  4 , Márk Juhász  1 , Pál Miheller  1 , Árpád V Patai  1 , Zsolt Tulassay  3 , Béla Molnár  3
Affiliations

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA

Kinga Tóth et al. PLoS One. .

Abstract

Background: Determination 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.

Methods: Plasma 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.

Results: The 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.

Conclusions: Methylated 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.

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Conflict of interest statement

Competing Interests: Hereby the authors would like to declare that at the time this study was performed the co-author Reinhold Wasserkort was employee of Epigenomics AG, and currently still is shareholder of this company. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. CT values of the assay for mSEPT9 in tissue and plasma samples.
Box-plot graphs of CT values for mSEPT9 from healthy (NED - no evidence of disease), adenoma (AD) and cancer (CRC) tissue and plasma samples. The upper and lower edges of each box plot represent the 25th percentile and the 75th percentile, respectively. The line across each box represents the median value for the variable. Individual values are plotted as red dots.
Figure 2
Figure 2. PMR values in plasma and tissue samples.
Percent of methylated reference (PMR) of mSEPT9 in healthy (NED - no evidence of disease), adenoma (AD) and cancer (CRC) tissue samples. All tissue and plasma samples are shown individually, and the order of the matching samples within each group is the same. Significance levels for groups comparisons: NED vs. CRC in plasma: p = 0.01; adenoma vs. CRC in plasma: p<0.01; NED vs. adenoma in tissue: p<0.001; NED vs. CRC in tissue: p<0.001.
Figure 3
Figure 3. SEPT9 methylation correlation in tissue, plasma and methylation correlation with cfDNA amounts in plasma cases.
A, Correlation of mSEPT9 levels between matched SEPT9 positive plasma and tissue cancer samples plotted with logarithmic scales with R2 = 0.008. B, Correlation of mSEPT9 levels between matched SEPT9 positive plasma samples from cancer group and cfDNA (circulating cell-free DNA) amounts plotted with logarithmic scales with R2 = 0.254 for stage I+II and R2 = 0.483 for stage III+IV.
Figure 4
Figure 4. Septin-9 immunohistochemistry in tissue samples.
Decreased epithelial expression of Septin-9 protein (brown cytoplasmic immunoreaction) in adenoma (B) and CRC (C) compared to the normal (A) samples (Digital microscope pictures, 20x relative magnification). This observation of Septin-9 protein expression level correlates with previous outcomes .
See this image and copyright information in PMC

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References

    1. Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, et al. (2007) Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 18:581–592. - PubMed
    1. Pignone M, Saha S, Hoerger T, Mandelblatt J (2002) Cost-effectiveness analyses of colorectal cancer screening: a systematic review for the U.S. Preventive Services Task Force. Ann Intern Med 137:96–104. - PubMed
    1. Siegel R, Desantis C, Virgo K, Stein K, Mariotto A, et al. (2012) Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62:220–241. - PubMed
    1. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, et al. (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319:525–532. - PubMed
    1. Smith RA, Cokkinides V, Brooks D, Saslow D, Brawley OW (2010) Cancer screening in the United States, 2010: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin 60:99–119. - PubMed

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