Abstract 1850: The detection of methylated Septin 9 in tissue and plasma of colorectal neoplasia and its relationship to the amount of free circulating DNA
Authors: Kalmar Alexandra , Toth Kinga , Wasserkort Reinhold , Sipos Ferenc , Wichmann Barnabas , Valcz Gabor , Tulassay Zsolt , Molnar Bela 
Published: May 2015

Abstract
Background
The determination of methylated Septin 9 (SEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, it has not been investigated how the methylated DNA detected in plasma relates to the occurrence of methylated DNA in colon tissue. The goal of this study was to quantitatively compare levels of methylated SEPT9 level in matched plasma and tissue samples of healthy, adenoma and CRC cases; and to determine the of amount of circulating free DNA (cfDNA) and the expression of Septin-9 protein in tissue.
Methods
Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 adenomas and 34 CRC. Following bisulfite conversion of DNA a commercial real time PCR assay was used to determine the total amount of DNA in each sample and the portion of DNA methylated at a specific locus of the Septin 9 gene. The presence of Septin-9 protein was determined using immunohistochemistry in healthy (n=10), adenoma (n=14) and CRC (n=13) samples.
Results
Similar concentrations of cfDNA were detected in NED (49.72 ng/ml), adenoma (45.39 ng/ml) and CRC (70.32 ng/ml) plasma cases. In tissue samples, percent methylated reference (PMR) values of SEPT9 above a selected 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. PMR differences found between NED vs. adenoma and NED vs. CRC comparisons were highly significant (p&lt;0.001). The protein expression of Septin-9 in tissues determined by IHC inversely correlated to SEPT9 methylation levels with abundant expression in normals and diminished expression levels in adenomas and tumors. In matching plasma samples SEPT9 PMR values, using a cut-off level higher than 0.01%, were detected in 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC cases. Highly significant PMR difference were observed in comparisons between NED vs. CRC (p&lt;0.01) and adenoma vs. CRC (p&lt;0.01).
Conclusions
Methylated SEPT9 was detected in all tissue samples, at very low levels in the NED group, but at significantly elevated levels in both adenoma and CRC. In plasma samples, elevated mSEPT9 values were only detected in the CRC group, but not in the adenoma group. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Parameters like the degree of vascularization of the lesions, the amount of cfDNA in plasma and probably additional factors seem to be equally important.
Citation Format: Alexandra Kalmar, Kinga Toth, Reinhold Wasserkort, Ferenc Sipos, Barnabas Wichmann, Gabor Valcz, Zsolt Tulassay, Bela Molnar. The detection of methylated Septin 9 in tissue and plasma of colorectal neoplasia and its relationship to the amount of free circulating DNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2014-1850