title: 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.
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.
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Kinga
authors.initials: K
authors.lastname: Tóth
authors.affiliation: Epigenomics AG, Berlin, Germany.
authors.forename: Reinhold
authors.initials: R
authors.lastname: Wasserkort
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Sipos
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary; Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kalmár
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Barnabás
authors.initials: B
authors.lastname: Wichmann
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Leiszter
authors.affiliation: Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Valcz
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Márk
authors.initials: M
authors.lastname: Juhász
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Pál
authors.initials: P
authors.lastname: Miheller
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Árpád V
authors.initials: ÁV
authors.lastname: Patai
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary; Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.affiliation: 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary; Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
mesh_terms: D000236:Adenoma / Q000097:blood / Q000235:genetics / Q000473:pathology*; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology*; D004247:DNA / Q000097:blood / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008745:Methylation; D008875:Middle Aged; D058112:Septins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D004247:DNA; C474315:SEPTIN9 protein, human; D058112:Septins