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Etablierung eines Verfahrens zum Nachweis epigenetischer Biomarker im peripheren Blut zur Stratifizierung der Therapie des Rektumkarzinoms

Fully-automated hypermethylation testing by One-Step-Real-Time-PCR of 6 different potential epigenetic biomarkers in peripheral blood for rectal cancer detection and follow-up.

Tobias Thormann
Doctoral thesis
Date of Examination: 2015-12-17
Date of issue: 2015-12-08
Advisor: Legler, Tobias Prof. Dr.
Referee: Legler, Tobias Prof. Dr.
Referee: Gaedcke, Jochen PD Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0028-8664-B

 

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Abstract

English

Bisulfite treatment based assays are mainly used to detect aberrant, tumor specific methylation of specific DNA regions either from tissue or from free circulating DNA in plasma or serum. Since bisulfate based protocols are difficult to fully automate the aim of this study was to develop a rapid protocol which allows the detection and follow-up of rectal cancer from the primary blood tube. Methods: In an one step methylation sensitive, restriction enzyme based quantitative Real-Time PCR, hypermethylation in the promoter regions of RUNX3, NEUROG1, SEPT9, HLTF, TMEFF2, SDC2 and RASSF1A was investigated. Sensitivity and Specificity was evaluated after DNA-isolation-process from tumor tissue and plasma from 25 patients and 48 voluntary blood donors for each gene. The diagnostic sensitivity as well as specificity was detected from plasma was up to 20 % and 100 % for RUNX3, 12 % and 96 % for NEUROG1, 53 % and 96 % for SEPTIN9, 16 % and 96 % for TMEFF2 and 56 % and 100 % for SDC2. Sensitivity evaluated from tumor tissue-DNA by similar specificity was 87 % (RUNX3), 9 % (NEUROG1), 100 % (SEPTIN9), 91 % (TMEFF2), 87 % (SDC2) and 35 % (RASSF1A). For these results this study-analysis revealed that a fully automatable test for hypermethylated promoter regions for the detection and follow-up of solid cancer can be developed. Further results of this study are retained and will published later on.    
Keywords: Rectal Cancer; RUNX3; NEUROG1; HLTF; SEPTIN9; TMEFF2; SDC2; RASSF1A; CpG-Island-Methylation; One-Step-Real-Time-PCR; Epigenetic Methylation; Hypermethylation
Schlagwörter: Ein-Schritt-Real-Time-PCR; Epigenetische Biomarker; Therapiestratifizierung; Rektumkarzinom; Restriktionsendonuklease; Bisulfit-Konversion
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