FU Berlin Digitale Dissertation
Florian Nikolaus Thilo : Detection and quantification of colorectal cancer cells in peripheral blood by real-time polymerase chain reaction. Nachweis und Quantifizierung von kolorektalen Tumorzellen in peripherem Blut mittels Real-time PCR
|Abstract| |Table of Contents| |More Information|

Abstract Background: quantifiable detection of circulating cancer cells by RT-PCR ist still laborious, and most markers, being used as targets for RT-PCR, are either not specific or not sensitive enough or both. We investigated two new targets and CEA in a real-time RT-PCR assay. Aims: To establish a quantifiable real-time reverse transcription (RT) polymerase chain reaction (PCR) assay for the detection of circulating colorectal cancer cells, and to assess, whether protease M or colon carcinoma specific gene 1 (ccsg 1) are superior to carcinoembryonic antigen (CEA). Methods: We developed an assay exclusively for protease M and CEA and improved this assay in titration experiments. The protocol was used to investigate blood (n=24) and neoplastic tissue (n=9) from patients with colorectal cancer. Specificy was determined by investigation of blood (n=7) and tissue of healthy volunteers (n=6). Results: In our titration experiments we were able to detect protease M a thousand times of cancer cells earlier than CEA. Of the seven samples of heathy volunteers, one was positive for CEA-mRNA (14%) while none was either positive for protease M (0%) or ccsg 1(0%). Expression levels of protease M and CEA spread widely in normal and neoplastic tissue. Expression of protease M-mRNA did not correlate with that of CEA-mRNA in patient blood samples (p=0,88). Internal standard was not superior to normalization with PBG-D. Conclusion: circulating colorectal cancer cells are detectable and quantifiable by real-time RT-PCR for protease M. Following studies have to show whether protease M is more specific or sensitive than CEA in this regard.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title 1. Einleitung 1.1 Das kolorektale Karzinom 1.2 Tumormarker 1.3 Problemstellung 2. Untersuchungsgut und Methodik 2.1 Verwendetes Untersuchungsgut 2.2 Verwendete Methoden 2.3 Statistische Methoden 3. Ergebnisse 3.1 Ergebnisse der Etablierungsphase 3.2 Ergebnisse an Patientenproben 3.3 Analyse der Schmelzkurve 3.4 Standardkurven 4. Diskussion 4.1 Durchgeführte Experimente 4.2 Methodenkritik 4.3 Methodische Verbesserungsvorschläge 4.4 mRNA-Tumormarker für den Nachweis von kolorektalen Tumorzellen 4.5 Prognostische Signifikanz der zirkulierenden Tumorzellen 4.6 Schlußfolgerung 5. Zusammenfassung 6. Literaturverzeichnis Abkürzungsverzeichnis Danksagung

More Information:
Online available:	http://www.diss.fu-berlin.de/2001/143/indexe.html
Language of PhDThesis:	german
Keywords:	real-time quantitation, cancer cells, rt-pcr, protease M, lightCycler
DNB-Sachgruppe:	33 Medizin
Date of disputation:	07-Sep-2001
PhDThesis from:	Fachbereich Humanmedizin, Freie Universität Berlin
First Referee:	Prof. Dr. med. Ulrich Keilholz
Second Referee:	Prof. Dr. med. Dr. h.c. Liang Chih Tung
Contact (Author):	fthilo@gmx.de
Contact (Advisor):	keilholz@ukbf.fu-berlin.de
Date created:	06-Aug-2001
Date available:	27-Aug-2001

 

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