title: Quantitative real-time RT-PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers.
abstract: The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer.
authors.affiliation: Department of Medicine III, University Hospital Benjamin Franklin, Free University Berlin, Berlin, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Schuster
authors.forename: Nicole
authors.initials: N
authors.lastname: Max
authors.forename: Benno
authors.initials: B
authors.lastname: Mann
authors.forename: Karin
authors.initials: K
authors.lastname: Heufelder
authors.forename: Florian
authors.initials: F
authors.lastname: Thilo
authors.forename: Jörn
authors.initials: J
authors.lastname: Gröne
authors.forename: Franziska
authors.initials: F
authors.lastname: Rokos
authors.forename: Heinz-Johannes
authors.initials: HJ
authors.lastname: Buhr
authors.forename: Eckhard
authors.initials: E
authors.lastname: Thiel
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keilholz
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D002272:Carcinoembryonic Antigen / Q000235:genetics; D016022:Case-Control Studies; D003106:Colon / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000473:pathology; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics; D007610:Kallikreins / Q000235:genetics; D053554:Keratin-20; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D011379:Prognosis; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D002272:Carcinoembryonic Antigen; D007381:Intermediate Filament Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C103551:KLK6 protein, human; D007610:Kallikreins