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. © 2003 Wiley-Liss, Inc. Key words: colorectal cancer; real-time RT-PCR; minimal residual disease; carcinoembryonic antigene; cytokeratin 20; protease MThe molecular monitoring of circulating tumor cells by RT-PCR, routinely applied in patients with certain leukemias and lymphomas, 1-4 is still under debate for patients with solid malignancies. In colorectal cancer, where indication for adjuvant therapy without metastasis is yet performed by histologic investigation of lymph nodes, 5 the immunocytochemical identification of epithelial cells in bone marrow 6 encouraged the detection of epithelial mRNA markers in blood and bone marrow by RT-PCR. 7 However, a series of subsequent investigations by a number of groups with conventional nested PCR led to conflicting reports on both the frequency of gene transcripts in blood of patients and the specificity of the method. 8 -20 The recent availability of real-time PCR equipment has obviously changed the situation. 21-23 The quantification of low-level background transcription allows the definition of cutoff values for marker expression in blood and thus improves specificity. 16,22,24,25 Furthermore, the reliable quantification of housekeeping gene expression allows excellent quality control on a per-sample basis and relates absolute marker concentration to sample quality.We now devel...

Prognostic significance of circulating tumour cells following surgical resection of colorectal cancers: a systematic review

“…Five studies were excluded as they were reviews or systematic reviews, which did not report on prognostic outcomes (Tsavellas et al, 2001;Sleijfer et al, 2007;Riethdorf et al, 2008;Sergeant et al, 2008;Tsouma et al, 2008). Ten studies were excluded as they did not report on prognostic outcomes (Denis et al, 1997;Nakamori et al, 1997;Douard et al, 2001;Guadagni et al, 2001;Patel et al, 2002;Silva et al, 2002;Yokoyama and Yamaue, 2002;Schuster et al, 2004;Yeh et al, 2006;Lagoudianakis et al, 2009). The outcomes measured in the 14 studies included in this review were too heterogeneous with regards to the methodology of data collection to allow quantitative analysis.…”

A Gene Expression Profile of Peripheral Blood in Colorectal Cancer

“…A recent review of real-time PCR-based assays with single molecular markers, such as CEA, CK19, and CK20, demonstrated low sensitivity, ranging from 4% to 35.9%, 25.9% to 41.9%, and 5.1% to 28.3%, respectively [13] . One study, performed with a newly identified molecular marker known as ProtM [27] , also attained unsatisfactory sensitivity. Circulating cancer cells from any cancer type are capable of disseminating from solid tumor tissues, penetrating and invading blood vessels, and circulating in the peripheral blood [28][29] .…”

Clinical Significance of Human Kallikrein Gene 6 Messenger RNA Expression in Colorectal Cancer

“…Because KLK6 has been found to be highly expressed in colorectal cancer cells as well as in ovarian cancer cells (12,15,16), we hypothesize that KLK6 may also have potential applications in the clinical diagnosis and monitoring of colorectal cancer. To the authors' knowledge, no information has been published concerning the clinical significance of KLK6 mRNA expression in colorectal cancer.…”

“…Several authors have reported that KLK6 mRNA is highly expressed in ovarian cancer tissues (6,12), and recent studies have indicated that hK6 could potentially be a biomarker for the diagnosis and monitoring of ovarian cancer (13,14). In colorectal cancer, KLK6, KLK8, and KLK10 have been found to be up-regulated in cancer tissues compared with normal tissues (15,16).…”