The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease

Abstract

The dissemination of cancer cells is a prerequisite in the development of micrometastases and solid metastases. Our previous examinations of these cells were based on immunocytological staining of tumor-associated antigens and cytokeratins. We have now developed a highly sensitive and specific detection method based on a nested reverse-transcriptasepolymerase-chain reaction (RT-PCR) of The main problem after surgical treatment of patients with colorectal, gastric or pancreatic cancer is the frequent recurrence of disease. Routine diagnostic methods fail to detect circulating tumor cells, distant micrometastases or early metastases (Gusterson, 1991). The detectability of single tumor cells would be a matter of urgent necessity, not least because adjuvant therapy concepts have been proposed for elimination of these malignant cells (Moertel et al., 1990; Riethmuller et al., 1994). To date, the detection of disseminated single tumor cells in the bone marrow of patients with gastrointestinal cancer is performed mainly by immunocytological methods (Schlimok et al., 1987(Schlimok et al., , 1991Juhl et al., 1994). In order to establish a method with increased sensitivity, our group, like others (Traweek et al., 1993; Gerhard et al., 1994), chose a PCR-based detection procedure.The use of cytokeratin-20 as a promising marker for the detection of epithelial tumor cells in the bone marrow and the peripheral blood has been proposed by Burchill et al. (1995). CK-20 is a member of the epithelial sub-group of the multigene family of the intermediate filament proteins, the cytokeratins. The expression of CK-20 mRNA and protein is almost entirely confined to gastric and intestinal epithelium, urothelium and Merkel cells. Nearly all colorectal adenocarcinomas tested and many adenocarcinomas of the stomach and the pancreas revealed CK-20 mRNA and protein expression (Moll et al., . 1993. With the CK-20-specific nested RT-PCR we have established a sensitive and specific method for the detection of disseminated epithelial tumor cells. Bone-marrow samples of patients with malignant gastrointestinal diseases were examined for CK-20 mRNA expression. To ensure that this finding of CK-20 mRNA at the molecular level is dependent on the presence of carcinoma cells, we investigated bone-marrow samples of a control group from healthy donors and of patients with benign gastrointestinal diseases, hematological neoplasms and infectious diseases. MATERIAL AND METHODS Cell linesThe cell lines and culture conditions have been described (Kalthoff et al., 1991(Kalthoff et al., ,1993 (Dr. T. Okabe, Tokyo, Japan) from pancreatic carcinoma; MKN-7, MKN-28, Kato 111 (Dr. Hirai, Tokyo, Japan) from gastric carcinoma; SW1116 and HT29 from colorectal carcinoma (Dr. W. Dippold, Mainz, Germany); Huh7 (Dr. P. Galle, Heidelberg, Germany) and MZ-1 (Dr. W. Dippold) were from liver carcinoma. Bone-marrow samplesHeparinized bone-marrow aspirates ( 5 ml) were obtained from patients with colorectal carcinoma (n = 57) and pancreatic carcinoma (n = 11). As a control,...

Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes

“…CK20 RT-PCR assays were performed as published (Soeth et al, 1996). Each sample was investigated twice and scored positive, if at least in one assay a specific amplification occurred.…”

“…In a number of clinical CK20 RT-PCR studies, comparable diagnostic specificities have been achieved for conventional (Burchill et al, 1995), nested RT-PCR (Soeth et al, 1996) and even double-nested RT-PCR (Funaki et al, 1998) systems. Recent studies have assigned clinical significance and prognostic value to the detection of CK20 mRNA-positive cells in bone marrow and peripheral blood samples of breast, gastric, pancreatic and colorectal cancer patients (Soeth et al, 1996(Soeth et al, , 1997Bostick et al, 1998;Weitz et al, 1998).…”

“…Recent studies have assigned clinical significance and prognostic value to the detection of CK20 mRNA-positive cells in bone marrow and peripheral blood samples of breast, gastric, pancreatic and colorectal cancer patients (Soeth et al, 1996(Soeth et al, , 1997Bostick et al, 1998;Weitz et al, 1998). Initiated by our interest in influential factors in RT-PCR diagnostics (Jung et al, 1997a(Jung et al, , 1998 and the observation of positive RT-PCR results from normal specimens, we have, in this study, systematically investigated the occurrence of illegitimate transcription of CK20 mRNA and its influence on the specificity of RT-PCR assays.…”

Differential expression of carcinoembryonic antigen (CEA) splice variants in whole blood of colon cancer patients and healthy volunteers: implication for the detection of circulating colon cancer cells

“…In the 1950's, cytological studies demonstrated that circulating cancer cells from solid tumors are detectable in the blood of cancer patients (Engell, 1955). In the 80's and 90's highly sophisticated techniques were developed, such as immunocytology (Redding et al, 1983;Schlimok et al, 1987;Juhl et al, 1994) and RT -PCR (Gerhard et al, 1994;Burchill et al, 1995;Soeth et al, 1996) enabling detection of circulating colon cancer cells in various body compartments. In colon cancer finding of minimal residual disease in bone marrow, peritoneal lavage and lymph node samples correlated with poor survival (Lindemann et al, 1992;Schott et al, 1998;Liefers et al, 1998).…”

Haematogenous cytokeratin 20 mRNA as a predictive marker for recurrence in oral cancer patients

“…Several groups have reported use of the RT-PCR method to detect prostatic cancer cells (Fadlon et al, 1996;Henke et al, 1997;Melchior et al, 1997), melanoma cell (Foss et al, 1995), and neuroblastoma cells (Burchill et al, 1995b) in peripheral blood by amplifying prostatic specific antigen, tyrosinase and tyrosine hydroxylase respectively. Some investigators used the epithelium-specific cytokeratin (CK8, CK19 or CK20) as a marker for detecting epithelial cancer cells which did not have any specific markers (Burchill et al, 1995a;Krismann et al, 1995;Soeth et al, 1996). However, Burchill et al (1995a) and Krisman et al (1995) reported that CK8 and CK19 were amplified in a high proportion of normal peripheral blood samples, but CK20 was not amplified in any normal blood samples.…”