Cytokeratin-20 reverse-transcriptase polymerase chain reaction as a new tool for the detection of circulating tumor cells in peripheral blood and bone marrow of bladder cancer patients.
OBJECTIVE
Systemic progression is the prevalent form of bladder tumor recurrence after radical cystectomy. The ability to detect circulating tumor cells in peripheral blood or bone marrow could be of prognostic value for the disease with the consequence of early adjuvant chemotherapy. We established a sensitive and specific method using a double cytokeratin-20 (CK-20) reverse-transcriptase polymerase chain reaction (RT-PCR) to detect circulating bladder cancer cells in venous blood and bone marrow


METHODS
The sensitivity of the detection method was determined by a serial dilution of bladder cancer cells from the cell line HT1376 in whole blood. Bone marrow from 20 bladder cancer patients was drawn prior to radical cystectomy and CK-20 cDNA was amplified by RT-PCR. Additionally, pre- and postoperative venous blood samples from 11 of these patients with bone marrow aspirates and 9 patients undergoing only transurethral resection of the bladder as well as blood samples of 25 healthy volunteers were investigated by CK-20 RT-PCR.


RESULTS
The detection limit of the method was 2 bladder cancer cells/ml whole blood containing one million peripheral blood mononuclear cells. The positive detection rate in bone marrow was 7 of 20 (35%) for bladder cancer patients of all stages. However, investigation of the preoperatively collected venous blood samples from 20 patients revealed onyl 2 positive findings, belonging to advanced tumor stages pT4pN0M0 and pT3pN2M0. In contrast, CK-20 was detected in 3 of 20 postoperatively collected venous blood samples from patients with low tumor stages (pTaNXM0 and pT1NXM0) as well as from 1 patient with pelvic lymph node metastases (pT3apN2M0). All venous blood samples of the control group (n = 25) were negative for CK-20.


CONCLUSIONS
The detection of circulating bladder tumor cells in venous blood and bone marrow by the CK-20 RT-PCR is a promising approach that could improve risk assessment and the identification of bladder cancer patients who would benefit from adjuvant chemotherapy.
Retz
M
M
Section of Experimental Urology, Department of Urology, Medical School, Christian Albrechts University of Kiel, Germany. mretz@urology.uni-kiel.de
Lehmann
J
J
Röder
C
C
Weichert-Jacobsen
K
K
Loch
T
T
Romahn
E
E
Lühl
C
C
Kalthoff
H
H
Stöckle
M
M
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000097:blood; D001853:Bone Marrow / Q000473:pathology*; D005260:Female; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000473:pathology
D014408:Biomarkers, Tumor; D007381:Intermediate Filament Proteins