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Letters to the Editor |
1 Department of Obstetrics, and Gynecology, University of Tübingen, Calwerstrasse 7, 72070 Tübingen, Germany
aAuthor for correspondence. Fax 49-7071-295424; e-mail sven.becker{at}med.uni-tuebingen.de.
To the Editor:
Despite histopathologic evidence of tumor-free margins, many breast cancer patients suffer from recurrence of their disease. This may reflect the presence of disseminated, premetastatic tumor cells at the time of initial diagnosis and therapy. The detection of disseminated tumor cells in bone marrow/blood has been associated with increased mortality in breast cancer patients (1). Reverse transcription-PCR (RT-PCR) for cytokeratin 19 (CK19) may be a useful alternative or complement to immunocytochemical detection (2). Unfortunately, results of RT-PCR studies vary widely. The reasons for this are conceptual as well as technical.
Because RNAses in samples can degrade RNA before analysis, we measured mRNA by RT-PCR in 15 sets of four samples each that were processed at different time points after collection and addition of a known amount of MCF-7 cells. One sample was processed immediately (
5 min after blood drawing); the other three blood samples were processed after being left in their original tubes at room temperature (10 sets of samples) and on ice (5 sets of samples) for 1, 2, and 8 h, respectively, to simulate routine handling.
After mRNA extraction, RT-PCR for CK19 was performed on the LightCycler® System. Primers and probes were specific for the CK19 gene (Tib Molbiol) and avoided amplification of the CK19 pseudogene (3). We used a calibration curve and the Fit Points Method of the LightCycler quantification software. Experimental details are available in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol50/issue4/. We repeated this set-up 10 times for experiments with samples kept at room temperature and 5 times for experiments with samples kept on ice.
The apparent concentration of tumor cells decreased with increasing time of storage (Fig. 1
). In 10 of 15 cases, the "1 h" samples yielded results that showed fewer or an equivalent amount of cells compared with the "0 h" results. In 12 of 15 cases, fewer cells were amplified in the "2 h" samples than in the "1 h" samples. Recovery of any cells was possible in only 4 of 15 "8 h" samples. We observed no difference in terms of recovery for samples that were kept at room temperature and those that were kept on ice at each time point.
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Our results demonstrate that the time between sample collection and RNA stabilization is an important factor when the amounts of cellular RNA to be detected are small, as in the case of disseminated tumor cells. Despite our expectations, results from experiments on samples stored on ice did not differ from results for those kept at room temperature. Detection of very small amounts of specific mRNA species, i.e., at the limits of detectability, might therefore be highly sensitive to even small amounts of RNase activity.
Most research groups use enrichment methods such as density gradient centrifugation over Ficoll or epithelial antigen magnetic bead enrichment before RNA isolation and/or freezing. These are lengthy procedures, which take at least 60–90 min before cells can be added to a RNA-stabilizing environment, and thus risk the loss of amplifiable RNA (4)(5). If RNA-based assays for the detection of minimal residual disease in blood or bone marrow are to be introduced into routine laboratory procedures with the potential to complement and/or substitute for immunocytochemical assays, the time between sample collection and processing in the laboratory must be taken into account. Techniques requiring less time, such as direct mRNA isolation and subsequent RT-PCR, might offer advantages to time-consuming enrichment strategies.
Acknowledgments
This work was supported by Angewandte Klinische Forschung Grant 75-0-0.
References
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