1 Department of Oncology, UZ Gasthuisberg, Leuven, Belgium; 2 National Cancer Institute, Cairo, Egypt; 3 Experimental Laboratory Medicine, UZ Gasthuisberg, Leuven; 4 Rega Institute, K.U. Leuven, Leuven, Belgium
Received 25 June 2002; revised 9 December 2002; accepted 4 April 2003
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Abstract |
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We previously developed a real-time quantitative RTPCR technique to detect breast carcinoma cells in peripheral blood (PB). The aim of the current study was to improve cytokeratin 19 (CK19) quantification using plasmid dilutions of cloned PCR fragments to obtain a more reliable and reproducible quantification of CK19 transcripts.
Materials and methods:
PB samples of 14 stage IV breast cancer patients and 23 healthy controls were examined with RTPCR using plasmid quantification.
Results:
Median CK19+ copy numbers of one and 11 were detected in the control group and stage IV breast cancer patients, respectively (MannWhitney, P 0.0001). When comparing the results obtained using cell line dilutions with those obtained using plasmid dilutions, a good correlation was observed (r2 = 0.98).
Conclusions:
Plasmid dilutions are more reliable than cell line dilutions for quantification of gene expression, and more objective criteria for positivity could be defined based on the characteristics of the standard curve (slope and intercept). A more universally accepted agreement on the definition of the cut-off value for positivity is needed.
Key words: breast cancer, cytokeratin 19, plasmid dilutions, quantitative RTPCR
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Introduction |
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We previously developed a real-time quantitative RTPCR technique to detect breast carcinoma cells in PB. This technique is sensitive and has a high reproducibility with many advantages over classic quantitative PCR methods. We detected significantly elevated levels of cytokeratin 19 positive (CK19+) cells in PB of <10% of the volunteers, in ±30% of stage IIII patients and in >70% of stage IV breast cancer patients [4]. We also applied the same technique to bone marrow samples of stage IIIIa breast cancer patients [5]. In the current study, the aim was to further validate our technique and to improve CK19 quantification using plasmid dilutions of the cloned PCR fragment to obtain a more reliable and reproducible quantification of CK19 transcripts.
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Materials and methods |
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Sample processing, RNA extraction and cDNA synthesis
We have previously described these procedures extensively [4]. In brief, starting from RNAzol lysates, total RNA was extracted using chloroform, precipitated with isopropanol and washed with 70% ethanol. The resulting pellet was redissolved in nuclease-free water. RNA concentrations were measured using a spectrophotometer (260 nm/280 nm). After heating at 65°C for 5 min to denature RNA and to inactivate RNases, 1 µg total RNA was subjected to reverse transcription using 300 U M-MLV Reverse Transcriptase (Life Technologies, Gaithersburg, MD, USA), 30 U RNasin RNase inhibitor (Promega, Madison, WI, USA), 25 µM random hexamer primers and RT buffer containing 250 mM TrisHCl pH 8.3, 375 mM KCl and 15 mM Mg2+ in a total volume of 40 µl at 37°C for 2 h. The reaction was terminated by heating at 65°C for 10 min.
Plasmid construction, amplification and purification
A 101-bp CK19-PCR fragment generated using primers, as described by Slade et al. [11], was cloned in the pGEM®-T Easy vector (Promega) and introduced in Escherichia coli TG1. From a selected transformant containing the desired construct, plasmid DNA was isolated using the Wizard® Plus SV Miniprep DNA purification system (Promega). The resulting CK19 plasmid was subsequently linearised by NcoI digestion. In addition, PCR fragments for two control genes, ß2-microglobulin (ß2m) and ß-glucuronidase (GUS), were cloned in the pCR®II-TOPO® vector (Invitrogen, Carlsbad, CA, USA; Life Technologies). To purify both plasmids, the same technique as for CK19 was applied. The resulting ß2m and GUS plasmids were linearised by BamHI and HindIII digestion, respectively.
Dilutions of linearised plasmids were prepared in 1 mM TrisHCl pH 8.0, 0.1 mM EDTA containing 50 µg/ml E.coli tRNA. Serial 10-fold dilutions were made in the range of 1 x 106 copies to one copy.
PCR
For each PCR, 6 µl cDNA (diluted 1:3 in nuclease-free water) or plasmid product (serial dilutions), 25 µl Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 900 nM forward primer, 900 nM reverse primer, 200 nM probe and nuclease-free water were added to a final volume of 50 µl. Amplification and detection were performed with the ABI Prism 7700 sequence detection system (Applied Biosystems). The thermal cycle used was 2 min at 50°C, 10 min at 95°C and 50 cycles of 15 s denaturation at 95°C and 1 min annealing at 60°C.
Quantification
Quantification was based on the Taqman principle. Before annealing to the target sequence, the fluorescence of the dual labelled (one fluorochrome, one quencher molecule) oligonucleotide probe is quenched. Upon hybridisation of the probe to the target sequence, the probe is hydrolysed by the 5'3' exonuclease activity of Taq polymerase. This results in an increase in fluorescence intensity proportional to the accumulation of PCR product. ROX, a rhodamine derivative, is present in the buffer solution as a passive reference label. A normalised signal is generated by the equation: Rn = (Rn+) (Rn), where Rn+ represents the ratio of reporter signal at any given time and the quencher baseline signal and Rn the ratio of the reporter baseline signal and the quencher baseline signal. Subsequently, the threshold is set at 10 times the standard deviation of the mean baseline emission calculated for the first cycles. The cycle threshold (Ct) is defined as the cycle number at which a samples
Rn fluorescence crossed this threshold, which represents a positive PCR result [4].
Standard curve
A standard curve was calculated using linear regression analysis. This standard curve displayed a linear relationship between Ct values and the logarithm of the initial number of positive cells or input plasmid copy number. The dynamic range of the standard curve spanned at least six orders of magnitude. The amount of product in a particular sample is determined by interpolation from a standard curve of Ct values generated from the plasmid dilution series.
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Results |
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When comparing the results obtained using cell line dilutions [4] with those obtained using plasmid dilutions, a good correlation was observed (r2 = 0.98). Equivalent results were obtained for both control genes (ß2m and GUS) after normalisation (r2 = 0.996) and their expression level was similar between the analysed samples (Figure 1).
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Table 2 shows a statistical comparison of the results obtained using either cell lines or plasmid dilutions for quantification.
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Discussion |
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Plasmid dilutions offer several advantages over dilutions based on cell lines. They are more reliable than cell line dilutions for the quantification of gene expression since variation between batches is minimal, whereas differences in expression levels might occur in cell lines grown at different time points or at different passages. Moreover, different cell lines express different levels of CK19, thus, making it difficult to estimate the significance of a certain expression level. Quantification using plasmids is more accurate since absolute copy numbers can be calculated based on concentration measurements. In addition, plasmid quantification is more reproducible since the variation between different PCR runs is extremely low. Our data showed that both ß2m and GUS could be used for normalisation of the PCR results.
Recently, Stathopoulou et al. [6] showed that the detection of CK19 mRNA-positive cells in the blood had prognostic implications for patients with stage I and II breast cancer, because positivity was associated with a reduced disease-free interval (P = 0.0007) and overall survival (P = 0.01). However, the PCR results were not quantitative, which might be necessary for the fine tuning of prognostic information. For chronic myeloid leukaemia it was shown that the actual level of positivity was related to the probability of relapse [7]. Moreover, in view of the significant level of false-positive results observed in healthy volunteers, non-quantitative PCR data might result in erroneous predictions. In another study, it was shown that it is possible to monitor disease response from PB samples using quantitative RTPCR in patients with metastatic breast cancer [8].
The detection of CK19-positive cells might thus be used as a surrogate marker in identifying patients who are at increased risk of relapse and could be candidates for further systemic adjuvant therapy. To the best of our knowledge, there are no prospective studies in the literature that have considered micrometastases as a factor in the basis of selecting treatment option for solid tumours.
A recurrent problem in several papers [1114] is the definition of a cut-off for positivity. Since the markers used for the detection of circulating tumour cells are generally not tumour-specific, and since background expression is often observed in the control groups, it is of great importance to precisely define the cut-off for positivity.
In our study, the upper limit of the 95% CI of the median of control group was used as the cut-off, and thus any sample containing greater than three copies was considered positive. Some studies used the highest value in the control group as the cut-off, and thus any values in the samples above this cut-off were considered positive for circulating tumour cells [911].
Shammas et al. [14] used as the cut-off definition the mean + 2 x standard deviation of the values of CK19 expression in their control group (healthy volunteers and patients without epithelial cancer).
Table 3 shows how applying these different definitions of cut-off on our results (both for quantification with cell line dilutions and with plasmid dilutions) resulted in significantly different numbers of positive samples compared with our original method.
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We conclude that plasmid dilutions are more reliable than cell line dilutions for the quantification of gene expression, and more objective criteria for positivity could be defined based on the characteristics of the standard curve (slope and intercept). A more universally accepted agreement on the definition of the cut-off value for positivity is needed to overcome the discrepancy between different studies. This would allow a better evaluation of the significance of detecting circulating tumour cells by RTPCR within each study and a better comparison between the respective studies.
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Acknowledgements |
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Footnotes |
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Correspondence to: Professor R. Paridaens, Gezwelziekten, Laboratorium Experimentele Oncologie, UZ Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. Tel: +32-1634-6900; Fax: +32-1634-6901; E-mail: robert.paridaens{at}uz.kuleuven.ac.be
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