Quantification of circulating cancer cells in whole blood samples by real time quantitative RT–PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT–PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT–PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P<0.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 79°C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 82°C melting temperature. The 82°C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 79°C to the 82°C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT–PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.
The main limitation of current cancer therapy is the development of metastatic disease which, in most patients, is not curable. In colon cancer, which counts for approximately one-third of all cancer related deaths in the US, cure depends on radical surgery prior the development of metastases. However, even after an apparent curative surgical intervention approximately 50% of the patients experience metastatic relapse that in most cases eventually leads to death.
Dissemination of tumor cells is an obligatory step toward progression to metastatic disease. 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).
Detection and quantification of circulating cancer cells in whole blood samples could serve as a unique and easy diagnostic tool to determine prognosis and therapeutic effectiveness of cancer therapy. So far, only RT–PCR has been shown to provide the sensitivity and practicability necessary to detect the low numbers of colon cancer cells present in whole blood samples.
In this study we evaluate CEA and CK20 as target molecules for the detection and quantification of circulating cancer cells by real-time RT–PCR because these are frequently used markers which have been successfully applied by several independent research groups (Gerhard et al., 1994; Burchill et al., 1995, Liefers et al., 1998; Mori et al., 1996; Soeth et al., 1997). In particular, we studied the phenomenon of CEA amplification from normal white blood cells. CEA is of special interest because it plays an active role in colon cancer progression and is used as a target molecule in a variety of clinical trials. Therefore, monitoring of the level of CEA expressing colon cancer cells is an attractive approach to determine therapeutic effectiveness in such trials. However, amplification has been reported in normal samples by using high cycle numbers indicating broad expression of these markers, in particular of CEA, at low levels (Mori et al., 1996; Ko et al., 1998; Goeminne et al., 2000). Quantification of the PCR signal would allow calculation of cell numbers. It would also solve problems arising from background signals by defining cut-off values in cancer patients. Because significant differences between patients with metastatic colon cancer and healthy volunteers are mandatory to assume any clinical benefit, we focused this clinical pilot study on these two groups.
Comparison Amplifluor® and SYBR®-Green forCEA RT–PCR
The Amplifluor® kit requires an annealing temperature not higher than 60°C. Because the CEA RT–PCR was established at annealing temperatures of 72°C for the B and C primer pair we first compared the Amplifluor® method and SYBR®-Green at both temperatures using CEA full length cDNA, HT29 and LS147T colon cancer cells, and samples from healthy volunteers. The specificity of the PCR reaction did not differ between 60°C and 72°C as demonstrated by gel electrophoresis, melting curve analysis and sequencing (data not shown). Both methods allowed reliable quantification of CEA cDNA. However, the sensitivity of the SYBR®-Green approach was approximately 1000-fold higher and, thus, was sensitive enough to detect signals from whole blood samples. Therefore, we chose this approach for further studies. Standard curves ranged from 10−9 g to 10−18 g of CEA cDNA, 10−6 to 10−11 g total RNA of HT29 cells for the detection of CK20 and from 107 to 101 copy numbers of GAPDH (Figure 1a–c). Dilution of total RNA from HT29 and LS147T colon cancer cells showed that real time RT–PCR was able to detect reliable signals from one cell (data not shown). The specificity was controlled by melt curve analysis (Figure 1d). The purity and length of the product was confirmed by gel electrophoresis and sequencing.
Analysis of clinical samples
As shown in Figure 2 both markers resulted in a significant background signal in the healthy control group suggesting low expression of CEA and CK20 in white blood cells. However, because of the extremely high values of a subset of patients the mean values in cancer patients were 10-fold higher for CEA (P>0.05) and 10 000-fold higher for CK20 (P<0.001). When we took the highest value of the control group as a cut-off level, two cancer patients (6%) had elevated CEA values. In contrast, using CK20 as a detection marker 12 cancer patients (38%) had at least 100-fold higher values (P<0.001). Nine of these patients (75%) had also high CEA levels, i.e. values which were in the upper range of the CEA real time PCR spectrum.
Assuming that a single HT29 cancer cell contains approximately 1 pg total RNA and that HT29 colon cancer cells and circulating cancer cells have similar CK20 levels, as many as 104 cancer cells/ml blood circulate in this group of metastatic cancer patients.
Detection of CEA splice variant
The melting curve analysis of PCR products revealed a surprising result for CEA. All samples of the control and cancer patients groups showed a CEA product at 79°C melting temperature instead of the expected 82°C. However, 30% of cancer patients had in addition the 82°C product (Figure 3a). Both products were indistinguishable by agaorse gel electrophoresis (not shown).
To further study this phenomenon, which indicates the presence of a different CEA-like product in white blood cells, we spiked LS147T cells in 10 ml whole blood of a healthy volunteer using a 10-fold dilution from 107 cells to 10 cells. From this we determined the melting point of the products of real-time RT–PCR. As shown in Figure 3b we found a major peak at 79°C but with increasing numbers of tumor cells the peak shifted stepwise to 82°C and completely replaced the 79°C peak when the tumor cells exceeded the number of white blood cells. This data strongly suggest that a competing target cDNA is present in white blood cells, which interferes with the measurement of the tumor cell CEA cDNA (lower panel).
We sequenced the real-time PCR products from three different control blood samples, three cancer patients' samples which showed both products, and from human HT29 and LS147T colon cancer cells. Figure 4a gives an overview of the CEA gene and the derived CEA cDNA (Figure 4b,c). The sequence of the PCR product from both cancer cell lines and the CEA cDNA control plasmid were homologous with the CEA-cDNA sequence obtained from the NCBI database (Figure 4b). In addition, the sequence of the PCR product of one cancer patient with the highest 82°C peak corresponded to the CEA cDNA sequence expressed by colon cancer cell lines. In contrast, all three samples from the control group and two of the cancer group resulted in a product which is composed by a truncated part of the M exon (Exon 9), followed by intron sequence starting shortly before the last exon (Exon 10). The following 3′-exon sequence (Exon 10) is identical to tumor cell CEA cDNA products (Figure 4c). Blast search (National Center for Biotechnology Information, NCBI) did not reveal any known corresponding protein.
The purpose of this study was to establish real-time RT–PCR using CEA and CK20 as detection markers for quantification of circulating tumor cells in whole blood of colon cancer patients. Previous studies indicate that both markers may serve as specific tumor cell markers in the analysis of whole blood samples (Gerhard et al., 1994; Burchill et al., 1995; Liefers et al., 1998; Mori et al., 1996; Soeth et al., 1997). However, according to our experience and reports from others (Mori et al., 1996; Ko et al., 1998; Goeminne et al., 2000) low CEA and CK20 levels are also present in normal blood samples. Thereby, standardization becomes difficult between various experiments and certainly between different research groups because a positive signal depends highly on minor assay condition in relation to cycle numbers. Quantification by real-time RT–PCR should circumvent the problem of ‘false positive’ signals by measuring background levels and defining cut-off values. Furthermore, this approach allows convenient follow-up measurements and, thus, may become a useful marker for therapeutic monitoring.
In the first series of experiments we compared the Amplifluor® and SYBR®-Green methods. The Amplifluor® approach was less sensitive and achieved only significant signals in patient's samples when used in a nested-PCR approach. Because a nested approach significantly altered the quality of the standard curve, reliable quantification was not possible. Because of its high sensitivity, SYBR®-Green was the staining method of choice. The higher risk of measuring unspecific fluorescence signals was controlled by melt curve analysis of each sample. Anyway, unspecific products did not appear in this study.
To demonstrate the ability of the method to distinguish between cancer and non-cancer we chose two extreme groups, i.e. metastatic colon cancer patients and healthy volunteers. A method which would not demonstrate differences in blood levels of these markers between these groups obviously would be of little clinical benefit.
We found CK20 to be a useful marker to define cancer cells because 38% of cancer patients showed at least 100-fold higher CK20 values compared to the highest value in the healthy control group. This finding is in accordance with studies using conventional nested RT–PCR, which demonstrated in stage IV colon cancer patients approximately 40% CK20 positivity of whole blood samples (Soeth et al., 1997). However, our study also clearly shows that CK20, an ‘epithelial cell marker’, is expressed at low levels by mesenchymal white blood cells and underscores the importance of signal quantification. Assuming that HT29 colon cancer cells and circulating cancer cells in patients have similar CK20 levels, we can roughly estimate that up to 104 cancer cells/ml blood circulate in metastatic cancer patients.
Only few clinical studies have analysed the usefulness of CEA for detection of circulating colon cancer cells in whole blood samples and none of them have quantified the PCR product by real-time RT–PCR. Recently, a large study was published which applied the primers used in our study to determine CEA mRNA level in whole blood cells before and after surgical therapy (Guadagni et al., 2001). In contrast to our study, CEA nested RT–PCR revealed a high number of CEA positive cancer patients but did not demonstrate a CEA signal in the healthy control group. It remains unclear why over a long postoperative period blood samples remained positive despite curative surgery. Other groups detected CEA in a significant number of normal blood samples (Ko et al., 1998) or in patients with inflammatory bowel disease (Castells et al., 1998) positive for CEA. It was also found that treatment with GM–CSF induced CEA expression in white blood cells (Jung et al., 1998). Overall, the usefulness of CEA as a PCR-based tumor cell detection marker remains questionable. In our study, there was only 10-fold increase in the CEA signal for cancer patients compared to the control group. Only two patients (6%) were responsible for this small and insignificant elevation of the mean value. Thereby, our detection rate was lower than in previous studies (Gerhard et al., 1994) but in accordance with studies which did not find significant differences between cancer patients and a control group (Ko et al., 1998). However, because we used a different method, i.e. real time RT–PCR, a direct comparison of our data with those studies is difficult.
Further analysis revealed that normal white blood cells express a splice variant of CEA in which an ‘intron’ sequence replaces part of the exon. This finding is highly surprising because such a splice variant has not been described in white blood cells yet. In addition to the splice variant, we found cancer patients also had the known CEA product found in colon cancer cells. A spiking experiment with tumor cells mimicked the results of the melt curve analysis of cancer patients indicating that the ‘cancer cell product’ competes with a different product in white blood cells. Most recently, we confirmed the presence of the splice variant using a primer, which binds to the intron sequence a few nucleotides before the 3′ exon starts. The combination of this primer with primer C produced the same sequence of the splice variant (data not shown). So far, splice variants for members of the CEA-family have been described for BGP (CEACAM1) (Hammarström, 1999) and CGM2 (CEACAM7) (Thompson et al., 1994) but not for CEA itself and it will be very interesting to define the corresponding protein. CEA is the most widely known tumor marker in colon cancer and high expression levels have been correlated with poor survival (Grem et al., 1997; Vogel et al., 2001). CEA contributes to the metastatic capacity of colon cancer cells by an adhesive function (Benchimol et al., 1989; Gangopadhyay et al., 1998) and, as shown most recently, by protecting tumor cells against apoptosis (Soeth et al., 2001; Wirth et al., 2002). Our findings may help to better understand its role in tumor biology. It is an interesting hypothesis that the CEA splice variant represents a physiological product, which is distinct from CEA expressed in malignancy. Clearly, further studies are needed to support this speculation, which is supported by the fact that CEA research has exclusively focused on cancer cell CEA. For example, the determination of the Exon/Intron structure of the CEA cDNA sequence has been derived from human colon cancer cells (Schrewe et al., 1990).
In summary, our study shows that circulating colon cancer cells can be detected and quantified in whole blood samples using real-time RT–PCR, in particular by CK20 measurement. Therefore, further studies are under way to determine its use as an intermediate endpoint marker to determine therapeutic effectiveness including surgically treated early stage cancer patients. Our data help to explain why ‘background signals’ have been frequently reported (Goeminne et al., 2000) despite the lack of CEA protein-expression by white blood cells. Subsequent redesign of the primer may help to overcome this problem. The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.
Materials and methods
Human HT29 and LS147T colon cancer cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and were maintained in continuous culture at 37°C/5% CO2 using modified IMEM (Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 1% glutamine and 10% heat-inactivated fetal bovine serum (FBS).
Following IRB approval by the board of Georgetown University, whole blood was obtained from 32 patients who were treated for metastatic colon cancer. In addition, we analysed blood samples from 17 healthy volunteers. The first 5 ml of blood was discarded to avoid contamination with skin cells. 20 ml of blood was collected in Vacutainer tubes with K3 EDTA (Becton Dickinson-Franklin Lakes, NJ, USA).
Mononuclear cells were separated using Ficoll-Paque Plus (Amersham, Piscataway, NJ, USA) density centrifugation as described (Juhl et al., 1994). Cells were resuspended in RNA-STAT60 (Tel-Test, Friendswood, TX, USA) and total RNA was prepared according to the manufacturer's manual modified by the use of 30 mg glycogen during isopropanol precipitation at −20°C for 30 min. Preparation of cDNA was performed as described (Gerhard et al., 1994) using random hexamer primer (PE Applied Biosystems, Foster City, CA, USA).
Primer and standard
Initially, we compared the sensitivity and reliability of the Amplifluor®- and the SYBR®-Green approach. Table 1a gives an overview about the primer sequences used in the PCR. Amplification of CEA was performed using previously published sequences, i.e. for the nested approach primer pair A/B and primer pair C/B, respectively (Gerhard et al., 1994). With respect to the Amplifluor®-method, the required Z-sequence (see protocol of manufacturer) was attached to the C-primer. CK20 PCR-amplification was performed using primers and protocol published by Burchill et al. (1995).
As a standard for CEA we used full length CEA cDNA ligated into the bluescript vector (Invitrogen) which was kindly provided by Dr M Neumaier, Dept. of Clinical Chemistry, Hamburg, Germany. As a CK20 standard we prepared an RNA stock of HT29 colon cancer cells which express high CK20 levels (Soeth et al., 1996). The relative amount of CK20 refers to the total RNA of HT29 cells.
Real-time RT–PCR using Amplifluor®
For CEA, cDNA was directly amplified (2 μg total RNA) using primer pair B and C (modified with Z-tail). Alternatively, a nested RT–PCR was performed as previously published (Gerhard et al., 1994) with 20 cycles using 2 μg total RNA and primer pair A and B preceding the real-time measurement. The protocol followed the manual of the ‘Amplifluor Universal Amplification and Detection Kit’ (Intergen, Purchase, NY, USA) using Platinum Taq DNA polymerase (Life Technologies, Rockville, MD, USA). The conditions for the PCR reactions are shown in Table 1b. Modification to the previously published protocol included an annealing temperature of 60°C instead of 72°C which is a requirement of the kit. Before doing so we confirmed that this had no impact on the specificity of the PCR reaction. For amplification, we used the ‘iCycler’ a real time PCR machine purchased from BioRad (Hercules, CA, USA).
Real-time RT–PCR using SYBR®-Green
All reagents except Platinum Taq polymerase (Life Technologies) were obtained from the ‘SYBR®-Green Core Reagents Kit’ (PE/Applied Biosystems). For CEA, we used the primer pair B and C as previously published. A modification of the published protocol (Gerhard et al., 1994) included an annealing temperature of 60°C instead of 72°C and the use of 40 cycles. The change of the annealing temperature allowed comparison with the Amplifluor® method. Analysis of CEA full length cDNA, colon cancer cells and blood samples showed no differences in specificity between both annealing temperatures.
GAPDH-Real time RT–PCR
To control for the RNA amount in the reaction mix we used GAPDH, a common housekeeping gene. An aliquot of each sample was analysed in parallel to CEA and CK20 RT–PCR using the ‘Amplifluor Direct Gene Systems Kit for GAPDH’ (Intergen) and Platinum Taq DNA polymerase (Life Technologies). This kit also provides a GAPDH cDNA template to create a standard curve. The procedure followed the protocol provided by the manufacturer. The threshold levels obtained from the CEA and CK20 PCR were adjusted to the threshold levels found in the GAPDH reaction to correct for minor variations in RNA loading.
PCR products were directly sequenced in the core facility of the Lombardi Cancer Center using the ‘Big Dye Terminator Sequencing Reaction Kit’ (PE/Applied Biosystems).
For statistical analysis the unpaired t-test was used.
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Dan P Hartman, Department of Pathology, Georgetown University Medical Center and Jeff M Smith, Bio-Rad Laboratories, for numerous helpful suggestions This work was supported by a grant of the National Institute of Health/National Cancer Institute (#R01-CA088972), and Lombardi Cancer Research Center Core Facility, U.S. Public Health Service Grant 2P30-CA-51008
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Hampton, R., Walker, M., Marshall, J. et al. 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. Oncogene 21, 7817–7823 (2002). https://doi.org/10.1038/sj.onc.1205906
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