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Clinical Cancer Research Vol. 6, 2741-2750, July 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Analytical Variables of Reverse Transcription-Polymerase Chain Reaction-based Detection of Disseminated Prostate Cancer Cells1

Alfred Zippelius, Ralf Lutterbüse, Gert Riethmüller and Klaus Pantel2

Institut für Immunologie der Universität München, Munich, 80336 Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early systemic spread of occult tumor cells that may develop into founders of incurable distant metastasis has been identified in prostate cancer patients by reverse transcription-PCR (RT-PCR) amplification of prostate-specific antigen (PSA) mRNA. Nevertheless, the introduction of this new staging tool into the clinical setting has been hampered by the disparate and contradictory data on the sensitivity and specificity of RT-PCR methods reported recently. We used PSA RT-PCR to examine the influence of analytical variables such as priming and enzyme of reverse transcriptase reaction, temperature and time of primer annealing, primer extension and denaturation, as well as the concentrations of magnesium chloride, Taq polymerase, deoxynucleotide triphosphate, primers and BSA on the amplification process. By systematically varying these chemical and physical components, we could demonstrate a significant increase in amplification yield and in stringency of primer annealing. This may explain the wide variety of published findings on molecular staging of prostate cancer, which currently impedes the clinical introduction of PSA RT-PCR assays in prostate cancer. Methodological analyses are needed for standardization and quality assurance to achieve reproducible molecular methods that can be used in clinical practice.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Million-fold amplification of DNA by the PCR is a powerful method to detect rare tumor-specific DNA (1) . This approach has been proven to be successful in the detection of residual tumor cells in malignant lymphomas and leukemias by amplification of tumor-specific nucleic acid sequences derived from chromosomal translocations or rearrangements of the immunoglobulin gene (2 , 3) . In contrast, the genomic characteristics of epithelial cancer cells are more heterogeneous (4) , except for certain mutations in the Ki-ras oncogene in colon or pancreatic carcinomas (5) . As a consequence, many investigations focused on the development of RT-PCR3 assays to screen for rare epithelial-specific or tumor-associated mRNA species in mesenchymal tissue, such as bone marrow, peripheral blood, or lymph node.

In prostate cancer, the most common current target transcripts of choice for detection of disseminated tumor cells are PSA and human glandular kallikrein, members of the kallikrein-like serine protease family (6 , 7) , and prostate-specific membrane antigen (PSM), a type II integral membrane protein (8) . As could be shown, expression is almost entirely restricted to prostatic epithelium, making these mRNA species interesting marker molecules in identifying prostate cancer cells (9 , 10) . However, the reported data on the sensitivity and specificity of these RT-PCR assays are still controversial, and the clinical relevance of positive findings in blood, bone marrow, and lymph nodes of patients with prostate cancer are also under debate (11) . One of the reasons for the discrepant findings might be the enormous variability of the applied assays. Even in the cases where the same primers for the amplification of PSA mRNA have been used, the RT-PCR conditions varied considerably, which might have caused the tremendous differences in the incidence of PCR-positive findings comparing patients at similar clinical and pathological stages (Tables 1Citation 2)Citation . Moreover some authors showed low-level illegitimate transcription of PSA mRNA in peripheral blood cells that may result in false-positive findings (12 , 13) . Many of the differences are probably caused by variabilities in the PCR conditions used by the various laboratories. At present, there is no consensus regarding the exact conditions, because they must be optimized for each set of primers.


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  		Table 1 Comparison of different components of reverse transcription in PSA RT-PCR assays used in studies on prostate cancer patients

 

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  		Table 2 Comparison of different components of PCR amplification in PSA RT-PCR assays used in studies on prostate cancer patients

 
The purpose of this work was to demonstrate the influence of different PCR conditions on the specificity (negatives identified/actual true negatives) and sensitivity (positives detected/actual true positives) of RT-PCR assays for PSA mRNA. This may form the basis for an international standardization of RT-PCR procedures used for the detection of occult cancer cells in blood, bone marrow, or lymph nodes. We used PSA RT-PCR as model for exemplifying the variability of PCR reactions, because PSA is the most predominant marker of prostate cancer cells, and there is increasing evidence that PSA RT-PCR assays may contribute to the molecular staging of prostate cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Extraction of mRNA.
Mononuclear cells from peripheral blood leukocytes and bone marrow aspirates were isolated by density gradient centrifugation through Ficoll-Hypaque (Pharmacia, Freiburg, Germany) at 400 g for 30 min. One million of these cells were removed for RT-PCR analyses and immediately spun for 5 min at 1000 x g. For purifying total RNA, the guanidinium thiocyanate method of Chomczynski and Sacchi (14) was modified as follows. The cell pellet was homogenized with 300 µl of solution D [4 M guanidine thiocyanate, 0.5% Sarkosyl (N-laurylsarcosine sodium salt), and 25 mM sodium citrate] by vigorous vortexing and stored at -20°C until needed. Sequentially, 30 µl of 2 M sodium acetate (pH 4.0), 300 µl of water-saturated phenol, and 90 µl of chloroform were added to the homogenate. After mixing thoroughly by vortexing, the samples were set on ice for 15 min and were then centrifuged at 13,000 rpm for 5 min. The aqueous phase was transferred to a fresh tube with 300 µl of a phenol-chloroform-isoamyl alcohol mixture (25:24:1, v/v/v). Again after vortexing and centrifugation (1 min at 13,000 rpm), the upper phase was reextracted with 300 µl of chloroform. Subsequently, the RNA was precipitated with 300 µl of isopropanol, 40 µl of 3 M sodium acetate (pH 5.0), and 20 µg of glycogen at -20°C overnight. After centrifugation, the RNA pellet was washed with 70% ethanol, dried for 4 min, and dissolved in 5 µl of HPLC-water. Cell lines used as positive controls for the PCR were prepared as described above.

RT-PCR.
Half of the total RNA was reverse transcribed using specific primers for PSA cDNA 5'-TGACGTGATACCTTGAAGCA-3' (nucleotides 970–989), as deduced from the cDNA sequence (15) ; the other half was reverse transcribed using random hexamer primers (Roche, Mannheim, Germany). The synthesis was carried out with a First-strand cDNA Synthesis kit (Life Technologies, Inc., Karlsruhe, Germany), including Superscript II in a final volume of 10 µl containing 50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM total dNTP, 0.2 µM of each of the specific primers, or 1.6 µg of the random primers and 100 units Superscript II. After the addition of RNA and initial denaturation at 70°C for 5 min to obtain single-strand mRNA, the samples were incubated at 37°C for 1 h and subsequently diluted with 10 µl of HPLC-water. Specific cDNA sequences were amplified in a reaction mix (10 µl) of 1 µl of cDNA, 1 µl of 10x PCR buffer [100 mM Tris (pH 8.3), 500 mM KCl, and 10 mM MgCl2], 100 µM dNTP, 0.4 µM of each of the two primers, 5 µg of BSA (Roche), and 0.5 unit of Taq DNA polymerase (Roche). The following PCR primers (Fig. 1)Citation were generated for detecting PSA cDNA: PSA sense, 5'-CTT GTA GCC TCT CGT GGC AG-3' (nucleotides 426–445); and PSA antisense, 5'-GAC CTT CAT AGC ATC CGT GAG-3' (nucleotides 699–719).



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  		Fig. 1. Schematic representation of a portion of the PSA gene showing the relative positions of the specific PCR primers.

 
Using genomic DNA as negative control, primers were chosen to hybridize on different exons of the targeted gene amplifying a PSA cDNA-specific product (293 bp; Fig. 4Citation ). The amplification was performed on a Hybaid Thermal Reactor (Biometra, Göttingen, Germany) with the following cycling profile using a plate control: denaturation at 94°C for 4 min, annealing at 64°C for 30 s, and extension at 72°C for 2 min for the first cycle; denaturation at 93°C for 40 s, annealing at 64°C for 30 s, and extension at 72°C for 20 s for 7 cycles; denaturation at 93°C for 40 s, annealing at 57°C for 30 s, and extension at 72°C for 20 s for 7 cycles; denaturation at 93°C for 40 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s for 50 cycles; and terminal extension at 72°C for 2 min and cooling at 30°C for 1 min. To check the integrity of the extracted mRNA, RT-PCR with recently published primers for EGP-40 (16) and primers for p53 was performed on the randomly primed cDNA. The primers for p53 were: p53 sense, 5'-GGA TGA CAG AAA CAC TTT TCG-3' (nucleotides 618–638); and p53 antisense, 5'-TCA GCT CTC GGA ACA TCT C-3' (nucleotides 1015–1033), amplifying a 415-bp fragment. PCR products were analyzed on agarose by gel electrophoresis using a 1-kb DNA ladder (Life Technologies, Inc.) and by direct visualization with ethidium bromide. The staining of PCR products on different gels was comparable by the addition of equal amounts of ethidium bromide (0.2 µg/ml) and equal concentrations of agarose (1.5%). Duplicate experiments demonstrated the reproducibility of the results. The specificity of the amplification products obtained was monitored with adequate restriction site analysis (SacI) and DNA sequencing. Mock RNA preparations were routinely processed as negative controls.



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  		Fig. 4. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with M-MLV reverse transcriptase and Superscript II. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–6, 5 LNCaP cells mixed in 106 MNCs: Lanes 3 and 4, M-MLV; Lanes 5 and 6, Superscript II.

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The PCR process consists of cycles of a three-step procedure, including denaturation, annealing, and extension. The principle of the RT-PCR technology is shown in Fig. 2Citation . PCR is carried out in a series of three-step cycles producing a detectable amount of target mRNA. Each cycle starts with a denaturation step at 92°C to 96°C to render the duplex DNA. This is followed by an annealing step during which the primers hybridize to a complementary site of the template. Finally DNA polymerase extends each primer by successive addition of dNTP forming complexes with Mg2+. As shown in Fig. 2Citation , DNA will continue to accumulate exponentially. The following analytical steps and chemical components are critical for the efficiency of cDNA amplification, and modification of these components can influence sensitivity and specificity of the RT-PCR assay: priming and enzyme of RT reaction, temperature and time of primer annealing, primer extension and denaturation, as well as the concentrations of magnesium chloride, Taq polymerase, dNTP, primers, and BSA.



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  		Fig. 2. Schematic diagram of PCR consisting of denaturation, annealing, and extension.

 
The following data will show the potential influence of each physical and chemical component on the amplification yield and the specific priming of the oligonucleotides. All experiments were performed with dilutions of LNCaP cells mixed in 1 x 106 MNCs. PCR optimization is decisive for amplification of low-abundance target mRNA species, such as PSA in bone marrow or peripheral blood samples of cancer patients. The consistent cellular expression of wt-p53 in the surrounding normal hematopoietic cells makes it an appropriate control to check the integrity of the input RNA. Moreover, we performed parallel RT-PCR assays specific for epithelial glycoprotein-40, which also is an appropriate positive control because of low level expression in hematopoietic cells (16) . However, the amplification rate of wt-p53 with the high cycle numbers used in our experiments reaches the plateau phase, resulting in a constantly high signal intensity. Therefore, variations of wt-p53 RT-PCR cannot be used to demonstrate any increase or decrease of amplification yield, i.e., assay sensitivity. Therefore, we used exogenous reconstruction experiments to assess the efficiency of reverse transcription and, after PCR amplification, by diluting cells of a prostate cancer cell line (LNCaP) in MNCs. In addition, MNCs from peripheral blood of healthy persons were used to control for nonspecific reactions. Mock preparations were routinely processed as negative controls for RNA purification and RT-PCR. The cycle parameters outlined here were found to be suitable for thermal cyclers with a metal block using a plate control and the particular primer sequences as described above. However, the exemplified variables affect RT-PCR-based assays in general.

Reverse Transcription.
First-strand synthesis of single-stranded mRNA in cDNA enables amplification with the Taq polymerase, a DNA-dependent polymerase. However, if the transcript of interest is only present in low abundancy, the variety of reverse transcriptase reaction (e.g., priming, RT enzyme) is a crucial issue concerning the assay sensitivity. As demonstrated in Fig. 3Citation , we could observe a higher signal intensity, representing a higher amplification yield, by using a specific primer, whereas the signal intensity was lower using oligo(dT) nucleotides or random hexanucleotides. In a similar manner, the introduction of a reverse transcriptase enzyme (Superscript II) featuring reverse transcriptase activity but lacking RNase H activity was, in our assay, more sensitive than the M-MLV reverse transcriptase with endonucleolytic activity (Fig. 4)Citation .



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  		Fig. 3. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with different priming of reverse transcriptase reaction. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–5, 5 LNCaP cells mixed in 106 MNCs: Lane 3, specific primers; Lane 4, random hexanucleotides; Lane 5, oligo dT.

 
Primer Annealing.
In the first step of the PCR reaction, the PSA gene sequence-specific primers anneal to the complementary sequences of the reverse transcribed cDNA of PSA and are extended by the Taq polymerase. In this fashion, the sequences serve as template, resulting in the specific extension of a 293-bp DNA fragment. Although the melting temperature of the primers (Tm) can be calculated by the Wallace formula (17) , the annealing temperature is usually optimized sequentially. As shown in Fig. 5Citation , the annealing temperature was one of the most critical components for optimization of the stringency of the PCR assay.



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  		Fig. 5. Amplification of the 293-bp PSA DNA fragment using MNCs and tumor cell dilutions with different annealing temperatures. Lane 1, DNA ladder. Lane 2, negative control; Lanes 3 and 4, 51°C, 106 MNCs, 50 LNCaP cells mixed in 106 MNCs; Lanes 5 and 6, 54°C, 106 MNCs, 50 LNCaP cells mixed in 106 MNCs; Lanes 7 and 8, 57°C, 106 MNCs, 50 LNCaP cells mixed in 106 MNCs; Lanes 9 and 10, 60°C, 106 MNCs, 50 LNCaP cells mixed in 106 MNCs.

 
Side products attributable to mispriming and consequently nonspecific amplification increase at a temperature of 51°C, whereas reduced annealing efficiency occurs at a temperature of 60°C (Fig. 5)Citation . Because low and high annealing temperatures can reduce the amplification yield, 57°C seemed to be optimal to minimize nonspecific annealing and maximize specific priming using peripheral blood. Nevertheless, the annealing temperature of 57°C causes nonspecific fragments in amplification of MNCs of control bone marrow (data not shown). Because in the first cycles primers must perform a screening on the reverse transcribed mRNA (cDNA), we could increase stringency by choosing high temperatures of 64°C first and then decrease temperatures to 57°C to attain higher efficiency, as described in "Materials and Methods."

Furthermore, the annealing step is influenced by the time of primer annealing. We chose a longer annealing time in the first cycle because of the genomic screening (18) , when the target copy number is low (data not shown). In the following cycles, we modified the annealing times from 10 to 50 s (Fig. 6)Citation . Although we could observe a lower signal intensity by shortening the annealing time, signal intensity increases at an annealing time of 50 s. Nevertheless, the overall cycle time also increases, resulting in a lower sensitivity of the PCR assay by reducing the half time (T0.5) of the Taq polymerase. Thirty s seemed to be adequate (Fig. 6)Citation .



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  		Fig. 6. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with different annealing times. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–6, 50 LNCaP cells mixed in 106 MNCs at 10, 20, 30, and 50 s, respectively.

 
Primer Extension.
The second step of the PCR reaction is characterized by the successive additions of dNTPs. DNA strand extension by DNA polymerase from Taq (Taq polymerase) occurs a temperature optimum of 72°C (data not shown) with a highly processive extension rate of >60 nucleotides/s. Because both features depend on the secondary structure of the particular target cDNA sequence, we modified extension time from 5 s to 50 s. Amplification of fragments shorter than 200–300 bp was most effective at an extension time of 30 s. Increasing or decreasing the extension time to 5 or 50 s, respectively, resulted in reduced sensitivity (Fig. 7)Citation .



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  		Fig. 7. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with different extension times. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–8, 50 LNCaP cells mixed in 106 MNCs at 5, 10, 20, 30, 40, and 50 s, respectively.

 
Denaturation Step.
To generate single-stranded DNA templates, the newly synthesized double-stranded DNA is heat denatured (melted). As shown in Fig. 8Citation , we varied the melting temperature from 89°C to 97°C. If the temperature is too low (e.g., 89°C), denaturation of the target cDNA is incomplete, which impedes primer annealing and prohibits amplification of the template sequence. In contrast, if the denaturation temperature is too high (e.g., 97°C), the enzyme activity is reduced preferentially in later cycles. Both causes decrease the efficiency of the amplification process and thus generate a weak signal intensity (Fig. 8)Citation . In our PCR assay, a temperature of 93°C was optimal for melting the double-stranded PSA cDNA.



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  		Fig. 8. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with different denaturation temperatures. Lane 1, DNA ladder; Lane 2, negative control. Lanes 3–7, 50 LNCaP cells mixed in 106 MNCs at 89°C, 91°C, 93°C, 95°C, and 97°C, respectively.

 
In a similar manner, the time of denaturation influences the melting efficiency of the cDNA fragments. We varied this time from 10 to 50 s. A denaturation time of a minimum of 30 s was optimal (Fig. 9)Citation ; shorter or longer times resulted in decreased signal intensity.



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  		Fig. 9. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions with different denaturation times. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–7, 50 LNCaP cells mixed in 106 MNCs at 10, 20, 30, 40, and 50 s, respectively.

 
Magnesium Chloride Concentration.
Magnesium is a critical component of the PCR process by influencing the enzyme activity and forming soluble complexes with dNTP that are essential for DNA synthesis (19) . The MgCl2 concentration was varied from 0.5 mM to 2.0 mM to find the optimum. As shown in Fig. 10Citation , MgCl2 was essential for the PCR reaction in a minimum concentration of 1 mM. Decreased concentrations of Mg2+ (e.g., 0.5 mM) yielded no amplification product, whereas increased concentrations (e.g., 2 mM) promoted nonspecific annealing of the primers, thus reducing the detection limit in tumor cell dilutional experiments (data not shown). Because dNTPs reduce free Mg2+, the optimal concentration of Mg2+ depends mainly on the amount of available dNTP.



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  		Fig. 10. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the MgCl2 concentration. Lane 1, DNA ladder. Lanes 2, 5, 8, 11, 14, 17, and 20, negative controls. Lanes 3–22, 50 LNCaP cells mixed in 106 MNCs at 0.5 mM (Lanes 3 and 4), 0.75 mM (Lanes 6 and 7), 1.0 mM (Lanes 9 and 10), 1.25 mM (Lanes 12 and 13), 1.5 mM (Lanes 15 and 16), 1.75 mM (Lanes 18 and 19), and 2.0 mM (Lanes 21 and 22).

 
Concentration of Taq Polymerase and dNTP Concentration.
Using blood or bone marrow as a source for template RNA, PCR amplification can be adversely affected by potential inhibitors [i.e., heme (20) , heparin (21) ]. The optimal amount of polymerase might be different in particular assays and is therefore an important factor to be optimized. We analyzed amounts of 0.01 to 2.0 units and could obtain a strong signal with 0.5 unit (Fig. 11)Citation .



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  		Fig. 11. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the amount of Taq polymerase. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–7, 50 LNCaP cells mixed in 106 MNCs at 0.01, 0.1, 0.5, 1.0, and 2.0 units, respectively.

 
The concentration of deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) yielded the highest signal intensity at a concentration of 10 µM (Fig. 12)Citation . By using high concentrations of dNTP, the amplification of the cDNA was reduced.



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  		Fig. 12. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the dNTP concentration. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–6, 50 LNCaP cells mixed in 106 MNCs at 5, 10, 50, and 100 µM, respectively.

 
Concentration of Primers and BSA.
During annealing to the target sequence, primers form a single-stranded template for the subsequent synthesis of a complementary strand that is carried out by the DNA polymerase. Testing different primer concentrations of 0.01 to 2.0 µM demonstrated that the optimal primer concentration was 0.4 µM (Fig. 13)Citation . Concentration must be high enough to allow rapid annealing to the single-strand target DNA, which explains why no amplification was observed if the amount of used primers was decreased to 0.1 µM. By increasing the concentration above the optimal value of 0.4 µM, a faint diffuse band, representing a single-stranded specific product, became visible (Fig. 13)Citation .



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  		Fig. 13. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the primer concentration. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–7, 50 LNCaP cells mixed in 106 MNCs at 0.01, 0.1, 0.4, 1.0, and 2.0 µM, respectively.

 
PCR assays can be influenced by numerous substances that promote or inhibit the reaction. We chose to add BSA in a concentration of 0.5 µg/µl and observed an increased sensitivity, as demonstrated by the higher signal intensity, as seen in Fig. 14Citation . A smear attributable to nonspecific amplification can occur when a higher concentration of BSA was used.



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  		Fig. 14. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by addition of BSA. Lane 1, DNA ladder. Lane 2, negative control. Lanes 3–5, 50 LNCaP cells mixed in 106 MNCs at 0, 5, and 10 µg, respectively.

 
Cycle Number and Sensitivity.
The modification of the PCR components, as described above, is necessary to avoid amplification of unwanted products and therefore optimize the stringency of the assay. Despite optimal PCR conditions, the amount of PCR product at each cycle eventually levels off at a plateau. Fig. 15Citation shows the influence of different cycle numbers on the sensitivity of the PCR assay, as determined by using dilutional experiments of MNCs mixed with LNCaP tumor cells. By increasing the cycles from 50 to 65 at highest stringency conditions, we were able to further increase the sensitivity from 25 to 5 tumor cells in 106 MNCs.



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  		Fig. 15. Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the number of PCR cycles. Lane 1, DNA ladder. Lanes 2 and 8, negative control. Lanes 3–7, 65 PCR cycles: 100 LNCaP cells mixed in 106 MNCs, 50 LNCaP cells mixed in 106 MNCs, 25 LNCaP cells mixed in 106 MNCs, 5 LNCaP cells mixed in 106 MNCs, and 0 LNCaP cells mixed in 106 MNCs, respectively; Lanes 9–13, 50 PCR cycles: 100 LNCaP cells mixed in 106 MNCs, 50 LNCaP cells mixed in 106 MNCs, 25 LNCaP cells mixed in 106 MNCs, 5 LNCaP cells mixed in 106 MNCs, and 0 LNCaP cells mixed in 106 MNCs, respectively.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Minimal residual disease in cancer patients is characterized by a low abundance of disseminated tumor cells that are undetectable by current tumor staging procedures (22) . Therefore, new technologies for molecular staging of cancer have been developed that are based on the detection of tumor-specific transcripts (1) . In situ hybridization and Northern blot analyses are molecular approaches to reveal tumor-specific RNA, but technical difficulties and lack of sensitivity limit their use for analysis of rare transcripts. The use of RT-PCR to detect low abundance of mRNA species combines high speed with efficiency and stringency. In prostate cancer, amplification of PSA mRNA has been widely applied to detect occult prostate cancer cells in blood, bone marrow, and regional lymph nodes. However, the data on the reported specificity and sensitivity are disparate, and the clinical relevance of this method still remains unclear (10 , 11 , 23) .

In our study, we evaluated some of the confounding factors of PCR-based assays, using PSA RT-PCR for setting-up a PCR. Although new PCR methodologies are under development that may identify and quantitate target sequences by monitoring the real-time progress of the PCR amplification (reviewed in Ref. 24 ), these assays require expensive equipment, and to our knowledge, they have not yet been used to detect disseminated prostate cancer cells. Beyond this question, optimization by varying the discussed physical and chemical components also remains a critical issue in standardization and validation of future approaches, such as real-time PCR. Tables 1Citation 2Citation summarize the potentially important parameters that are different between RT-PCR-based assays used to look for disseminated cells. Because the efficiency of RNA preparation, reverse transcription, and PCR amplification can vary between 10 and 90% (25) , we consistently performed dilutional experiments as internal standard to document the sensitivity. Thus, using the described first-strand cDNA synthesis, the efficiency of the reverse transcriptase reaction was higher, with a reverse transcriptase enzyme lacking RNase H activity (Superscript II) than is applied in most published assays (Table 1)Citation than the M-MLV reverse transcriptase with endonucleolytic activity. Besides differences with regard to the amount of used RNA and buffer systems, we observed a slight advantage of initiating reverse transcription with specific primers, although random hexamers have also been used successfully by other groups (reviewed in Ref. 23 ). As a prerequisite of PCR amplification, we tested a series of primers specific for a region that is common to the three different PSA-specific variant cDNAs overspanning exons 2 and 3 (Fig. 7)Citation . Although all of these primers had balanced distribution of G/C- and A/T-rich domains and were characterized by similar Tm of 55°C to 65°C, the combination of the used primer sequences empirically showed highest efficiency. Moreover, the optimization of the annealing temperature was one of the most critical components of the PCR process, because nonspecific annealing can dramatically reduce amplification yield of the PCR process. To guarantee high stringency in the first few cycles, we chose increased temperatures for specific screening and reduced temperatures for higher yield. In an analysis of bone marrow in particular, we found that nonspecific annealing could be reduced and time consuming. Southern Blots for identifying specific PCR bands might be avoided. Hot-start amplifications using chemically modified form of Taq Polymerase (AmpliTaq Gold DNA polymerase) may furthermore improve the amplification yield and stringency (26) .

In contrast to other published assays (Tables 1Citation 2Citation ; Refs. 9 , 13 , 16, and 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ), our sets of primers appear to work better with lower magnesium concentration. As demonstrated in Fig. 10Citation , increase of Mg2+ causes nonspecific annealing that is attributable to influencing the melting temperature (Tm) of DNA. In contrast, a decrease of Mg2+ lowers the sensitivity by reduced dNTP incorporation and enzyme activity. We yielded a PCR with minimal nonspecific amplification that enables routine detection by agarose gel electrophoresis. There appears to be a stoichiometric interaction between the dNTPs and magnesium. Because high amounts of nucleosides might reduce the available magnesium concentration, it was necessary to decrease the concentration of dNTP in parallel to magnesium. This is in contrast to other published assays (Table 2)Citation . Furthermore, we observed an increase of specificity by reducing the reaction volume to 10 µl, whereas in most studies, the volume was 5–10 times higher (Table 2)Citation . Therefore, we could decrease the time for the reaction mixture to reach the denaturation, annealing, and extension temperatures (i.e., ramp time). This probably causes a decrease in nonspecific annealing and shortened the length of the PCR assay. Moreover, we chose the lowest possible times for extension, denaturation, and annealing steps, resulting in enhanced sensitivity of the assay, as controlled by dilutional experiments (Fig. 15)Citation . In contrast to most other authors, we minimized the cycle time and could therefore preserve enzyme activity. In the same manner, high temperatures shorten the half-time (T0.5) of the enzyme. As demonstrated in Fig. 8Citation , the signal intensity was reduced if denaturation temperatures reached 97°C. On the other hand, at lower temperatures of 89°C, the DNA melted only partially, which prohibits the access of the primers to the DNA, resulting in the reduction of the amount of amplified products. Overall, the absence of nonspecific annealing and short cycle times are prerequisites for the efficient amplification of low-abundance target transcripts. We could demonstrate a higher yield of the PCR by establishing stringent PCR conditions with minimal amplification of unspecific products and then by increasing cycle numbers to a maximum of 65 to achieve optimal amplification of the specific product.

Although in most studies nested PCR is performed, we could achieve a similar sensitivity of one tumor cell/106 normal cells by using an optimized one-round RT-PCR. Nested PCR assays may have the major drawback of introducing a high contamination risk (37) . Aerosols containing amplified DNA from previous PCR assays, in particular, might be the most potent source of false-positive results. Expensive precautions regarding laboratory space and analytical equipment can minimize but not prevent such cross-contamination. As demonstrated in Fig. 15Citation , the described optimization of the analytical steps resulted in a RT-PCR assay with high stringency. PCR-generated fragments of isolated tumor cells could be routinely detected by agarose gel electrophoresis, even in a background of 6 x 106 MNCs, without need of a confirmatory Southern Blot analysis. Time-consuming methods for identification of PCR products might hamper the introduction of PCR assays in clinical practice.

It is essential to consider the potential PCR-inhibitory activities of various substances present in blood or bone marrow. Heparin, frequently used as an anticoagulant, can cause attenuation of target DNA amplification (21) . Furthermore, porphyrin compounds derived from heme are regarded as strong inhibitors of the PCR (20 , 38) . As seen in Fig. 11Citation , an increase of Taq concentration might overcome these inhibitory influences. In addition, we decided to add BSA to the reaction mixture and could demonstrate an increase in the PCR yield (Fig. 14)Citation , which is probably attributable to the binding of BSA to the heme compound that is not completely extracted by organic solvents used for RNA purification. Moreover, BSA might stabilize the Taq polymerase at high temperatures during the denaturation step (20) .

By using the outlined approach, we could detect PSA-producing cells in the bone marrow aspirates of 28% of the patients with clinically localized prostate cancer (16) . Although other patients were analyzed with similar pathological stages, the reported RT-PCR PSA positivities vary tremendously (23) . Similarly, particular reports on the ectopic expression (39) of PSA resulting in false-positive controls (12) appear inconsistent and difficult to interpret. By performing a nested PCR with primers specific for exons 3 to 5, Henke et al. (13) could demonstrate a frequent amplification of PSA mRNA in hematopoietic tissue. In contrast, in the one-round RT-PCR described here and also in most of recently published assays, PSA target mRNA was consistently absent in control samples. Because the limit of detection in published assays is 10-5 to 10-6 and therefore surprisingly similar, decreased diagnostic specificity caused by increased analytical sensitivity (40) cannot sufficiently explain this discrepancy. In a similar manner, an inconsistent rate of ectopic expression in normal hematopoietic tissue is also common for CK19 mRNA, one of the most frequently used epithelial markers for PCR assays in breast cancer (reviewed in Ref. 41 ). Although Fields et al. (42) demonstrated a high specificity of CK19 RT-PCR and a correlation between PCR positivity in bone marrow aspirates and tumor progression, other authors found CK19 transcripts in the peripheral blood and bone marrow of healthy volunteers and noncarcinoma patients (43, 44, 45) . Interestingly, sample processing and biological parameters concerning the patient (inflammation and granulocyte-colony stimulating factor mobilization) can contribute to false-positive results (46) . However, it needs to be stressed that the clinical performance of the assays including cycle number and primer localization on different exons varies greatly. We have demonstrated that variations in PCR procedures may have large impact on the obtained data, and the heterogeneity of procedures, as listed in Tables 1Citation 2Citation , may explain, in part, the differences in these contradictory findings.

In summary, RT-PCR is a powerful tool to detect disseminated prostate carcinoma cells at low frequencies of 10-5 to 10-6. The specific expression of PSA in prostate-derived cancer cells opens up a new opportunity for quantification of the residual tumor-load using real time PCR, which will become less expensive and therefore more widespread in the future. The present work may help to establish standardized laboratory conditions that may lead to the introduction of RT-PCR techniques into current tumor staging protocols.


   ACKNOWLEDGMENTS
 
We are grateful for the technical assistance of Simone Baier and Tanja Siart and thank Dr. Marcus Otte for critically reading the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung and the Deutsche Forschungsgemeinschaft, Bonn, Germany. Back

2 To whom requests for reprints should be addressed, at Frauenklinik, Universitätsklinikum Eppendorf, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany; E-mail: Pantel{at}uke.uni-hamburg.de Back

3 The abbreviations used are: RT-PCR, reverse transcription-PCR; PSA, prostate-specific antigen; dNTP, deoxynucleotide triphosphate; MNC, mononuclear cell; wt, wild type; M-MLV, Moloney murine leukemia virus; CK19, cytokeratin 19. Back

Received 9/ 8/99; revised 3/27/00; accepted 3/30/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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