Analytical Variables of Reverse Transcription-Polymerase ... · Analytical Variables of Reverse...
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Analytical Variables of Reverse Transcription-Polymerase Chain Reaction-based Detection of Disseminated Prostate Cancer Cells 1 Alfred Zippelius, Ralf Lutterbu ¨ se, Gert Riethmu ¨ ller, and Klaus Pantel 2 Institut fu ¨r Immunologie der Universita ¨t Mu ¨nchen, Munich, 80336 Germany ABSTRACT 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 tran- scription-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 tran- scriptase reaction, temperature and time of primer anneal- ing, primer extension and denaturation, as well as the con- centrations of magnesium chloride, Taq polymerase, deoxynucleotide triphosphate, primers and BSA on the am- plification process. By systematically varying these chemical and physical components, we could demonstrate a signifi- cant 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 Million-fold amplification of DNA by the PCR is a pow- erful method to detect rare tumor-specific DNA (1). This ap- proach 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 im- munoglobulin gene (2, 3). In contrast, the genomic characteris- tics of epithelial cancer cells are more heterogeneous (4), except for certain mutations in the Ki-ras oncogene in colon or pan- creatic carcinomas (5). As a consequence, many investigations focused on the development of RT-PCR 3 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 tran- scripts 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 mem- brane 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 consid- erably, which might have caused the tremendous differences in the incidence of PCR-positive findings comparing patients at similar clinical and pathological stages (Tables 1 and 2). More- over 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 vari- ous laboratories. At present, there is no consensus regarding the exact conditions, because they must be optimized for each set of primers. The purpose of this work was to demonstrate the influence of different PCR conditions on the specificity (negatives iden- tified/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, be- cause PSA is the most predominant marker of prostate cancer Received 9/8/99; revised 3/27/00; accepted 3/30/00. 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. 2 To whom requests for reprints should be addressed, at Frauenklinik, Universita ¨tsklinikum Eppendorf, Universita ¨t Hamburg, Martinistrasse 52, 20246 Hamburg, Germany; E-mail: [email protected]. 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. 2741 Vol. 6, 2741–2750, July 2000 Clinical Cancer Research Research. on December 11, 2020. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from
Transcript of Analytical Variables of Reverse Transcription-Polymerase ... · Analytical Variables of Reverse...
Analytical Variables of Reverse Transcription-Polymerase ChainReaction-based Detection of Disseminated ProstateCancer Cells1
Alfred Zippelius, Ralf Lutterbuse,Gert Riethmuller, and Klaus Pantel2
Institut fur Immunologie der Universita¨t Munchen, Munich,80336 Germany
ABSTRACTEarly systemic spread of occult tumor cells that may
develop into founders of incurable distant metastasis hasbeen identified in prostate cancer patients by reverse tran-scription-PCR (RT-PCR) amplification of prostate-specificantigen (PSA) mRNA. Nevertheless, the introduction of thisnew staging tool into the clinical setting has been hamperedby the disparate and contradictory data on the sensitivityand specificity of RT-PCR methods reported recently. Weused PSA RT-PCR to examine the influence of analyticalvariables such as priming and enzyme of reverse tran-scriptase reaction, temperature and time of primer anneal-ing, primer extension and denaturation, as well as the con-centrations of magnesium chloride, Taq polymerase,deoxynucleotide triphosphate, primers and BSA on the am-plification process. By systematically varying these chemicaland physical components, we could demonstrate a signifi-cant increase in amplification yield and in stringency ofprimer annealing. This may explain the wide variety ofpublished findings on molecular staging of prostate cancer,which currently impedes the clinical introduction of PSART-PCR assays in prostate cancer. Methodological analysesare needed for standardization and quality assurance toachieve reproducible molecular methods that can be used inclinical practice.
INTRODUCTIONMillion-fold amplification of DNA by the PCR is a pow-
erful method to detect rare tumor-specific DNA (1). This ap-proach has been proven to be successful in the detection ofresidual tumor cells in malignant lymphomas and leukemias byamplification of tumor-specific nucleic acid sequences derived
from chromosomal translocations or rearrangements of the im-munoglobulin gene (2, 3). In contrast, the genomic characteris-tics of epithelial cancer cells are more heterogeneous (4), exceptfor certain mutations in the Ki-ras oncogene in colon or pan-creatic carcinomas (5). As a consequence, many investigationsfocused on the development of RT-PCR3 assays to screen forrare epithelial-specific or tumor-associated mRNA species inmesenchymal tissue, such as bone marrow, peripheral blood, orlymph node.
In prostate cancer, the most common current target tran-scripts of choice for detection of disseminated tumor cells arePSA and human glandular kallikrein, members of the kallikrein-like serine protease family (6, 7), and prostate-specific mem-brane antigen (PSM), a type II integral membrane protein (8).As could be shown, expression is almost entirely restricted toprostatic epithelium, making these mRNA species interestingmarker molecules in identifying prostate cancer cells (9, 10).However, the reported data on the sensitivity and specificity ofthese RT-PCR assays are still controversial, and the clinicalrelevance of positive findings in blood, bone marrow, andlymph nodes of patients with prostate cancer are also underdebate (11). One of the reasons for the discrepant findings mightbe the enormous variability of the applied assays. Even in thecases where the same primers for the amplification of PSAmRNA have been used, the RT-PCR conditions varied consid-erably, which might have caused the tremendous differences inthe incidence of PCR-positive findings comparing patients atsimilar clinical and pathological stages (Tables 1 and 2). More-over some authors showed low-level illegitimate transcription ofPSA mRNA in peripheral blood cells that may result in false-positive findings (12, 13). Many of the differences are probablycaused by variabilities in the PCR conditions used by the vari-ous laboratories. At present, there is no consensus regarding theexact conditions, because they must be optimized for each set ofprimers.
The purpose of this work was to demonstrate the influenceof different PCR conditions on the specificity (negatives iden-tified/actual true negatives) and sensitivity (positives detected/actual true positives) of RT-PCR assays for PSA mRNA. Thismay form the basis for an international standardization of RT-PCR procedures used for the detection of occult cancer cells inblood, bone marrow, or lymph nodes. We used PSA RT-PCR asmodel for exemplifying the variability of PCR reactions, be-cause PSA is the most predominant marker of prostate cancer
Received 9/8/99; revised 3/27/00; accepted 3/30/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported by a grant from the Deutsche Krebshilfe/Dr.Mildred-Scheel-Stiftung and the Deutsche Forschungsgemeinschaft,Bonn, Germany.2 To whom requests for reprints should be addressed, at Frauenklinik,Universitatsklinikum Eppendorf, Universitat Hamburg, Martinistrasse52, 20246 Hamburg, Germany; E-mail: [email protected].
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 leukemiavirus; CK19, cytokeratin 19.
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cells, and there is increasing evidence that PSA RT-PCR assaysmay contribute to the molecular staging of prostate cancer.
MATERIALS AND METHODSExtraction of mRNA. Mononuclear cells from periph-
eral blood leukocytes and bone marrow aspirates were isolatedby density gradient centrifugation through Ficoll-Hypaque(Pharmacia, Freiburg, Germany) at 400 g for 30 min. Onemillion of these cells were removed for RT-PCR analyses andimmediately spun for 5 min at 10003 g. For purifying totalRNA, the guanidinium thiocyanate method of Chomczynski andSacchi (14) was modified as follows. The cell pellet was ho-mogenized with 300ml of solution D [4 M guanidine thiocya-nate, 0.5% Sarkosyl (N-laurylsarcosine sodium salt), and 25 mM
sodium citrate] by vigorous vortexing and stored at220°C untilneeded. Sequentially, 30ml of 2 M sodium acetate (pH 4.0), 300ml of water-saturated phenol, and 90ml of chloroform wereadded to the homogenate. After mixing thoroughly by vortex-ing, the samples were set on ice for 15 min and were thencentrifuged at 13,000 rpm for 5 min. The aqueous phase wastransferred to a fresh tube with 300ml of a phenol-chloroform-isoamyl alcohol mixture (25:24:1, v/v/v). Again after vortexingand centrifugation (1 min at 13,000 rpm), the upper phase wasreextracted with 300ml of chloroform. Subsequently, the RNAwas precipitated with 300ml of isopropanol, 40ml of 3 M
sodium acetate (pH 5.0), and 20mg of glycogen at220°Covernight. After centrifugation, the RNA pellet was washedwith 70% ethanol, dried for 4 min, and dissolved in 5ml ofHPLC-water. Cell lines used as positive controls for the PCRwere prepared as described above.
RT-PCR. Half of the total RNA was reverse transcribedusing specific primers for PSA cDNA 59-TGACGTGATACCT-TGAAGCA-39 (nucleotides 970–989), as deduced from thecDNA sequence (15); the other half was reverse transcribedusing random hexamer primers (Roche, Mannheim, Germany).The synthesis was carried out with a First-strand cDNA Syn-thesis kit (Life Technologies, Inc., Karlsruhe, Germany), includ-ing Superscript II in a final volume of 10ml containing 50 mM
Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM
total dNTP, 0.2mM of each of the specific primers, or 1.6mg ofthe random primers and 100 units Superscript II. After theaddition of RNA and initial denaturation at 70°C for 5 min to
obtain single-strand mRNA, the samples were incubated at 37°Cfor 1 h and subsequently diluted with 10ml of HPLC-water.Specific cDNA sequences were amplified in a reaction mix (10ml) of 1 ml of cDNA, 1 ml of 103 PCR buffer [100 mM Tris (pH8.3), 500 mM KCl, and 10 mM MgCl2], 100 mM dNTP, 0.4mM
of each of the two primers, 5mg of BSA (Roche), and 0.5 unitof Taq DNA polymerase (Roche). The following PCR primers(Fig. 1) were generated for detecting PSA cDNA: PSA sense,59-CTT GTA GCC TCT CGT GGC AG-39 (nucleotides 426–445); and PSA antisense, 59-GAC CTT CAT AGC ATC CGTGAG-39 (nucleotides 699–719).
Using genomic DNA as negative control, primers werechosen to hybridize on different exons of the targeted geneamplifying a PSA cDNA-specific product (293 bp; Fig. 4). Theamplification was performed on a Hybaid Thermal Reactor(Biometra, Gottingen, Germany) with the following cyclingprofile 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 forthe first cycle; denaturation at 93°C for 40 s, annealing at 64°Cfor 30 s, and extension at 72°C for 20 s for 7 cycles; denatur-ation at 93°C for 40 s, annealing at 57°C for 30 s, and extensionat 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 50cycles; and terminal extension at 72°C for 2 min and cooling at30°C for 1 min. To check the integrity of the extracted mRNA,RT-PCR with recently published primers for EGP-40 (16) andprimers for p53 was performed on the randomly primed cDNA.The primers for p53 were: p53 sense, 59-GGA TGA CAG AAACAC TTT TCG-39 (nucleotides 618–638); and p53 antisense,59-TCA GCT CTC GGA ACA TCT C-39(nucleotides 1015–1033), amplifying a 415-bp fragment. PCR products were ana-lyzed on agarose by gel electrophoresis using a 1-kb DNAladder (Life Technologies, Inc.) and by direct visualization with
Fig. 1 Schematic representation of a portion of thePSAgene showingthe relative positions of the specific PCR primers.
Table 1 Comparison ofdifferent components of reverse transcriptionin PSA RT-PCR assays used in studies on prostate cancer patients
Ref. Priming
Concentrationof MgCl2
(mM)
Concentrationof KCl(mM)
Concentrationof dNTP
(mM) Enzyme
Amount ofRNA used
(mg)IsolatedRNA
Position ofRT primer
Coreyet al. (9) Random 6.0 150 0.25 Superscript II/200 units 5.0 TotalDeguchiet al. (35) Random 3.0 75 0.4 M-MLV/150 units 0.2 mRNAGhosseinet al. (33) Specific 2.0 50 0.2 AMV/2 units 0.4 Total Exon 4Henkeet al. (13) Oligo(dT) 2.5 50 0.5 Superscript II/200 units 1.0 TotalIsraeli et al. (28) Random 2.5 50 0.5 Superscript II/200 units 5.0 TotalKatz et al. (27) Oligo(dT) 3.0 75 1.0 Superscript II/200 units 1.0 TotalMelchior et al. (36) Random 2.5 50 1.0 Superscript II/200 units 5.0 TotalSeidenet al. (29) Specific 2.5 50 0.5 Superscript II/200 units NR Total Exon 3Sokoloff et al. (34) Oligo(dT) 2.0 50 0.2 AMV/2 units 1.0 TotalWood et al. (30–32) Random 3.5 50 0.2 AMV/2.5 units 3.0 TotalZippeliuset al. (16) Specific 3.0 75 0.5 Superscript II/100 units 2.0 Total Exon 5
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ethidium bromide. The staining of PCR products on differentgels was comparable by the addition of equal amounts ofethidium bromide (0.2mg/ml) and equal concentrations of aga-rose (1.5%). Duplicate experiments demonstrated the reproduc-ibility of the results. The specificity of the amplification prod-ucts obtained was monitored with adequate restriction siteanalysis (SacI) and DNA sequencing. Mock RNA preparationswere routinely processed as negative controls.
RESULTSThe PCR process consists of cycles of a three-step proce-
dure, including denaturation, annealing, and extension. Theprinciple of the RT-PCR technology is shown in Fig. 2. PCR iscarried out in a series of three-step cycles producing a detectableamount of target mRNA. Each cycle starts with a denaturationstep at 92°C to 96°C to render the duplex DNA. This is followedby an annealing step during which the primers hybridize to acomplementary site of the template. Finally DNA polymeraseextends each primer by successive addition of dNTP formingcomplexes with Mg21. As shown in Fig. 2, DNA will continueto accumulate exponentially. The following analytical steps andchemical components are critical for the efficiency of cDNAamplification, and modification of these components can influ-ence sensitivity and specificity of the RT-PCR assay: primingand enzyme of RT reaction, temperature and time of primerannealing, primer extension and denaturation, as well as theconcentrations of magnesium chloride, Taq polymerase, dNTP,primers, and BSA.
The following data will show the potential influence ofeach physical and chemical component on the amplificationyield and the specific priming of the oligonucleotides. All ex-periments were performed with dilutions of LNCaP cells mixedin 1 3 106 MNCs. PCR optimization is decisive for amplifica-tion of low-abundance target mRNA species, such as PSA inbone marrow or peripheral blood samples of cancer patients.The consistent cellular expression of wt-p53 in the surroundingnormal hematopoietic cells makes it an appropriate control tocheck the integrity of the input RNA. Moreover, we performedparallel RT-PCR assays specific for epithelial glycoprotein-40,which also is an appropriate positive control because of lowlevel expression in hematopoietic cells (16). However, the am-plification rate of wt-p53 with the high cycle numbers used inour experiments reaches the plateau phase, resulting in a con-stantly high signal intensity. Therefore, variations of wt-p53RT-PCR cannot be used to demonstrate any increase or decreaseof amplification yield,i.e.,assay sensitivity. Therefore, we usedexogenous reconstruction experiments to assess the efficiencyof reverse transcription and, after PCR amplification, by dilutingcells of a prostate cancer cell line (LNCaP) in MNCs. Inaddition, MNCs from peripheral blood of healthy persons wereused to control for nonspecific reactions. Mock preparationswere routinely processed as negative controls for RNA purifi-cation and RT-PCR. The cycle parameters outlined here werefound to be suitable for thermal cyclers with a metal block usinga plate control and the particular primer sequences as describedabove. However, the exemplified variables affect RT-PCR-based assays in general.
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Reverse Transcription. First-strand synthesis of single-stranded mRNA in cDNA enables amplification with the Taqpolymerase, a DNA-dependent polymerase. However, if thetranscript of interest is only present in low abundancy, thevariety of reverse transcriptase reaction (e.g.,priming, RT en-zyme) is a crucial issue concerning the assay sensitivity. Asdemonstrated in Fig. 3, we could observe a higher signal inten-sity, representing a higher amplification yield, by using a spe-cific primer, whereas the signal intensity was lower using oli-go(dT) nucleotides or random hexanucleotides. In a similarmanner, the introduction of a reverse transcriptase enzyme (Su-perscript II) featuring reverse transcriptase activity but lackingRNase H activity was, in our assay, more sensitive than theM-MLV reverse transcriptase with endonucleolytic activity(Fig. 4).
Primer Annealing. In the first step of the PCR reaction,the PSAgene sequence-specific primers anneal to the comple-mentary sequences of the reverse transcribed cDNA of PSA andare extended by the Taq polymerase. In this fashion, the se-
quences serve as template, resulting in the specific extension ofa 293-bp DNA fragment. Although the melting temperature ofthe primers (Tm) can be calculated by the Wallace formula (17),the annealing temperature is usually optimized sequentially. Asshown in Fig. 5, the annealing temperature was one of the mostcritical components for optimization of the stringency of thePCR assay.
Side products attributable to mispriming and conse-quently nonspecific amplification increase at a temperatureof 51°C, whereas reduced annealing efficiency occurs at atemperature of 60°C (Fig. 5). Because low and high anneal-ing temperatures can reduce the amplification yield, 57°Cseemed to be optimal to minimize nonspecific annealing andmaximize specific priming using peripheral blood. Neverthe-less, the annealing temperature of 57°C causes nonspecificfragments in amplification of MNCs of control bone marrow(data not shown). Because in the first cycles primers mustperform a screening on the reverse transcribed mRNA(cDNA), we could increase stringency by choosing high
Fig. 2 Schematic diagram of PCRconsisting of denaturation, annealing,and extension.
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temperatures of 64°C first and then decrease temperatures to57°C to attain higher efficiency, as described in “Materialsand Methods.”
Furthermore, the annealing step is influenced by the time ofprimer annealing. We chose a longer annealing time in the firstcycle because of the genomic screening (18), when the targetcopy number is low (data not shown). In the following cycles,we modified the annealing times from 10 to 50 s (Fig. 6).Although we could observe a lower signal intensity by shorten-ing the annealing time, signal intensity increases at an annealingtime of 50 s. Nevertheless, the overall cycle time also increases,resulting in a lower sensitivity of the PCR assay by reducing thehalf time (T0.5) of the Taq polymerase. Thirty s seemed to beadequate (Fig. 6).
Primer Extension. The second step of the PCR reactionis characterized by the successive additions of dNTPs. DNAstrand extension by DNA polymerase from Taq (Taq polymer-ase) 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 theparticular target cDNA sequence, we modified extension timefrom 5 s to 50 s. Amplification of fragments shorter than200–300 bp was most effective at an extension time of 30 s.Increasing or decreasing the extension time to 5 or 50 s, respec-tively, resulted in reduced sensitivity (Fig. 7).
Denaturation Step. To generate single-stranded DNAtemplates, the newly synthesized double-stranded DNA is heatdenatured (melted). As shown in Fig. 8, we varied the meltingtemperature 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 ofthe template sequence. In contrast, if the denaturation temper-ature is too high (e.g.,97°C), the enzyme activity is reducedpreferentially in later cycles. Both causes decrease the efficiencyof the amplification process and thus generate a weak signalintensity (Fig. 8). In our PCR assay, a temperature of 93°C wasoptimal for melting the double-stranded PSA cDNA.
In a similar manner, the time of denaturation influences themelting efficiency of the cDNA fragments. We varied this timefrom 10 to 50 s. A denaturation time of a minimum of 30 s wasoptimal (Fig. 9); shorter or longer times resulted in decreasedsignal intensity.
Fig. 3 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions with different priming of reverse transcriptase reaction.Lane 1,DNA ladder. Lane 2,negative control.Lanes 3–5,5 LNCaPcells mixed in 106 MNCs: Lane 3,specific primers;Lane 4,randomhexanucleotides;Lane 5,oligo dT.
Fig. 4 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions with M-MLV reverse transcriptase and Superscript II.Lane 1,DNA ladder.Lane 2,negative control.Lanes 3–6,5 LNCaPcells mixed in 106 MNCs: Lanes 3and 4, M-MLV; Lanes 5and 6,Superscript II.
Fig. 5 Amplification of the 293-bp PSA DNA fragment using MNCsand tumor cell dilutions with different annealing temperatures.Lane 1,DNA ladder.Lane 2,negative control;Lanes 3and4, 51°C, 106 MNCs,50 LNCaP cells mixed in 106 MNCs; Lanes 5and6, 54°C, 106 MNCs,50 LNCaP cells mixed in 106 MNCs; Lanes 7and8, 57°C, 106 MNCs,50 LNCaP cells mixed in 106 MNCs; Lanes 9and10,60°C, 106 MNCs,50 LNCaP cells mixed in 106 MNCs.
Fig. 6 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions with different annealing times.Lane 1,DNA ladder.Lane2, negative control.Lanes 3–6,50 LNCaP cells mixed in 106 MNCs at10, 20, 30, and 50 s, respectively.
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Magnesium Chloride Concentration. Magnesium is acritical component of the PCR process by influencing the en-zyme activity and forming soluble complexes with dNTP thatare essential for DNA synthesis (19). The MgCl2 concentrationwas varied from 0.5 mM to 2.0 mM to find the optimum. Asshown in Fig. 10, MgCl2 was essential for the PCR reaction ina minimum concentration of 1 mM. Decreased concentrations ofMg21 (e.g.,0.5 mM) yielded no amplification product, whereasincreased concentrations (e.g.,2 mM) promoted nonspecificannealing of the primers, thus reducing the detection limit intumor cell dilutional experiments (data not shown). BecausedNTPs reduce free Mg21, the optimal concentration of Mg21
depends mainly on the amount of available dNTP.Concentration of Taq Polymerase and dNTP Concen-
tration. Using blood or bone marrow as a source for templateRNA, PCR amplification can be adversely affected by potentialinhibitors [i.e.,heme (20), heparin (21)]. The optimal amount ofpolymerase might be different in particular assays and is there-fore an important factor to be optimized. We analyzed amountsof 0.01 to 2.0 units and could obtain a strong signal with 0.5 unit(Fig. 11).
The concentration of deoxynucleoside triphosphates(dATP, dCTP, dGTP, and dTTP) yielded the highest signalintensity at a concentration of 10mM (Fig. 12). By using highconcentrations of dNTP, the amplification of the cDNA wasreduced.
Concentration of Primers and BSA. During annealingto the target sequence, primers form a single-stranded templatefor the subsequent synthesis of a complementary strand that iscarried out by the DNA polymerase. Testing different primerconcentrations of 0.01 to 2.0mM demonstrated that the optimalprimer concentration was 0.4mM (Fig. 13). Concentration mustbe high enough to allow rapid annealing to the single-strandtarget DNA, which explains why no amplification was observedif the amount of used primers was decreased to 0.1mM. Byincreasing the concentration above the optimal value of 0.4mM,a faint diffuse band, representing a single-stranded specificproduct, became visible (Fig. 13).
PCR assays can be influenced by numerous substances thatpromote or inhibit the reaction. We chose to add BSA in aconcentration of 0.5mg/ml and observed an increased sensitiv-ity, as demonstrated by the higher signal intensity, as seen inFig. 14. A smear attributable to nonspecific amplification can
occur when a higher concentration of BSA was used.Cycle Number and Sensitivity. The modification of the
PCR components, as described above, is necessary to avoidamplification of unwanted products and therefore optimize thestringency of the assay. Despite optimal PCR conditions, theamount of PCR product at each cycle eventually levels off at aplateau. Fig. 15 shows the influence of different cycle numberson the sensitivity of the PCR assay, as determined by usingdilutional experiments of MNCs mixed with LNCaP tumorcells. By increasing the cycles from 50 to 65 at highest strin-gency conditions, we were able to further increase the sensitivityfrom 25 to 5 tumor cells in 106 MNCs.
DISCUSSIONMinimal residual disease in cancer patients is characterized
by a low abundance of disseminated tumor cells that are unde-tectable by current tumor staging procedures (22). Therefore,new technologies for molecular staging of cancer have beendeveloped that are based on the detection of tumor-specifictranscripts (1).In situ hybridization and Northern blot analysesare molecular approaches to reveal tumor-specific RNA, buttechnical difficulties and lack of sensitivity limit their use foranalysis of rare transcripts. The use of RT-PCR to detect lowabundance of mRNA species combines high speed with effi-ciency and stringency. In prostate cancer, amplification of PSAmRNA has been widely applied to detect occult prostate cancer
Fig. 7 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions with different extension times.Lane 1,DNA ladder.Lane2, negative control.Lanes 3–8,50 LNCaP cells mixed in 106 MNCs at5, 10, 20, 30, 40, and 50 s, respectively.
Fig. 8 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions with different denaturation temperatures.Lane 1,DNAladder;Lane 2,negative control.Lanes 3–7,50 LNCaP cells mixed in106 MNCs at 89°C, 91°C, 93°C, 95°C, and 97°C, respectively.
Fig. 9 Amplification of the 293-bp PSA DNA fragment using tumorcell 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.
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cells in blood, bone marrow, and regional lymph nodes. How-ever, the data on the reported specificity and sensitivity aredisparate, and the clinical relevance of this method still remainsunclear (10, 11, 23).
In our study, we evaluated some of the confounding factorsof PCR-based assays, using PSA RT-PCR for setting-up a PCR.Although new PCR methodologies are under development thatmay identify and quantitate target sequences by monitoring thereal-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 disseminatedprostate cancer cells. Beyond this question, optimization byvarying the discussed physical and chemical components alsoremains a critical issue in standardization and validation offuture approaches, such as real-time PCR. Tables 1 and 2summarize the potentially important parameters that are differ-ent between RT-PCR-based assays used to look for dissemi-nated cells. Because the efficiency of RNA preparation, reversetranscription, and PCR amplification can vary between 10 and90% (25), we consistently performed dilutional experiments as
Fig. 10 Amplification of the 293-bp PSA DNA fragment using tumor cell dilutions by varying the MgCl2 concentration. Lane 1,DNA ladder.Lanes2, 5, 8, 11, 14, 17,and20, negative controls.Lanes 3–22,50 LNCaP cells mixed in 106 MNCs at 0.5 mM (Lanes 3and4), 0.75 mM (Lanes 6and7), 1.0 mM (Lanes 9and10), 1.25 mM (Lanes 12and13), 1.5 mM (Lanes 15and16), 1.75 mM (Lanes 18and19), and 2.0 mM (Lanes 21and22).
Fig. 11 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions by varying the amount of Taq polymerase.Lane 1,DNAladder.Lane 2,negative control.Lanes 3–7,50 LNCaP cells mixed in106 MNCs at 0.01, 0.1, 0.5, 1.0, and 2.0 units, respectively.
Fig. 12 Amplification of the 293-bp PSA DNA fragment using tumorcell 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 100mM, respectively.
Fig. 13 Amplification of the 293-bp PSA DNA fragment using tumorcell 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 2.0mM, respectively.
Fig. 14 Amplification of the 293-bp PSA DNA fragment using tumorcell dilutions by addition of BSA.Lane 1,DNA ladder.Lane 2,negativecontrol.Lanes 3–5,50 LNCaP cells mixed in 106 MNCs at 0, 5, and 10mg, respectively.
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internal standard to document the sensitivity. Thus, using thedescribed first-strand cDNA synthesis, the efficiency of thereverse transcriptase reaction was higher, with a reverse tran-scriptase enzyme lacking RNase H activity (Superscript II) thanis applied in most published assays (Table 1) than the M-MLVreverse transcriptase with endonucleolytic activity. Besides dif-ferences with regard to the amount of used RNA and buffersystems, we observed a slight advantage of initiating reversetranscription with specific primers, although random hexamershave also been used successfully by other groups (reviewed inRef. 23). As a prerequisite of PCR amplification, we tested aseries of primers specific for a region that is common to thethree different PSA-specific variant cDNAs overspanning exons2 and 3 (Fig. 7). Although all of these primers had balanceddistribution of G/C- and A/T-rich domains and were character-ized by similarTm of 55°C to 65°C, the combination of the usedprimer sequences empirically showed highest efficiency. More-over, the optimization of the annealing temperature was one ofthe most critical components of the PCR process, because non-specific annealing can dramatically reduce amplification yieldof the PCR process. To guarantee high stringency in the first fewcycles, we chose increased temperatures for specific screeningand reduced temperatures for higher yield. In an analysis ofbone marrow in particular, we found that nonspecific annealingcould be reduced and time consuming. Southern Blots for iden-tifying specific PCR bands might be avoided. Hot-start ampli-fications using chemically modified form of Taq Polymerase(AmpliTaq Gold DNA polymerase) may furthermore improvethe amplification yield and stringency (26).
In contrast to other published assays (Tables 1 and 2; Refs.9, 13, 16, and 27–36), our sets of primers appear to work betterwith lower magnesium concentration. As demonstrated in Fig.10, increase of Mg21 causes nonspecific annealing that is at-tributable to influencing the melting temperature (Tm) of DNA.In contrast, a decrease of Mg21 lowers the sensitivity by re-duced dNTP incorporation and enzyme activity. We yielded aPCR with minimal nonspecific amplification that enables rou-tine detection by agarose gel electrophoresis. There appears tobe a stoichiometric interaction between the dNTPs and magne-
sium. Because high amounts of nucleosides might reduce theavailable magnesium concentration, it was necessary to decreasethe concentration of dNTP in parallel to magnesium. This is incontrast to other published assays (Table 2). Furthermore, weobserved an increase of specificity by reducing the reactionvolume to 10ml, whereas in most studies, the volume was 5–10times higher (Table 2). Therefore, we could decrease the timefor the reaction mixture to reach the denaturation, annealing,and extension temperatures (i.e.,ramp time). This probablycauses a decrease in nonspecific annealing and shortened thelength of the PCR assay. Moreover, we chose the lowest pos-sible times for extension, denaturation, and annealing steps,resulting in enhanced sensitivity of the assay, as controlled bydilutional experiments (Fig. 15). In contrast to most other au-thors, we minimized the cycle time and could therefore preserveenzyme activity. In the same manner, high temperatures shortenthe half-time (T0.5) of the enzyme. As demonstrated in Fig. 8,the signal intensity was reduced if denaturation temperaturesreached 97°C. On the other hand, at lower temperatures of 89°C,the DNA melted only partially, which prohibits the access of theprimers to the DNA, resulting in the reduction of the amount ofamplified products. Overall, the absence of nonspecific anneal-ing and short cycle times are prerequisites for the efficientamplification of low-abundance target transcripts. We coulddemonstrate a higher yield of the PCR by establishing stringentPCR conditions with minimal amplification of unspecific prod-ucts and then by increasing cycle numbers to a maximum of 65to achieve optimal amplification of the specific product.
Although in most studies nested PCR is performed, wecould achieve a similar sensitivity of one tumor cell/106 normalcells by using an optimized one-round RT-PCR. Nested PCRassays may have the major drawback of introducing a highcontamination risk (37). Aerosols containing amplified DNAfrom previous PCR assays, in particular, might be the mostpotent source of false-positive results. Expensive precautionsregarding laboratory space and analytical equipment can mini-mize but not prevent such cross-contamination. As demon-strated in Fig. 15, the described optimization of the analyticalsteps resulted in a RT-PCR assay with high stringency. PCR-
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 2and8, negative control.Lanes 3–7,65 PCR cycles: 100 LNCaP cells mixed in 106 MNCs, 50 LNCaP cells mixed in 106 MNCs, 25 LNCaPcells 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.
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generated fragments of isolated tumor cells could be routinelydetected by agarose gel electrophoresis, even in a background of6 3 106 MNCs, without need of a confirmatory Southern Blotanalysis. Time-consuming methods for identification of PCRproducts might hamper the introduction of PCR assays in clin-ical practice.
It is essential to consider the potential PCR-inhibitoryactivities of various substances present in blood or bone mar-row. Heparin, frequently used as an anticoagulant, can causeattenuation of target DNA amplification (21). Furthermore, por-phyrin compounds derived from heme are regarded as stronginhibitors of the PCR (20, 38). As seen in Fig. 11, an increase ofTaq concentration might overcome these inhibitory influences.In addition, we decided to add BSA to the reaction mixture andcould demonstrate an increase in the PCR yield (Fig. 14), whichis probably attributable to the binding of BSA to the hemecompound that is not completely extracted by organic solventsused for RNA purification. Moreover, BSA might stabilize theTaq polymerase at high temperatures during the denaturationstep (20).
By using the outlined approach, we could detect PSA-producing cells in the bone marrow aspirates of 28% of thepatients with clinically localized prostate cancer (16). Althoughother patients were analyzed with similar pathological stages,the reported RT-PCR PSA positivities vary tremendously (23).Similarly, particular reports on the ectopic expression (39) ofPSA resulting in false-positive controls (12) appear inconsistentand difficult to interpret. By performing a nested PCR withprimers specific for exons 3 to 5, Henkeet al. (13) coulddemonstrate a frequent amplification of PSA mRNA in hema-topoietic tissue. In contrast, in the one-round RT-PCR describedhere and also in most of recently published assays, PSA targetmRNA was consistently absent in control samples. Because thelimit of detection in published assays is 1025 to 1026 andtherefore surprisingly similar, decreased diagnostic specificitycaused by increased analytical sensitivity (40) cannot suffi-ciently explain this discrepancy. In a similar manner, an incon-sistent rate of ectopic expression in normal hematopoietic tissueis also common for CK19 mRNA, one of the most frequentlyused epithelial markers for PCR assays in breast cancer (re-viewed in Ref. 41). Although Fieldset al. (42) demonstrated ahigh specificity of CK19 RT-PCR and a correlation betweenPCR positivity in bone marrow aspirates and tumor progression,other authors found CK19 transcripts in the peripheral blood andbone marrow of healthy volunteers and noncarcinoma patients(43–45). Interestingly, sample processing and biological param-eters concerning the patient (inflammation and granulocyte-colony stimulating factor mobilization) can contribute to false-positive results (46). However, it needs to be stressed that theclinical performance of the assays including cycle number andprimer localization on different exons varies greatly. We havedemonstrated that variations in PCR procedures may have largeimpact on the obtained data, and the heterogeneity of proce-dures, as listed in Tables 1 and 2, may explain, in part, thedifferences in these contradictory findings.
In summary, RT-PCR is a powerful tool to detect dissem-inated prostate carcinoma cells at low frequencies of 1025 to1026. The specific expression of PSA in prostate-derived cancercells opens up a new opportunity for quantification of the
residual tumor-load using real time PCR, which will becomeless expensive and therefore more widespread in the future. Thepresent work may help to establish standardized laboratoryconditions that may lead to the introduction of RT-PCR tech-niques into current tumor staging protocols.
ACKNOWLEDGMENTSWe are grateful for the technical assistance of Simone Baier and
Tanja Siart and thank Dr. Marcus Otte for critically reading the manu-script.
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2000;6:2741-2750. Clin Cancer Res Alfred Zippelius, Ralf Lutterbüse, Gert Riethmüller, et al. Cancer CellsChain Reaction-based Detection of Disseminated Prostate Analytical Variables of Reverse Transcription-Polymerase
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