Application of Real-Time Reverse Transcriptase-Polymerase Chain Reaction in Urological Oncology

0022-5347/03/1695-1858/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 169, 1858 –1864, May 2003 Printed in U.S.A.

DOI: 10.1097/01.ju.0000047363.03411.6b

Review Article APPLICATION OF REAL-TIME REVERSE TRANSCRIPTASEPOLYMERASE CHAIN REACTION IN UROLOGICAL ONCOLOGY ANDRES JAN SCHRADER, JOERG LAUBER, OSKAR LECHNER, AXEL HEIDENREICH,* RAINER HOFMANN AND JAN BUER From the Department of Urology, Philipps-University of Medicine, Marburg, Department of Cell Biology and Immunology, German Research Center for Biotechnology, Braunschweig and Institute of Medical Microbiology, Medizinische Hochschule Hannover, Hannover, Germany

ABSTRACT

Purpose: During the last decade numerous different reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been described. However, the lack of highly sensitive, quantitative and reliable methodology has been responsible for its limited use in modern urology. Early semiquantitative RT-PCR techniques often proved not to produce consistent results and have a high failure rate due to complicated working models. In this article we provide a comprehensive and intelligible description of real-time PCR technology, which is a novel quantitative methodology to analyze gene expression. In addition, we report the first preclinical and clinical applications in molecular urology. Materials and Methods: The current literature was reviewed in regard to different current real-time RT-PCR protocols and their use in modern urological oncology. Results: Real-time RT-PCR is a reliable, rapid and relatively inexpensive technique that can be easily adapted for standardized preclinical and clinical applications at different centers. Its sensitivity equals at least that of conventional RT-PCR and the option of exact quantification of gene expressions allows proper differentiation among high, low and illegitimate RNA transcription. It eliminates post-PCR processing of PCR products, thereby, increasing throughput and decreasing the chance of carryover contamination. Conclusions: Although the application of real-time RT-PCR has gained wide acceptance in urological research, its routine clinical use is still in its infancy. However, due to its high sensitivity and exact quantitation real-time RT-PCR may be the method of choice for modern preclinical and clinical studies in the future. KEY WORDS: urologic neoplasms, reverse transcriptase polymerase chain reaction, gene expression profiling, RNA

Reverse transcriptase-polymerase chain reaction (RTPCR) is the most sensitive method for detecting low abundance mRNA. However, in its classic configuration it cannot provide any quantitative information on target mRNA and, thus, the equivalent number of transcriptionally active cells and/or amount of gene expression per cell. However, this information may often be of central interest in in vitro gene expression studies and clinical applications, such as the detection of disseminated or (during therapy) residual and relapsing disease in patients with cancer. Today the advent of real-time RT-PCR has revolutionized gene expression studies. In conventional PCR methodology a specific DNA sequence (template) is amplified by a definite number of reaction cycles and the formed PCR products (amplicon) are visualized, for example by ethidium bromide staining on gel. In real-time PCR methodology the visualization and documentation of increased DNA copies are performed during each cycle. A number of suitable instruments now on the market is capable of performing this technique (table 1). These machines monitor the fluorescence emitted

throughout the reaction as an indicator of amplicon production during each PCR cycle, in contrast to end point detection by most conventional quantitative PCR methods. We discuss different real-time PCR protocols. We reviewed the currently available literature focusing on the application of this technique in urological oncology. THE METHOD

Real-time PCR is based on the detection and quantification of a fluorescent reporter. The signal increases in direct proportion to the amount of PCR product formed in the reaction. By recording the amount of fluorescence emission at each cycle it is possible to monitor the PCR reaction online during the exponential phase, in which the first significant increase in the amount of PCR product correlates with the initial amount of template. Quantification is based on the threshold cycle, defined as the cycle number at which fluorescence and, thus, the PCR product become clearly detectable above background noise. The threshold cycle value can than be translated into a quantitative result by generating a standard curve. The standard curve for a certain gene is compiled by plotting individual threshold cycle values of a series of dilutions against the logarithm of the corresponding volumes (figs. 1 and 2). An equivalent calculation, as for the specific

Supported by the German National Merit Foundation and Deutsche Krebshilfe. * Financial interest and/or other relationship with Hoffmann La Roche, Novartis Pharmaceuticals, American Medical Systems, Yamanouchi, Urotech GmBH and Aventis Pharmaceuticals. 1858

Majority of consumables supplied by manufacturer only

Colors Colors Colors Colors 4 2 4 4 Halogen lamp/photo multiplier tubes Light emitting diode/photo multiplier tubes Light emitting diode/photo multiplier tubes Light emitting diode/photodetectors

Rapid/majority of consumables supplied by manufacturer only Highly sensitive detection Gradient function Halogen lamp/charge coupled device Light emitting diode/photo multiplier tubes

Peltier/air Peltier/air Air Peltier/air Stratagene MJ Research Corbett Research Cepheid Mx4000 Opticon Rotor-Gene Smart Cycler

96 96 36–72 16–96

Peltier/air Air 96 32 BioRad Roche iCycler Light Cycler

* Maximum number of samples analyzed simultaneously.

Highly sensitive detection High throughput

Advantages/Disadvantages Multiplex

2 Colors 4 Colors 2 Colors Detection range 500–660 nm. 2–6 Colors 3 Colors

Excitation/Detection

Halogen lamp/charge coupled device Halogen lamp/cooled charge coupled device Laser/charge coupled device Laser/cooled charge coupled device Pressure cooler/heating Peltier/air Pressure cooler/heating Peltier/air

Temperature Control Format*

96 96 96 384 Biosystems Biosystems Biosystems Biosystems

Manufacturer

Applied Applied Applied Applied

Machine

1859

FIG. 1. Currently available detection methods for real-time RTPCR use dsDNA specific intercalating agents such as SYBRGreen (A) or different techniques based on sequence specific labeled probes (B to F).

5700 7000 7700 7900

TABLE 1. Current instruments available for real-time RT-PCR

No longer available

QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN UROLOGY

genes of interest, should be done for a housekeeping gene, for example ␤-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal protein S9, to normalize for differences in the amount of total RNA/cDNA starting material. There are 2 general methods for the quantitative detection of the amplicon, including dsDNA intercalating agents or fluorescent probes, such as dual hybridization probes, hydrolysis probes, molecular beacons, scorpions, duplex scorpions and g-quenching probes. The use of fluorescent dsDNA intercalating agents such as SYBR Green (Applied Biosystems, PerkinElmer, Urayasu, Japan) or ethidium bromide is the simplest and most cost-effective method since no amplicon specific labeled hybridization probes are required (fig. 1, A). For example, SYBR Green only fluoresces when bound to dsDNA. Thus, the intensity of the fluorescence signal depends on the amount of dsDNA present in the reaction. The major drawback of this method is that it is not specific because the dye binds to all dsDNAs formed during the PCR reaction, that is also to nonspecifically amplified dsDNA. However, choosing stringent PCR conditions avoids the formation of nonspecific dsDNA products and the specificity of the reaction can easily be confirmed using melting curve analysis (fig. 2, C). Fluorescent probes offer the best possibility to quantify

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FIG. 2. FAS and ribosomal protein S9 (RPS9) gene expression and quantification by real-time RT-PCR using SYBR Green DNA binding dye methodology in 5 renal cell carcinoma cell lines A498 (a), CAKI-1 (b), CAKI-2 (c), HA7-RCC (d) and LB1047-RCC (e), human embryonic kidney cell line 293 (f) and RPTEC (g). A, amplification plot of fluorescence versus cycle number. I, increase in fluorescence/amplicon during each PCR cycle. II, chosen threshold of 0.5, which is important to determine threshold cycle value of each sample. B, standard curves with threshold cycle values plotted against input cDNA copy number. C, melting curves of temperature versus fluorescence with 1 sharp and overlapping melting peak for all reactions, indicating amplification specificity.

different target sequences in 1 reaction (multiplex PCR) just by using different primers and probes with different dyes having distinct emission wavelengths. Dual hybridization probe technology is based on fluorescence resonance energy transfer. Two fluorescently labeled probes are used, including 1 labeled with a donor dye at the 3⬘ end and the other labeled with an acceptor dye at the 5⬘ end (fig. 1, B). The probes are designed to bind within the amplified fragment and adjacent to each other in a head-totail arrangement. When the amplicon is produced, the probes hybridize and bring the 2 dyes next to each other, allowing energy transfer between the 2 dyes and leading to a change in the fluorescent signal. Hydrolysis probes, for example Taqman (ABI, Foster City, California) technology, make use of an oligonucleotide hybridization probe with a reporter (5⬘ end) and a quencher (3⬘ end) dye attached (fig. 1, C). During PCR the probe is cleaved by the 5⬘ nuclease activity of, for example Taq DNA polymerase, separating the reporter dye from the quencher dye. Thus, fluorescence resonance energy transfer no longer occurs. It generates a sequence specific fluorescent signal that increases with each cycle. Molecular beacons also have a fluorescent reporter and quencher at their 5⬘ and 3⬘ ends, respectively (fig. 1, D). The probes have complementary ends that form a hairpin structure, bringing fluorescent dye and quencher into close proximity. When the probe hybridizes to its target sequence of the amplicon, the hairpin loop opens, separating dye and quencher, and generating a fluorescent signal. Molecular beacons remain intact during PCR and they must rehybridize to the target sequence each cycle for fluorescence emission. Like molecular beacons, scorpions are PCR primers with a stem loop tail containing a fluorophore and quencher (fig. 1, E). However, the stem loop tail is attached to the PCR primer sequence by a so-called PCR stopper, a chemical modification that prevents PCR from copying the stem loop sequence of the scorpion primer. During PCR scorpion primers are extended to form amplicon. At the appropriate stage in the PCR cycle (annealing phase) the probe sequence in the scorpion tail curls back to hybridize to the target sequence in the amplicon. Because the tail of the scorpion and the amplicon are now part of the same strand of DNA, the interaction is intramolecular. Duplex scorpions are a modification of scorpions (fig. 1, F). However, in contrast to molecular beacons or scorpions, flu-

orophore and quencher dye are separated onto different and complementary oligonucleotides. The advantage of duplex scorpions is greatly increased separation between quencher and fluorophore, which decreases fluorophore quenching when the probe is bound to the target, resulting in better signal intensity compared with molecular beacons and scorpions, respectively.1 Bustin2 and Giulietti et al3 have reported further details regarding the different chemistries and real-time PCR equipment. We present a simple and cost-effective real-time protocol that can easily be adapted at many urological research laboratories. It is based on cDNA detection by its incorporation of intercalating agents such as SYBR Green without the need of a specific probe. As mentioned, the major drawback when using SYBR Green is that nonspecific amplified dsDNAs cannot easily be distinguished from specific ones. Thus, primers must be intensively tested to avoid nonspecific amplification or primer-dimer complex formation. Optimal primer length is 17 to 24 bases and annealing temperature is 52C to 65C. Amplicon length should be 80 to 250 bp. Shorter amplicons have the advantage of amplifying more efficiently than longer ones. To avoid unspecific products the optimal concentration of each primer must be determined in a so-called primer matrix. Usually a 900, 300 and 50 mM. concentration of a 5⬘ primer is tested with a 900, 300 and 50 mM. concentration of the corresponding 3⬘ primer with and without template (that is 2 ⫻ 9 reactions), at least in duplicates. By performing this crucial optimization step one can raise the difference in the threshold cycle for specific and nonspecific amplicons. As in conventional RT-PCR, primers binding to target sequences located on different exons should be chosen to avoid false-positive results arising from the amplification of (genomic) DNA contamination. However, if the target is an intronless gene, the RNA must be treated with ribonucleasefree deoxyribonuclease. Figures 2 and 3 show an example of a simple study of conventional versus real-time RT-PCR, in this case using a GeneAmp 5700 (Applied Biosystems, Foster City, California) machine. FAS and ribosomal protein S9 (RPS9) mRNA expression in the 5 human kidney cancer cell lines A498, CAKI-1, CAKI-2, HA7-RCC and LB1047-RCC, the human embryonic kidney cell line 293 and RPTEC, which is derived from proximal tubulus epithelium, were evaluated by conventional RT-PCR using 30 amplification cycles and by realtime RT-PCR using 40 amplification cycles (fig. 3). Each

QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN UROLOGY

FIG. 3. Characterization of FAS and ribosomal protein S9 (RPS9) mRNA expression in 5 renal cell carcinoma cell lines A498, CAKI-1, CAKI-2, HA7-RCC and LB1047-RCC, human embryonic kidney cell line 293 and RPTEC. A, conventional RT-PCR. B, quantitative RTPCR with FAS expression values corrected for ribosomal protein S9 mRNA expression. Error bars indicate maximal variations in 4 PCR tests performed. Values in columns represent ratios based on ribosomal protein S9 normalized FAS expression in RPTEC, for example (FAS/RPS9)A498/(FAS/RPS9)RPTEC ⫽ factor 43.

method was able to detect differences in mRNA expression levels among the cell lines tested. However, only real-time RT-PCR enabled the precise quantification of gene expression mandatory for an exact comparison of target gene expression in each cell line, for example (FAS/RPS9)A498/(FAS/ RPS9)RPTEC ⫽ factor 43 (fig. 3, B). CLINICAL APPLICATION IN UROLOGICAL ONCOLOGY

Until today the use of real-time RT-PCR in urological research has been in its infancy. However, early promising studies have been performed and published recently, of which the majority focuses on urological tumor research. Prostate cancer. One of the first applications for conventional RT-PCR in urology was the detection of prostate specific antigen (PSA) mRNA as a specific marker of circulating prostatic cells in patients with metastatic prostate cancer.4 In the following years the analytical sensitivity of this technique was substantially improved by increasing the number of PCR cycles, using nested PCR or enhancing sensitivity to detect amplification products.5 However, the majority of PSA mRNA assays have been only qualitative. It has caused considerable controversy regarding the usefulness of the RTPCR test for the molecular staging of prostate cancer since highly sensitive RT-PCR protocols have led to the irritating detection of PSA mRNA in the blood of patients with hyperplasia, healthy individuals and even female donors.6 With the advent of semiquantitative RT-PCR for PSA mRNA7⫺9 and finally real-time RT-PCR new hope arose that discriminating prostatic and nonprostatic source of PSA mRNA could simply be enabled by quantification. Recently Gelmini et al reported a real-time RT-PCR procedure measuring PSA mRNA in peripheral blood by a Taqman protocol, that is specific primers plus hydrolysis probes, and an ABI Prism Sequence Detector (Applied Biosystems) (fig.1, C).10 They reported the specific detection of PSA mRNA in 14 of 44 blood samples (32%) from patients with organ confined prostate cancer before therapy with a broad expression range of 20 to 70,000 PSA mRNA molecules per ml. blood. This method was at least as sensitive as conventional nested RT-PCR and using a proper cutoff level none of the 30 control samples from healthy donors tested positive.

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Gelmini et al reported no significant shedding of prostate cells into peripheral blood during radical prostatectomy and 1 week after surgery PSA mRNA was dramatically decreased. Straub et al used a Light-Cycler System (Roche Molecular Systems, Basel, Switzerland) combined with a SYBR Green protocol to quantify PSA mRNA in the peripheral blood of 87 patients with locally confined prostate cancer.11 Samples were obtained before and 1 week after radical retropubic prostatectomy. PSA mRNA could be detected preoperatively in 48 patients (40%) with stage pT2 tumors and in 39 (72%) with disease greater than stage pT2. Moreover, significant quantitative differences were observed in stages pT2 versus greater than pT2 disease (factor 7.6). Only limited intraoperative shedding of prostate cells into the bloodstream could be observed, which was more prominent in tumors of greater than stage pT2 than in stage pT2 tumors. In 27 patients who underwent transurethral resection of the prostate for benign prostatic hyperplasia only 8% presented preoperatively with a PSA mRNA positive blood sample, while mean PSA mRNA expression was greater than 17 and greater than 130 times lower than in patients with stages pT2 and greater than pT2 disease, respectively (for all groups ANOVA p ⬍0.001). Other current clinical real-time RT-PCR studies in the field of urology support the advantages of using this quantitative method. Linja et al used a Light Cycler and dual hybridization probes to quantify androgen receptor gene expression in androgen dependent and independent prostate cancer.12 All refractory tumors expressed androgen receptor with a median expression of 6-fold higher in hormone refractory than in primary tumors (Kruskal-Wallis test p ⬍0.001). Helenius et al used the same approach to detect increased urokinase-type plasminogen activator mRNA in a fraction of hormone refractory prostate carcinomas.13 Bladder cancer. For transitional cell carcinoma of the bladder and upper urinary tract several noninvasive molecular diagnostic tests have been developed in recent years with heterogeneous success since they lack sensitivity or specificity. Inoue et al were the first to use a Light-Cycler based real-time RT-PCR assay with specific primers and dual hybridization probes to quantify cytokeratin 20 expression in urine sediment to predict the existence, stage and grade of bladder cancer.14 Mean urine cytokeratin 20 mRNA values corrected for GAPDH mRNA expression in 47 patients with transitional cell cancer were significantly higher than in 19 without carcinoma and in 27 controls (factor greater than 22 and greater than 74, respectively; Kruskal-Wallis test p ⬍0.001). In the carcinoma group values significantly correlated with tumor grade, urinary cytological class and depth of tumor invasion. The sensitivity and specificity of real-time RT-PCR with proper cutoff levels were 81% and 83%, whereas those of conventional cytology were 28% and 100%, respectively. All cytologically positive cases were PCR positive. Inoue et al concluded that quantitative detection of exfoliated cancer cells by real-time RT-PCR was more sensitive than cytological detection of transitional cell carcinoma cells in urine and more specific than conventional RT-PCR protocols with comparable sensitivity. In addition, they claimed that this method required less than half the time needed to perform conventional RT-PCR and excellently showed the importance of using a housekeeping gene to normalize calculated gene expression values. CYP4B1 gene product, which is a member of the P450 family, is a potential activator of procarcinogens, such as aromatic amines, in the human bladder. Using real-time RT-PCR and hydrolysis probes Imaoka et al detected enhanced expression of CYP4B1 mRNA in normal bladder tissues from patients with bladder tumors compared with tissues from those without bladder tumors, suggesting that high expression of this gene increases the risk of bladder cancer.15

Ca cells in blood Ca cells in blood Ca Ca Ca Ca cell lines Ca Ca Ca Ca

Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate Prostate

Prostate Ca

Transitional cell Ca cells in urine Transitional cell Ca/bladder Transitional cell Ca

Renal cell Ca Renal cell Ca Renal cell Ca Nephroblastoma

Renal cell Ca

Gelmini et al10 Straub et al11 Linja et al12 Helenius et al13 Span et al22 Peirce et al23 Calvo et al24 de Kok et al25 Savinainen et al26 Burger et al27

Bieche et al28

Inoue et al14 Imaoka et al15 De Kok et al16

Paradis et al17 Chuanzhong et al18 Tricarico et al19 Efferth et al20, 21

Schrader et al29 Gene Amp 5700

ABIPrism 7700 ABIPrism 7700 ABIPrism 7700 iCycler iQ

LightCycler ABIPrism 7700 ABIPrism 7700

ABIPrism 7700

ABIPrism 7700 LightCycler LightCycler LightCycler ABIPrism 7700 ABIPrism 7700 iCycler iQ ABIPrism 7700 LightCycler Rotor-Gene

Machine

SYBR Green

Hydrolysis probes Hydrolysis probes Hydrolysis probes SYBR Green

Dual hybridization probes Hydrolysis probes Hydrolysis probes

Hydrolysis probes

Hydrolysis probes* SYBR Green Dual hybridization probes Dual hybridization probes Hydrolysis probes/molecular beacon Hydrolysis probes SYBR Green Hydrolysis probes Dual hybridization probes SYBR Green

Methodology

Human telomerase RT G250 MN/CA IX Vascular endothelial growth factor Lung resistance protein/multidrug resistance related protein 1 CX-chemokine receptor 4/CXchemokine ligand 12 (SDF-1) 9D7

Cytokeratin 20 CYP4B1 Human telomerase RT

PSA PSA Androgen receptor/PSA Urokinase-type plasminogen activator Human chorionic gonadotropin ␤ Human prolactin receptor Selenoprotein-P DD3/human telomerase RT ERBB2 (HER-2/neu) ␦-Catenin/prostate specific membrane antigen CGA

Target Gene

TABLE 2. Real-time RT-PCR studies with focus on urological oncology

Klade et Renal cell Ca ABIPrism 7700 Hydrolysis probes * For example, Taqman. † For example, housekeeping gene used to normalize gene expression for sample-to-sample differences in RNA output and quality, and RT efficiency.

al30

Specimen

References

GAPDH

Ribosomal protein S9

Human acidic ribosomal phosphoprotein P0 GAPDH — ␤2-microglobulin

GAPDH ␤-Actin 18s rRNA

Human acidic ribosomal phosphoprotein P0/Cytokeratin 18

— — TATA box binding protein TATA box binding protein Adenosine triphosphate-synthase 6 ␤-Actin 28s rRNA 18s rRNA TATA box binding protein ␤2-microglobulin

Normalizer†

1862 QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN UROLOGY

QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN UROLOGY

De Kok et al applied a Taqman protocol to quantify the expression of human telomerase RT, which is the catalytic subunit of human telomerase, in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia.16 Human telomerase RT mRNA was detected in all tumor tissue samples but not in normal bladder samples. An increase in pathological tumor grade and clinical stage correlated with increased human telomerase RT expression. Kidney cancer. Using Taqman technology Paradis et al quantified human telomerase RT mRNA expression in 41 renal cell carcinomas.17 They reported significantly higher human telomerase RT mRNA expression in 73% of tumor samples compared with corresponding normal tissue. In a subgroup of 29 patients with conventional renal cell carcinoma human telomerase RT mRNA expression correlated significantly with tumor stage (p ⫽ 0.01). Chuanzhong et al developed a quantitative RT-PCR to measure G250 (MN/CA IX) mRNA expression (a renal cell carcinoma associated antigen) using a real-time procedure based on the use of hydrolysis probes and an ABI Prism 7700 Sequence Detection System (Perkin-Elmer, Foster City, California).18 In contrast to 6 normal kidney tissues all 31 renal cell carcinoma samples tested positive for G250 mRNA expression. Moreover, G250 expression inversely correlated with tumor grade, that is G250 expression in high grade carcinoma was significantly lower than in low grade tumors. However, G250 mRNA expression did not correlate with tumor stage. Tricarico et al used Taqman methodology to confirm the over expression of vascular endothelial growth factor in renal cell carcinoma tissue compared with nonadjacent, nonneoplastic renal tissue in the same subject.19 Furthermore, using an iCycler iQ (BioRad Instruments, Munich, Germany) real-time PCR system Efferth et al successfully identified the expression of lung resistance protein, a potential multidrug resistance gene, in Wilms tumors (table 1).20 Lung resistance protein mRNA expression was significantly associated with chemotherapeutic pretreatment and tumor stage. In a similar study they quantified the expression of multidrug resistance related protein 1, which inversely correlated with the survival of patients with nephroblastoma.21 Table 2 lists further studies using real-time technology. CONCLUSIONS

Real-time RT-PCR technique is by far more reliable and less complicated than earlier semiquantitative RT-PCR protocols. First applications have proved that it is a highly promising methodology for future preclinical and clinical use in molecular urology. K. Steube and H. G. Drexler, DSMZ Braunschweig provided the cell lines and P. Gatzlaff and T. Toepfer, German Research Center for Biotechnology provided technical assistance. REFERENCES

1. Solinas, A., Brown, L. J., McKeen, C., Mellor, J. M., Nicol, J., Thelwell, N. et al: Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res, 29: E96, 2001 2. Bustin, S. A.: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 25: 169, 2000 3. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. and Mathieu, C.: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25: 386, 2001 4. Moreno, J. G., Croce, C. M., Fischer, R., Monne, M., Vihko, P., Mulholland, S. G. et al: Detection of hematogenous micrometastasis in patients with prostate cancer. Cancer Res, 52: 6110, 1992 5. Polascik, T. J., Oesterling, J. E. and Partin, A. W.: Prostate specific antigen: a decade of discovery—what we have learned

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igenicity and down-regulation of selenoprotein-P in mouse and human tumors. Cancer Res, 62: 5325, 2002 25. de Kok, J. B., Verhaegh, G. W., Roelofs, R. W., Hessels, D., Kiemeney, L. A., Aalders, T. W. et al: DD3(PCA3), a very sensitive and specific marker to detect prostate tumors. Cancer Res, 62: 2695, 2002 26. Savinainen, K. J., Saramaki, O. R., Linja, M. J., Bratt, O., Tammela, T. L., Isola, J. J. et al: Expression and gene copy number analysis of ERBB2 oncogene in prostate cancer. Am J Pathol, 160: 339, 2002 27. Burger, M. J., Tebay, M. A., Keith, P. A., Samaratunga, H. M., Clements, J., Lavin, M. F. et al: Expression analysis of deltacatenin and prostate-specific membrane antigen: their poten-

tial as diagnostic markers for prostate cancer. Int J Cancer, 100: 228, 2002 28. Bieche, I., Latil, A., Parfait, B., Vidaud, D., Laurendeau, I., Lidereau, R. et al: CGA gene (coding for the alpha subunit of glycoprotein hormones) overexpression in ER alpha-positive prostate tumors. Eur Urol, 41: 335, 2002 29. Schrader, A. J., Lechner, O., Templin, M., Dittmar, K. E., Machtens, S., Mengel, M. et al: CXCR4/CXCL12 expression and signalling in kidney cancer. Br J Cancer, 86: 1250, 2002 30. Klade, C. S., Dohnal, A., Furst, W., Sommergruber, W., Heider, K. H., Gharwan, H. et al: Identification and characterization of 9D7, a novel human protein overexpressed in renal cell carcinoma. Int J Cancer, 97: 217, 2002