This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Kok, J. B. Articles by Schalken, J. A. Search for Related Content PubMed PubMed Citation Articles by de Kok, J. B. Articles by Schalken, J. A.

DD3^PCA3, a Very Sensitive and Specific Marker to Detect Prostate Tumors

Departments of Clinical Chemistry [J. B. d. K., R. W. R., D. W. S.], Experimental Urology [G. W. V., D. H., L. A. K., T. W. A., J. A. S.], and Epidemiology and Biostatistics [L. A. K.], University Medical Centre Nijmegen, 6500 HB Nijmegen, the Netherlands

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We identified DD3^PCA3 as one of the most prostate cancer-specific genes at present (M. J. Bussemakers et al. Cancer Res., 59: 5975–5979, 1999). Consequently, DD3^PCA3 is an interesting candidate for use as a diagnostic and/or prognostic marker. In this study we developed a method for the accurate quantification of DD3^PCA3 mRNA, using real-time quantitative reverse transcription-PCR. DD3^PCA3 was expressed at low levels in normal prostate but not in 21 selected other normal tissues, blood, or 39 tumor samples other than prostate. The diagnostic and prognostic value of DD3^PCA3 in normal, hyperplastic, and malignant prostate tissues was determined and compared with another promising tumor marker for prostate cancer, telomerase reverse transcriptase (hTERT gene), the expression of which is related to telomerase activity. Sensitivity and specificity estimates for both genes were calculated as the area under the receiver-operating characteristics curve (AUC-ROC). DD3^PCA3 (AUC-ROC, 0.98) demonstrated better diagnostic efficacy than hTERT (AUC-ROC, 0.88). Moreover, the median increase in mRNA expression in tumor tissues compared with nonmalignant prostate tissues was much higher for DD3^PCA3 (34-fold) than for hTERT (6-fold). In tumor tissues, the median expression of DD3^PCA3 was much higher than hTERT (5849 versus 10 normalized mRNA copies). A significant relationship was observed only between tumor stage and hTERT gene expression. We conclude that expression of the DD3^PCA3 gene is a very sensitive and specific marker for the detection of prostate tumor cells in a high background of normal (prostate) cells. Consequently, DD3 measurements may be used for clinical application in prostate needle biopsies or bodily fluids such as blood, ejaculate, urine, or prostate massage fluid.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Prostate cancer is the most frequently diagnosed cancer in the Western male population and the second leading cause of cancer mortality. Patients with organ-confined prostate cancer are generally treated with radical surgery or radiation therapy. Alternatively, if the tumor has spread locally or distantly, androgen ablation is the standard therapy used. Unfortunately, most of the latter patients will develop progressive disease, and there is no other effective treatment available. Therefore, methods are necessary that can detect the tumor early, at a time when the tumor is still locally confined and potentially curable by radical prostatectomy.

A significant contribution to the early detection of prostate cancer has been the discovery of PSA² and subsequent development of various immunological assays in serum. Serum PSA is now recognized as the premier marker for detection of prostate cancer and can be used for screening selected populations of patients and for monitoring patients after therapy (1) . However, serum PSA levels are regularly elevated in men with BPH, prostatitis, and other nonmalignant disorders, resulting in reduced specificity (1) .

Novel cancer-specific markers have been identified that may aid early diagnosis and help to differentiate between tumor and nonmalignant growth. Telomerase activity is one of the most promising markers. High activity has been detected in the majority (90%) of prostate tumors (2 , 3) , whereas only low or absent activity was observed in normal and BPH tissues (3, 4, 5) . An alternative for telomerase activity measurement is the quantification of telomerase reverse transcriptase mRNA (hTERT gene) by use of real-time quantitative PCR (6) . Because hTERT expression is the rate-limiting determinant of the telomerase enzyme (7) , accurate quantification of hTERT mRNA copies may better differentiate between malignant and benign prostate growth than semiquantitative telomerase activity measurements.

Next to hTERT gene expression, other genes, such as PSGR and PCGEM1, have recently been identified that have more prostate-specific expression (8 , 9) . We identified and characterized DD3^PCA3 (DD3), a new prostate-specific gene (10) . Northern blot analysis showed that DD3 mRNA is expressed at low levels in normal prostate and is abundantly present in prostate tumor tissues (10) . A more quantitative, reproducible, and sensitive assay is necessary to test DD3 as a diagnostic or prognostic marker in clinical samples, which often contain only small amounts of mRNA.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tissues.
Normal tissue samples were obtained from autopsies with a postmortem delay of <4 h. These tissues, together with 39 frozen tissues from nine different types of tumors (lung, esophagus, ileum, colon, pancreas, testis, breast, bladder, and melanoma), were randomly selected from the tissue bank of the Department of Pathology (University Medical Centre Nijmegen). Prostate tissue sections (normal, BPH, and tumor) were carefully selected and removed by a pathologist from fresh prostates of patients after radical prostatectomy at the Canisius-Wilhelmina Hospital (Nijmegen, the Netherlands) and the University Medical Centre Nijmegen. The stages (TNM) of these tumors were determined at the Department of Pathology of both hospitals. Frozen tumor, BPH, and normal prostate tissues were step-sectioned with evaluation by H&E staining at regular intervals. This evaluation included determination of percentage of normal, BPH, and tumor cells, together with Gleason score of the tumor sections.

RNA Isolation and cDNA Synthesis.
Total RNA from healthy and tumor tissues was isolated by disruption of 10–25 frozen, 20-µm sections in 1 ml of RNAzolB (Biotex Laboratories Inc, Houston, TX) or TRIzol (Life Technologies, Inc., Breda, the Netherlands) using a sterile pestle. After the manufacturer’s protocols were completed, RNA was further purified using the RNeasy kit (Qiagen, Hilden, Germany). RNA was quantified spectrophotometrically.

Purified RNA (0.2–1.0 µg) was added to RNase-free water to a final volume of 10 µl, denatured for 5 min at 90°C, and cooled immediately on ice. Reverse transcription mixture (10 µl) was added, containing first strand buffer (Life Technologies, Inc.), 200 units of Moloney murine leukemia virus (Life Technologies, Inc.), 20 units of RNasin (Promega, Madison Wisconsin), 10 mM DTT, 4.75 µM random hexamers, and 600 µM deoxynucleotides. After annealing of the hexanucleotides for 10 min at 20°C, cDNA synthesis was performed for 45 min at 42°C, followed by an enzyme inactivation step for 5 min at 95°C. cDNA was stored at -20°C until use.

Quantitative Measurement of PCA3, hTERT, and 18S rRNA.
The gene sequence of DD3 is presented in GenBank accession no. AF103907. The structure of the DD3 gene with exon-intron boundaries was described previously (10) . Primers and a TaqMan probe for the cDNA-specific real-time quantitative PCR assay were designed: (a) DD3-95F (located in exon 1), 5'-GGTGGGAAGGACCTGATGATAC-3', (b) DD3-521R (located in exon 4a), 5'-GGGCGAGGCTCATCGAT-3', and probe DD3-FAM (located in exon 4a), 5'-FAM-AGAAATGCCCGGCCGCCATC-TAMRA-3'. In the quantitative PCR assay we added 30 pmol of each primer, 20 pmol of TaqMan probe, 25 µl of Universal Master Mix (Applied Biosystems, Foster City, CA), and 1 µl of cDNA in a total reaction volume of 50 µl. After enzyme inactivation for 10 min at 95°C, 40 two-step cycles were performed (30 s at 95°C, 75 s at 60°C). Because 95% of the DD3 mRNA splice products consist of exons 1, 3, and 4a, the size of the majority of PCR amplicons was 262 bp (10) . When no increase in fluorescence was detected after 40 PCR cycles, samples were regarded as negative for DD3 expression.

For quantitative hTERT mRNA measurements, we used 45 pmol of primer LT5 (5'-CGGAAGAGTGTCTGGAGCAA-3'), 15 pmol of primer LT6 (5'-GGATGAAGCGGAGTCTGGA-3'), 10 pmol of probe (5'-FAM-TTGCAAAGCATTGGAATCAGACAGCACT-TAMRA 3'), 25 µl of Universal Master Mix, and 1 µl of cDNA in a 50-µl reaction volume. PCR cycling parameters were 10 min at 95°C, followed by 40 cycles of 30 s at 95°C and 60 s at 60°C.

The level of 18S rRNA (rRNA) expression was measured in all samples to normalize DD3 and hTERT expression for sample-to-sample differences in RNA input, RNA quality, and reverse transcription efficiency. For quantification of rRNA we used a Pre Developed Assay Reagent and followed the accompanying instructions (Applied Biosystems). To prevent low Ct values from interfering with calculations of the threshold baseline, cDNA samples were diluted 20-fold in water. Amplification parameters were identical to those for hTERT, but only 25 PCR cycles were performed.

The principle of the real-time PCR is described by Gibson et al. (11) . PCR reactions were performed in the ABI Prism 7700 Sequence Detection System (Applied Biosystems), and Ct values for DD3, hTERT, and 18S rRNA were derived by the computer. To be able to transform the Ct values into absolute mRNA copy numbers, we used a calibration curve (5-log range) that was prepared from a dilution series of linearized plasmid containing the DD3, hTERT, or rRNA amplicon insert.

Statistical Analysis.
The distributions of normalized hTERT and normalized DD3 were characterized by their median values and ranges. Differences in these markers between normal and tumor tissue were tested for statistical significance with the nonparametric Mann-Whitney U test. The nonparametric Kruskal-Wallis test was used to test differences between prostate cancer patients with different tumor stages and Gleason scores.

To visualize the efficacy of the two markers to discriminate tumor tissue from normal tissue (in the absence of an arbitrary cutoff value), we summarized the data in a ROC curve. This curve plots the sensitivity (true positives) on the Y axis against 1 - the specificity (false positives) on the X axis, considering each observed value as a possible cutoff value. The AUC was calculated as a single measure for the discriminative efficacy of a marker. When a marker has no discriminative value, the ROC curve will lie close to the diagonal and the AUC is close to 0.5. When a test has strong discriminative value, the ROC curve will move up to the upper left-hand corner and the AUC will be close to 1.0. The Statistical Package for Social Sciences (SPSS) was used for analyses.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Prostate Specificity of DD3.
Absolute copy numbers of DD3 mRNA were determined from cDNA from 22 different normal tissues and peripheral blood leukocytes. These absolute copy numbers were normalized to the expression of rRNA (Table 1) . Normal prostate tissue expressed DD3 mRNA at a low level. DD3 was absent in all other tissues, except for kidney, in which DD3 was expressed at an insignificant level.

For DD3, median normalized expression was also significantly different between nonmalignant (median, 174; range, 0–593) and malignant prostate tissues (median, 5,849; range, 334–39,456; P < 0.0001). Median up-regulation of DD3 from normal to tumor tissues was 34-fold. Even in tissues containing 10% tumor cells, normalized DD3 expression was clearly higher than that in nonmalignant tissues (Table 3 , samples 38–47). No significant correlation was found between DD3 expression and tumor stage or Gleason score.

A ROC curve was constructed for both markers (Fig. 1) . The AUC-ROC represents the diagnostic efficacy of the continuous test result. The AUC-ROC was 0.98 (95% confidence interval, 0.96–1.00) for DD3 and 0.88 (95% confidence interval, 0.78–0.97) for hTERT, indicating good discrimination power for both tests.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Normalized expression of hTERT mRNA (A) and DD3 mRNA (B) in normal prostate, BPH, and tumor tissues. ROC curves (insets) are shown to demonstrate diagnostic efficacy of each marker.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously detected a statistically significant relationship between the expression of hTERT mRNA and tumor stage and grade in urothelial cell carcinomas (15) . Others found a similar relationship for breast tumors and Wilms’ tumors (16 , 17) . In the present study in prostate tumors, this correlation was observed only for stage, but not for Gleason score (grade). The 21 analyzed prostate tissues (with tumor cells 20%) were derived from radical prostatectomies, with only little variation in stage and grade. This low number may have caused the lack of correlation with grade. In contrast, Latil et al. (18) did not detect a correlation between hTERT expression and pathological parameters for 33 prostate tumor samples with a broader range of stage and Gleason grade.

Typically, both we (present study) and Latil et al. (18) detected low hTERT expression in the majority of samples from normal prostate tissues and BPH. This contrasts with findings using the telomeric repeat amplification protocol, with which telomerase activity could not be detected in most normal and BPH tissues (3, 4, 5) . The difference can be explained by the increased sensitivity of the real-time reverse transcription-PCR assay (16 , 18) . Moreover, low hTERT expression in normal prostate tissues corroborates findings that prostatic tissues contain stem cells (19) , which generally express telomerase activity.

Recently, DD3 has been described as one of the most prostate cancer-specific genes (10) . Unfortunately, the biological function of DD3 has not been unraveled, and no homology to any gene present in the computer databases has been found (10) . Because of the lack of extensive open reading frames, we suspect that the gene functions as a noncoding RNA (10) . Therefore, a mRNA-based method was necessary to quantify the expression of DD3 in tissues. Our quantitative gene expression data showed that DD3 is regulated by a unique prostate cancer-specific transcriptional mechanism. However, no correlation between transcriptional activity and tumor stage or grade was detected. For future clinical applications, our real-time, quantitative reverse transcription-PCR test will provide a very sensitive and specific tool to detect prostate tumor cells in tissue biopsies and bodily fluids.

	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 To whom requests for reprints should be addressed, at 564 AKC, UMC Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, the Netherlands. Fax: 31-24-3541743; E-mail: J.dekok{at}ckcl.azn.nl

² The abbreviations used are: PSA, prostate-specific antigen; BPH, benign prostate hyperplasia; hTERT, human telomerase reverse transcriptase; DD3, prostate cancer antigen 3 (PCA3, according to HUGO nomenclature); FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; Ct, threshold cycle; AUC, area under the curve; ROC, receiver-operating characteristic.

Received 9/17/01. Accepted 2/26/02.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Pannek J., Partin A. W. Prostate-specific antigen: what’s new in 1997. Oncology, 11: 1273-1278, 1997.[Medline]
2. Zhang W., Kapusta L. R., Slingerland J. M., Klotz L. H. Telomerase activity in prostate cancer, prostatic intraepithelial neoplasia, and benign prostatic epithelium. Cancer Res., 58: 619-621, 1998.[Abstract/Free Full Text]
3. Lin Y., Uemura H., Fujinami K., Hosaka M., Harada M., Kubota Y. Telomerase activity in primary prostate cancer. J. Urol., 157: 1161-1165, 1997.[Medline]
4. Sommerfeld H. J., Meeker A. K., Piatyszek M. A., Bova G. S., Shay J. W., Coffey D. S. Telomerase activity: a prevalent marker of malignant human prostate tissue. Cancer Res., 56: 218-222, 1996.[Abstract/Free Full Text]
5. Caldarera E., Crooks N. H., Muir G. H., Pavone-Macaluso M., Carmichael P. L. An appraisal of telomerase activity in benign prostatic hyperplasia. Prostate, 45: 267-270, 2000.[Medline]
6. de Kok J. B., Zendman A. J., van de Locht L. T., Ruers T. J. M., van Muijen G. N., Mensink E., Swinkels D. W. Real-time hTERT quantification: a promising telomerase-associated tumor marker. Lab. Investig., 79: 911-912, 1999.[Medline]
7. Meyerson M., Counter C. M., Eaton E. N., Ellisen L. W., Steiner P., Caddle S. D., Ziaugra L., Beijersbergen R. L., Davidoff M. J., Liu Q., Bacchetti S., Haber D. A., Weinberg R. A. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell, 90: 785-795, 1997.[Medline]
8. Xu L. L., Stackhouse B. G., Florence K., Zhang W., Shanmugam N., Sesterhenn I. A., Zou Z., Srikantan V., Augustus M., Roschke V., Carter K., McLeod D. G., Moul J. W., Soppett D., Srivastava S. PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer. Cancer Res., 60: 6568-6572, 2000.[Abstract/Free Full Text]
9. Srikantan V., Zou Z., Petrovics G., Xu L., Augustus M., Davis L., Livezey J. R., Connell T., Sesterhenn I. A., Yoshino K., Buzard G. S., Mostofi F. K., McLeod D. G., Moul J. W., Srivastava S. PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer. Proc. Natl. Acad. Sci. USA, 97: 12216-12221, 2000.[Abstract/Free Full Text]
10. Bussemakers M. J., van Bokhoven A., Verhaegh G. W., Smit F. P., Karthaus H. F., Schalken J. A., Debruyne F. M., Ru N., Isaacs W. B. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res., 59: 5975-5979, 1999.[Abstract/Free Full Text]
11. Gibson U. E., Heid C. A., Williams P. M. A novel method for real time quantitative RT-PCR. Genome Res., 6: 995-1001, 1996.[Abstract/Free Full Text]
12. Shay J. W., Bacchetti S. A survey of telomerase activity in human cancer. Eur. J. Cancer, 33: 787-791, 1997.
13. Broccoli D., Young J. W., de Lange T. Telomerase activity in normal and malignant hematopoietic cells. Proc. Natl. Acad. Sci. USA, 92: 9082-9086, 1995.[Abstract/Free Full Text]
14. Liu K., Schoonmaker M. M., Levine B. L., June C. H., Hodes R. J., Weng N. P. Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes. Proc. Natl. Acad. Sci. USA, 96: 5147-5152, 1999.[Abstract/Free Full Text]
15. de Kok J. B., Schalken J. A., Aalders T. W., Ruers T. J., Willems H. L., Swinkels D. W. Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas. Int. J. Cancer, 87: 217-220, 2000.[Medline]
16. Dome J. S., Chung S., Bergemann T., Umbricht C. B., Saji M., Carey L. A., Grundy P. E., Perlman E. J., Breslow N. E., Sukumar S. High telomerase reverse transcriptase (hTERT) messenger RNA level correlates with tumor recurrence in patients with favorable histology Wilms’ tumor. Cancer Res., 59: 4301-4307, 1999.[Abstract/Free Full Text]
17. Bieche I., Nogues C., Paradis V., Olivi M., Bedossa P., Lidereau R., Vidaud M. Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay. Clin. Cancer Res., 6: 452-459, 2000.[Abstract/Free Full Text]
18. Latil A., Vidaud D., Valeri A., Fournier G., Vidaud M., Lidereau R., Cussenot O., Biache I. hTERT expression correlates with MYC over-expression in human prostate cancer. Int. J. Cancer, 89: 172-176, 2000.[Medline]
19. Foster C. S., Ke Y. Stem cells in prostatic epithelia. Int. J. Exp. Pathol., 78: 311-329, 1997.[Medline]

This article has been cited by other articles:

				B. Laxman, D. S. Morris, J. Yu, J. Siddiqui, J. Cao, R. Mehra, R. J. Lonigro, A. Tsodikov, J. T. Wei, S. A. Tomlins, et al. A First-Generation Multiplex Biomarker Analysis of Urine for the Early Detection of Prostate Cancer Cancer Res., February 1, 2008; 68(3): 645 - 649. [Abstract] [Full Text] [PDF]

				D. Hessels, F. P. Smit, G. W. Verhaegh, J. A. Witjes, E. B. Cornel, and J. A. Schalken Detection of TMPRSS2-ERG Fusion Transcripts and Prostate Cancer Antigen 3 in Urinary Sediments May Improve Diagnosis of Prostate Cancer Clin. Cancer Res., September 1, 2007; 13(17): 5103 - 5108. [Abstract] [Full Text] [PDF]

				I. M. Thompson and D. P. Ankerst Prostate-specific antigen in the early detection of prostate cancer Can. Med. Assoc. J., June 19, 2007; 176(13): 1853 - 1858. [Abstract] [Full Text] [PDF]

				M. P.M.Q. van Gils, D. Hessels, O. van Hooij, S. A. Jannink, W. P. Peelen, S. L.J. Hanssen, J. A. Witjes, E. B. Cornel, H. F.M. Karthaus, G. A.H.J. Smits, et al. The Time-Resolved Fluorescence-Based PCA3 Test on Urinary Sediments after Digital Rectal Examination; a Dutch Multicenter Validation of the Diagnostic Performance Clin. Cancer Res., February 1, 2007; 13(3): 939 - 943. [Abstract] [Full Text] [PDF]

				K. V. Prasanth and D. L. Spector Eukaryotic regulatory RNAs: an answer to the 'genome complexity' conundrum Genes & Dev., January 1, 2007; 21(1): 11 - 42. [Abstract] [Full Text] [PDF]

				J. Groskopf, S. M.J. Aubin, I. L. Deras, A. Blase, S. Bodrug, C. Clark, S. Brentano, J. Mathis, J. Pham, T. Meyer, et al. APTIMA PCA3 Molecular Urine Test: Development of a Method to Aid in the Diagnosis of Prostate Cancer Clin. Chem., June 1, 2006; 52(6): 1089 - 1095. [Abstract] [Full Text] [PDF]

				P. Thelen, J.-G. Scharf, P. Burfeind, B. Hemmerlein, W. Wuttke, B. Spengler, V. Christoffel, R.-H. Ringert, and D. Seidlova-Wuttke Tectorigenin and other phytochemicals extracted from leopard lily Belamcanda chinensis affect new and established targets for therapies in prostate cancer Carcinogenesis, August 1, 2005; 26(8): 1360 - 1367. [Abstract] [Full Text] [PDF]

				R. Foley, D. Hollywood, and M. Lawler Molecular pathology of prostate cancer: the key to identifying new biomarkers of disease Endocr. Relat. Cancer, September 1, 2004; 11(3): 477 - 488. [Abstract] [Full Text] [PDF]

				S. Zelivianski, R. Glowacki, and M.-F. Lin Transcriptional activation of the human prostatic acid phosphatase gene by NF-{kappa}B via a novel hexanucleotide-binding site Nucleic Acids Res., July 7, 2004; 32(12): 3566 - 3580. [Abstract] [Full Text] [PDF]

				S. M. O'Hara, J. G. Moreno, D. R. Zweitzig, S. Gross, L. G. Gomella, and L. W.M.M. Terstappen Multigene Reverse Transcription-PCR Profiling of Circulating Tumor Cells in Hormone-Refractory Prostate Cancer Clin. Chem., May 1, 2004; 50(5): 826 - 835. [Abstract] [Full Text] [PDF]

				A. Latil, I. Bieche, L. Chene, I. Laurendeau, P. Berthon, O. Cussenot, and M. Vidaud Gene Expression Profiling in Clinically Localized Prostate Cancer: A Four-Gene Expression Model Predicts Clinical Behavior Clin. Cancer Res., November 15, 2003; 9(15): 5477 - 5485. [Abstract] [Full Text] [PDF]

				O. Gandini, M. Santulli, M. R. Cardillo, A. Stigliano, and V. Toscano Correspondence re: J. B. de Kok et al., DD3, A Very Sensitive and Specific Marker to Detect Prostate Tumors. Cancer Res., 62: 2695-2698, 2002. Cancer Res., August 1, 2003; 63(15): 4747 - 4747. [Full Text] [PDF]

				G. W. Verhaegh, J. B. de Kok, D. Hessels, F. Smit, and J. A. Schalken Reply Cancer Res., August 1, 2003; 63(15): 4748 - 4749. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Kok, J. B. Articles by Schalken, J. A. Search for Related Content PubMed PubMed Citation Articles by de Kok, J. B. Articles by Schalken, J. A.