We identified DD3PCA3 as one of the most prostate cancer-specific genes at present (M. J. Bussemakers et al. Cancer Res., 59: 5975–5979, 1999). Consequently, DD3PCA3 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 DD3PCA3 mRNA, using real-time quantitative reverse transcription-PCR. DD3PCA3 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 DD3PCA3 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). DD3PCA3 (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 DD3PCA3 (34-fold) than for hTERT (6-fold). In tumor tissues, the median expression of DD3PCA3 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 DD3PCA3 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.

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 DD3PCA3 (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.

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.

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.

In addition, DD3 mRNA expression was measured in 39 tumor samples representing nine different tumor types (Table 2). DD3 mRNA could not be detected in all tested tumor samples. The absence of expression of DD3 in the vast majority of both normal and tumor tissues other than prostate demonstrated the high prostate specificity of this novel tumor marker.

Diagnostic Performance of hTERT and DD3 as Prostate Tumor Markers.

DD3 and hTERT mRNA expression was quantitatively measured in 11 normal prostate tissues, 5 BPH samples, and 31 prostate adenocarcinomas (Table 3). No significant differences in hTERT or DD3 mRNA expression were detected between normal and BPH tissues. Therefore, normal and BPH tissues together were defined as one group of nonmalignant tissues.

Median hTERT mRNA expression levels between nonmalignant (median, 1.7; range, 0–5.2) and tumor tissues (median, 10.1; range, 0.7–76.1) were significantly different (P < 0.0001). Median up-regulation of hTERT expression from normal to tumor tissues was 6-fold. A significant relationship was found between hTERT expression and tumor stage (P = 0.02), but not with Gleason score.

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.

We developed a method for the accurate quantification of DD3 mRNA expression and showed that DD3 was exclusively expressed in the prostate. For diagnosis of tumor, DD3 expression was compared with the expression of hTERT mRNA. hTERT gene expression is the rate-limiting determinant of telomerase activity (7) and at present the most common tumor marker (12). Sensitivity and specificity estimates for both genes were calculated as the AUC-ROC. Although both hTERT and DD3 could differentiate between malignant and nonmalignant prostate tissues, DD3 showed much better diagnostic characteristics than hTERT, with AUC-ROC values of 0.98 for DD3 and 0.88 for hTERT. The median increase in mRNA expression from nonmalignant to malignant tissues was only 6-fold for hTERT, whereas the median DD3 increase was 34-fold. A large increase is important for the detection of a few malignant cells in a high background of normal prostate cells. This was clearly shown for 10 tissue samples containing ≤10% tumor cells, in which DD3 showed higher sensitivity than hTERT. Moreover, in prostate tumors, the median normalized expression of DD3 mRNA was ∼580-fold higher than hTERT mRNA (5849 versus 10 normalized copies, respectively). This high expression in tumor cells may also be advantageous for the detection of the few malignant prostate cells shed into blood, urine, prostatic massage fluid, or ejaculate. In addition, DD3 mRNA expression could not be detected in leukocytes, which are regularly present in these bodily fluids, whereas hTERT mRNA is expressed in leukocytes and may cause false positivity (13, 14).

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.

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.

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.

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.

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.

Close modal
Table 1

DD3 expression in normal tissues

TissuerRNAa (×108)DD3              aNormalized DD3b
Skin 4.8 
Skeletal muscle 48.0 
Heart 12.0 
Lymph gland 26.0 
Spleen 38.0 
Bone marrow 4.6 
Lung 12.0 
Stomach 20.0 
Ileum 13.0 
Colon 16.0 
Liver 40.0 
Pancreas 24.0 
Testis 28.0 
Bladder 34.0 
Kidney 52.0 390 
Uterus 44.0 
Thyroid gland 2.8 
Brain 14.0 
Esophagus 7.2 
Ovaries 7.8 
Breast 0.6 
Prostate 4.6 699 152 
Leukocytes 34.0 
TissuerRNAa (×108)DD3              aNormalized DD3b
Skin 4.8 
Skeletal muscle 48.0 
Heart 12.0 
Lymph gland 26.0 
Spleen 38.0 
Bone marrow 4.6 
Lung 12.0 
Stomach 20.0 
Ileum 13.0 
Colon 16.0 
Liver 40.0 
Pancreas 24.0 
Testis 28.0 
Bladder 34.0 
Kidney 52.0 390 
Uterus 44.0 
Thyroid gland 2.8 
Brain 14.0 
Esophagus 7.2 
Ovaries 7.8 
Breast 0.6 
Prostate 4.6 699 152 
Leukocytes 34.0 
a

Ct values were transformed to absolute gene copy numbers by calibration curves (5-log range) of rRNA and DD3 plasmid calibrators. The functions of the calibration curves were: f(CtrRNA) = −3.40 log [rRNA copies/20] + 40.80; and f(CtDD3) = −3.84 log [DD3 copies] + 45.32.

b

Normalized DD3 = (DD3/rRNA) × 108.

Table 2

Normalized DD3 expression in pooled tumor tissues

See descriptions of rRNA, DD3, and normalized DD3 in footnotes for Table 1.

Tumor poolnrRNA (×108)DD3Normalized DD3
Lung 13.0 
Esophagus 7.6 
Ileum 15.0 
Colon 18.0 
Pancreas 92.0 
Testis 15.0 
Breast 8.0 
Bladder 13.0 
Melanoma 8.8 
Tumor poolnrRNA (×108)DD3Normalized DD3
Lung 13.0 
Esophagus 7.6 
Ileum 15.0 
Colon 18.0 
Pancreas 92.0 
Testis 15.0 
Breast 8.0 
Bladder 13.0 
Melanoma 8.8 
Table 3

Normalized DD3 and hTERT expression in normal prostate, BPH, and adenocarcinoma tissue samples

Sample no.PathologicalTumor in tissue sample (%)ExpressionbNormalized hTERTfNormalized DD3g
TissueStageaGleason scorearRNAc (× 108)hTERT              dDD3e (× 103)
Normal    5.0 17 0.9 3.4 187 
Normal    11.0 54 0.0 4.9 
Normal    6.9 1.1 1.0 155 
Normal    6.1 10 1.6 1.6 264 
Normal    8.6 15 3.1 1.7 364 
Normal    6.6 12 3.9 1.8 593 
Normal    5.1 0.1 0.0 11 
Normal    3.5 0.6 1.4 181 
Normal    4.3 0.9 0.0 212 
10 Normal    4.2 0.7 2.1 166 
11 Normal    3.9 1.5 1.5 372 
12 BPH    5.5 18 1.8 3.3 326 
13 BPH    12.0 0.6 0.6 13 
14 BPH    6.1 32 0.1 5.2 10 
15 BPH    4.3 21 0.04 4.9 
16 BPH    11.0 18 0.4 1.6 40 
17 Tumor pT3 70 4.6 39 49.6 8.5 10,783 
18 Tumor pT3 50 5.7 60 30.4 10.5 5,340 
19 Tumor pT3 70 8.4 75 169.0 8.9 20,095 
20 Tumor pT3 80 7.5 278 111.0 37.1 14,865 
21 Tumor pT3 50 11.0 433 255.0 39.4 23,190 
22 Tumor pT3 65 7.0 266 63.0 38.0 8,998 
23 Tumor pT3 20 7.2 120 146.0 16.7 20,292 
24 Tumor pT2 40 7.5 13 34.5 1.7 4,599 
25 Tumor pT3 80 8.8 165 4.7 18.8 531 
26 Tumor pT3 75 4.3 306 16.2 71.2 3,779 
27 Tumor pT3 80 6.1 92 241.0 15.1 39,456 
28 Tumor pT2 70 7.2 202 226.0 28.1 31,457 
29 Tumor pT3 80 7.8 425 39.2 54.5 5,025 
30 Tumor pT3 90 10.0 471 197.0 47.1 19,716 
31 Tumor pT2 20 5.7 43.4 0.7 7,620 
32 Tumor pT3 70 5.9 76 41.9 12.9 7,101 
33 Tumor pT3 20 5.0 10 2.5 2.0 492 
34 Tumor pT2 40 4.2 1.5 1.4 356 
35 Tumor pT3 30 4.2 54 5.8 12.9 1,380 
36 Tumor pT2 30 4.9 43 1.6 8.8 334 
37 Tumor pT3 80 2.8 62 16.4 22.1 5,849 
38 Tumor   1.7 12 25.6 7.1 15,059 
39 Tumor   10 7.7 51 14.9 6.6 1,941 
40 Tumor   5 (+ 70% PIN)h 5.6 426 23.1 76.1 4,131 
41 Tumor   10 (+ 55% PIN) 4.8 15 56.5 3.1 11,776 
42 Tumor   10 4.2 29 8.6 6.9 2,038 
43 Tumor   5.2 34 38.5 6.5 7,404 
44 Tumor   <1 6.4 6.3 0.9 988 
45 Tumor   10 7.2 35 20.0 4.9 2,778 
46 Tumor   9.3 94 94.4 10.1 10,172 
47 Tumor   <1 10.0 47 9.6 4.7 962 
Sample no.PathologicalTumor in tissue sample (%)ExpressionbNormalized hTERTfNormalized DD3g
TissueStageaGleason scorearRNAc (× 108)hTERT              dDD3e (× 103)
Normal    5.0 17 0.9 3.4 187 
Normal    11.0 54 0.0 4.9 
Normal    6.9 1.1 1.0 155 
Normal    6.1 10 1.6 1.6 264 
Normal    8.6 15 3.1 1.7 364 
Normal    6.6 12 3.9 1.8 593 
Normal    5.1 0.1 0.0 11 
Normal    3.5 0.6 1.4 181 
Normal    4.3 0.9 0.0 212 
10 Normal    4.2 0.7 2.1 166 
11 Normal    3.9 1.5 1.5 372 
12 BPH    5.5 18 1.8 3.3 326 
13 BPH    12.0 0.6 0.6 13 
14 BPH    6.1 32 0.1 5.2 10 
15 BPH    4.3 21 0.04 4.9 
16 BPH    11.0 18 0.4 1.6 40 
17 Tumor pT3 70 4.6 39 49.6 8.5 10,783 
18 Tumor pT3 50 5.7 60 30.4 10.5 5,340 
19 Tumor pT3 70 8.4 75 169.0 8.9 20,095 
20 Tumor pT3 80 7.5 278 111.0 37.1 14,865 
21 Tumor pT3 50 11.0 433 255.0 39.4 23,190 
22 Tumor pT3 65 7.0 266 63.0 38.0 8,998 
23 Tumor pT3 20 7.2 120 146.0 16.7 20,292 
24 Tumor pT2 40 7.5 13 34.5 1.7 4,599 
25 Tumor pT3 80 8.8 165 4.7 18.8 531 
26 Tumor pT3 75 4.3 306 16.2 71.2 3,779 
27 Tumor pT3 80 6.1 92 241.0 15.1 39,456 
28 Tumor pT2 70 7.2 202 226.0 28.1 31,457 
29 Tumor pT3 80 7.8 425 39.2 54.5 5,025 
30 Tumor pT3 90 10.0 471 197.0 47.1 19,716 
31 Tumor pT2 20 5.7 43.4 0.7 7,620 
32 Tumor pT3 70 5.9 76 41.9 12.9 7,101 
33 Tumor pT3 20 5.0 10 2.5 2.0 492 
34 Tumor pT2 40 4.2 1.5 1.4 356 
35 Tumor pT3 30 4.2 54 5.8 12.9 1,380 
36 Tumor pT2 30 4.9 43 1.6 8.8 334 
37 Tumor pT3 80 2.8 62 16.4 22.1 5,849 
38 Tumor   1.7 12 25.6 7.1 15,059 
39 Tumor   10 7.7 51 14.9 6.6 1,941 
40 Tumor   5 (+ 70% PIN)h 5.6 426 23.1 76.1 4,131 
41 Tumor   10 (+ 55% PIN) 4.8 15 56.5 3.1 11,776 
42 Tumor   10 4.2 29 8.6 6.9 2,038 
43 Tumor   5.2 34 38.5 6.5 7,404 
44 Tumor   <1 6.4 6.3 0.9 988 
45 Tumor   10 7.2 35 20.0 4.9 2,778 
46 Tumor   9.3 94 94.4 10.1 10,172 
47 Tumor   <1 10.0 47 9.6 4.7 962 
a

Only adenocarcinomas samples that contained ≥20% tumor cells were used for statistical analysis for a relationship between DD3 or hTERT expression and pathological stage or Gleason score. Gleason score was defined as the sum of the two most common histological grades detected in the tissue sections by pathological review.

b

Ct values for all samples were transformed to absolute gene copy numbers, using calibration curves (5-log range) of plasmid calibrators.

c

f(CtrRNA) = −3.38 log [rRNA copies/20] + 38.71 (r2 = 1.0).

d

f(CthTERT) = −3.39 log [hTERT copies] + 40.43 (r2 = 0.99).

e

f(CtDD3) = −3.74 log [DD3 copies] + 40.19 (r2 = 0.99).

f

Normalized hTERT = (hTERT/rRNA) × 108.

g

Normalized DD3 = (DD3/rRNA) × 108.

h

PIN, prostatic intraepithelial neoplasia.

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