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Clinical Chemistry 48: 1265-1271, 2002;
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(Clinical Chemistry. 2002;48:1265-1271.)
© 2002 American Association for Clinical Chemistry, Inc.

Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease

Alice Ylikoski1, Kim Pettersson1, Jussi Nurmi1, Kerttu Irjala2, Matti Karp1, Hans Lilja3, Timo Lövgren1a and Martti Nurmi4

1 Department of Biotechnology, University of Turku, 20520 Turku, Finland.
Departments of
2 Clinical Chemistry and
4 Urology, Turku University Hospital, 20520 Turku, Finland.

3 Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, 20502 Malmö, Sweden.

aAddress correspondence to this author at: Department of Biotechnology, University of Turku, Tykistökatu 6 A 6th Floor, 20520 Turku, Finland. Fax 358-2-3338050; e-mail Timo.Lovgren{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease.

Methods: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1–pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 102–106 mRNA copies.

Results: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann–Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (<=5) were negative for both PSA and hK2 (P = 0.02, Mann–Whitney U-test).

Conclusions: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate-specific antigen (PSA)1 and human glandular kallikrein 2 (hK2) have important applications in prostate cancer (PC) diagnostics. In healthy prostate tissue, the hK2 mRNA concentrations have been estimated to be from 10% to 50% of the PSA mRNA concentrations (1)(2)(3). Both proteins are secreted into seminal fluid (4)(5), although the concentration of hK2 is approximately two orders of magnitude lower than that of PSA (6). PSA and hK2 are also expressed at very low concentrations in the endometrium (7), the pituitary gland (8), and nonpathologic and pathologic breast tissues and secretions (6)(9)(10). Furthermore, in vitro studies have shown that hK2 converts the inactive PSA zymogen form into enzymatically active PSA (11)(12). Measurements of different molecular forms of PSA (total, free, and complexed) are widely used to diagnose and monitor PC patients (13)(14)(15)(16). Further improvements in the discrimination of patients with PC from individuals with benign prostatic hyperplasia (BPH) (17) as well as in preoperative staging (18) have been obtained with assays specific for hK2, either by use of the hK2 concentration alone or in combination with, especially, the free fraction of PSA (19)(20)(21).

Demonstration of circulating PC cells in blood has generally been expected to give information on the extraprostatic spread of the disease and the aggressiveness of the cancer. Approximately 20–30% of patients with clinically localized PC are found to have locally extensive or metastatic disease subsequent to radical surgery (22)(23). Recurrent disease may be attributable to PC cells in peripheral blood or bone marrow that were undetectable at the time of diagnosis. Radical prostatectomy represents an inappropriate treatment for patients with metastatic disease, which usually is established by lymphadenectomy. This is an invasive procedure, and therefore, there is a need for noninvasive tests that can reliably distinguish metastatic and aggressive cancers from those that are confined to the prostate gland. Circulating PC cells can be detected by reverse transcription-PCR (RT-PCR) for the measurement of prostate-specific mRNA markers. The most common mRNA markers for prostatic cells are PSA (24)(25)(26)(27)(28), hK2 (23)(29)(30), and prostate-specific membrane antigen (31)(32)(33)(34). However, studies to date have produced contradictory results regarding the utility of RT-PCR to predict PC progression and aggressiveness. In some studies, a preoperatively positive PSA RT-PCR result has been found to predict the final pathologic stage and prognosis in patients undergoing radical prostatectomy (27)(31)(35)(36)(37), whereas other studies have not shown such a correlation (32)(33)(38). In addition, detection of PSA and hK2 mRNA by highly sensitive RT-PCR methods in blood from healthy individuals demonstrated the need for quantitative and standardized RT-PCR procedures to discriminate between PSA and hK2 expression by circulating prostate and other cells (e.g., blood cells) (7)(38)(39)(40)(41). Recent reports on RT-PCR detection of hK2 mRNA have suggested that hK2 may be a useful marker for the detection of progressive PC (29)(30) and may provide supplementary information when combined with PSA RT-PCR results (23).

We recently developed a truly quantitative, internally standardized RT-PCR assay for the detection of PSA (42)(43) and hK2 (44) mRNA in blood samples from PC patients. The preliminary evaluation of a multiplexed quantitative RT-PCR assay for PSA and hK2 mRNA (44) showed stronger expression of hK2 mRNA than of PSA mRNA in patients with aggressive PC and with metastasized PC. We have now further evaluated the multiplexed quantitative RT-PCR assay. We measured the number of PSA and hK2 mRNA copies in blood samples from patients with benign disease and PC to evaluate how the copy numbers of the target mRNAs separate between the different stages of PC and the benign condition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
blood samples
We collected 10-mL blood samples (EDTA) from patients with PC and benign disease at Turku University Hospital. The procedures followed were approved by the local ethics committee and were in accordance with the Helsinki Declaration of 1975, as revised in 1996. Each blood sample was aliquoted into two 5-mL portions, and the nucleated blood cells with the possible circulating cancer cells were collected by dextran sedimentation of the red blood cells (43). The cell pellets were snap-frozen in liquid nitrogen and then stored at -72 °C until RNA extraction. The blood samples were obtained before physical examinations and surgical procedures, and the samples were processed immediately so that the time from venipuncture to obtaining the cell pellet was no more than 1.5–2 h. A total of 70 specimens from men with PC (age range, 54–86 years) and men with benign disease [BPH, high-grade prostatic intraepithelial neoplasia, and prostatitis; age range, 57–90 years] were selected and delivered to the laboratory "blinded". The blood samples were collected from consecutive patients who were referred to the Department of Urology for BPH or PC. The patients included 17 patients with confirmed BPH [12 found by transurethral resection of the prostate (TURP); 2 by biopsy, and 3 by adenomectomy]; 1 patient with high-grade prostatic intraepithelial neoplasia and BPH found by TURP; 1 patient with prostatitis; 10 patients with confirmed organ-confined PC (1 with pT1a, 4 with pT2a, and 5 with pT2b); 6 patients with non-organ-confined PC (5 with pT3a and 1 with pT3b); 8 patients with PC with regional lymph node involvement (4 found by radical prostatectomy and 4 by lymphadenectomy; pT1N1–pT4N1); 6 patients with distant metastatic PC (bone scan, M1), still hormone responsive; 11 patients with distant, hormone-independent bone metastases; and 10 patients with clinically staged PC (1 with T1c, 1 with T2, 3 with T2a, 2 with T2b, 1 with T3a, 1 with T3b, and 1 with T4). Each of the patients diagnosed with PC had the diagnosis confirmed by histologic examination of either biopsied or surgically resected material. Serum free- and total-PSA values were measured with a DELFIA ProstatusTM PSA F/T Dual assay (Perkin-Elmer Life Sciences, Wallac Oy) according to manufacturer’s instructions at the University Hospital Turku; values were available for 15 patients with benign disease and all the PC patients. The PSA values were analyzed from a serum sample taken at the same time as the blood sample for the RT-PCR analysis.

multiplexed quantitative rt-pcr assay for psa and HK2
Simultaneous quantification of PSA and hK2 mRNA was performed by a multiplexed RT-PCR assay (44) based on the use of target-like synthetic internal standard mRNA (IS-PSA and IS-hK2) and an external calibration curve. Known amounts of IS-PSA and IS-hK2 mRNA were added to each sample at the beginning of the RNA extraction. The calibration curve covered the range from 50 (i.e., the analytical detection limit) to 106 copies of PSA and hK2 mRNA and contained 5000 copies of IS-PSA and IS-hK2 mRNA mixed with the dilutions of synthetic calibrator (PSA and hK2) mRNA. The IS and calibrator mRNA was produced in vitro and purified as described previously (44). The functional detection limit of the assay was 100 copies of PSA and hK2 mRNA, with an interassay CV <23%. For the quantification of PSA and hK2 mRNA in a sample, 50 000 copies of each IS mRNA were added to the denatured cell pellet just before RNA extraction. The RNA pellet was dissolved in 80 µL of RNase-free, sterile water. One-tenth (8 µL) of each sample (corresponding in theory to 5000 copies of each internal standard mRNA) was then analyzed by RT-PCR. After the extraction, the target and the IS mRNA in the sample and the mRNA in the external calibration curve were coamplified by RT-PCR (in the same amplification mixture).

The PCR mixture contained two pairs of primers for the specific amplification of hK2 and IS-hK2 and of PSA and IS-PSA, respectively. The 3' primers were labeled with biotin to produce biotinylated amplification products. After 30 cycles of PCR, the amplification products were quantified by dual-label solution hybridization assays on streptavidin-coated microtitration wells and time-resolved fluorometry using specific europium and terbium chelate-labeled detection probes for the target (PSA and hK2) and the ISs (IS-PSA and IS-hK2), respectively. The amount of the target mRNA in the sample was calculated by comparing the target-to-IS fluorescence ratio in the sample with that in the calibration curve. The use of ISs enables the control of variations during the whole assay procedure from RNA extraction to detection of the amplification products by solution hybridization. No cross-reactivity has been detected; assay conditions allow only specific amplification of the PCR templates with their correct primers and specific detection of the PCR products by the hybridization probes.

statistical analysis
The nonparametric Mann–Whitney U-test was used to determine differences between the benign and cancer cases with regard to PSA mRNA, hK2 mRNA, serum total PSA, and serum free PSA concentrations and the percentage of serum free PSA. Samples with undetectable PSA or hK2 mRNA copies were assigned a value of 99 copies (below the functional detection limit of 100 mRNA copies) in statistical analyses of the data. Statview 4.53 for Windows was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
psa and HK2 MRNA
Detectable amounts of PSA and hK2 mRNA copies were found in 41 of 51 (80%) and 43 of 51 (84%) PC patients, respectively. Fig. 1 shows the number of patients who had detectable amounts of PSA and/or hK2 mRNA in their blood. In patients with organ-confined disease (pT1a–pT2b), PSA and hK2 were detected in 6 of 10 patients, whereas 3 patients were negative for both PSA and hK2 mRNA. Interestingly, PSA or hK2 mRNA-negative patients with organ-confined PC had Gleason scores <=5, which can be considered to represent less aggressive cancers than those with Gleason scores >5. Patients with organ-confined PC and both positive PSA and hK2 mRNA results had Gleason scores >5. The Gleason scores were significantly different between the target mRNA-positive and -negative patients (P = 0.02, Mann–Whitney U-test). However, tumor grade did not correlate with PSA mRNA, hK2 mRNA, or tumor stage when all the cancer cases were compared. PSA and hK2 mRNA was detected in all patients with non-organ-confined (pT3a–pT3b; n = 6) and lymph node-positive (pT1N1–pT4N1; n = 8) PC. Furthermore, PSA and hK2 were detected in five of six and six of six patients with distant metastatic PC, respectively. In patients with hormone-independent metastatic PC, both PSA and hK2 were detected in 7 of 11 patients, and the remaining 4 patients were positive for either PSA (n = 2) or hK2 (n = 2) mRNA. In patients with clinically staged PC (T1c–T4), PSA and hK2 were detected in 7 of 10 and 8 of 10, respectively, with 2 patients negative for both markers. One BPH patient of the 19 patients with benign disease had detectable amounts of PSA (1500 copies) and hK2 (3100 copies) mRNA.



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  		Figure 1. Number of patients with either positive or negative RT-PCR results.

PSA- & hK2- denotes patients negative for both PSA and hK2 mRNA, PSA+ denotes patients positive for PSA mRNA, hK2+ denotes patients positive for hK2 mRNA, and PSA+ & hK2+ denotes patients positive for both PSA and hK2 mRNA. The categories include patients with benign disease (BPH, high-grade prostatic intraepithelial neoplasia, and prostatitis), confirmed organ-confined PC (Oc), confirmed non-organ-confined PC (Noc), PC involving regional lymph nodes (LN+), metastatic PC (Met), hormone-independent PC (Horm. ind.), and clinically staged PC (Clin. staged).

All RNA samples were analyzed twice to confirm the results, and the results were concordant. Table 1 shows the median values and the interquartile ranges for PSA mRNA, hK2 mRNA, serum free PSA, serum total PSA, and the percentage of free PSA among the patients with benign disease and PC. Box plots representing the PSA and hK2 mRNA copy numbers, serum total- and free-PSA concentrations, and the percentage of free PSA among the patients with benign disease and PC is shown in Fig. 2 . The difference between the PSA and hK2 mRNA copy numbers did not reach significance.


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  		Table 1. Medians (interquartile ranges) for PSA mRNA, hK2 mRNA, serum total PSA, serum free PSA, and percentage of serum free PSA for patients with benign disease or PC.1



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  		Figure 2. Box plots showing the distribution of PSA mRNA and hK2 mRNA copy numbers (A), serum total- and free-PSA concentrations (B), and the percentage of serum free PSA (C) among patients with benign disease () and PC ({square}).

Circles indicate outliers.

discriminative power of psa and HK2 MRNA
PSA and hK2 mRNA strongly discriminated between benign disease and PC (P <0.0001, Mann–Whitney U-test) as shown in Table 1Up . PSA and hK2 mRNA copy numbers were able to differentiate the benign disease from the organ-confined PC (P = 0.021 for PSA and 0.019 for hK2, Mann-Whitney U-test; data not shown), although four patients were negative for either PSA or hK2 mRNA. Serum PSA values analyzed at two different time points were available for the one BPH patient with detectable PSA and hK2 mRNA. This patient was suspected to have cancer because of the low percentages of serum free PSA (3.9% and 5.5%) with total PSA concentrations of 14 and 13 µg/L, respectively, at the two time points. This patient had BPH as confirmed by TURP at the time when the blood sample was taken for the RT-PCR analysis. Biopsies were later taken on two occasions from the same patient, and no cancerous tissue was found. Of the other variables tested, serum total and free PSA discriminated between benign disease and PC (P = 0.03 and 0.02, respectively, Mann-Whitney U-test). The percentage of free serum PSA differentiated patients with benign disease from those with organ-confined PC (P = 0.006, Mann–Whitney U-test; data not shown).

Neither PSA nor hK2 mRNA copy numbers differentiated between the different stages of PC when the PC patients were divided into subgroups based on the stage of the cancer. However, the number of patients in each subgroup was relatively small, making a reliable comparison difficult. Of the other variables, serum total PSA differentiated patients with organ-confined disease from patients with lymph node-positive PC (P = 0.01, Mann–Whitney U-test) and metastatic PC (P = 0.005, Mann–Whitney U-test; data not shown). Serum free PSA differentiated patients with organ-confined PC from patients with lymph node-positive PC (P = 0.009, Mann–Whitney U-test), metastatic PC (P = 0.008, Mann–Whitney U-test), and metastatic hormone-independent PC (P = 0.008, Mann–Whitney U-test; data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of their high sensitivity, RT-PCR assays incorporating prostate cell-derived mRNA markers such as PSA, prostate-specific membrane antigen, and hK2 have been used for the detection of PC cells. However, results on the clinical utility of RT-PCR assays in staging of PC have been controversial. This may be attributable to the variable, nonstandardized assay procedures and the presence of low expression of the target mRNA transcripts in nonprostatic cells (e.g., nondiseased blood cells) (7)(34)(38)(39)(40)(41). For these reasons, there has been a need for truly quantitative and standardized RT-PCR assays. We recently developed a quantitative RT-PCR assay for highly specific and reproducible detection of PSA and hK2 mRNA simultaneously from the same sample (44). A critical feature of this assay is the use of ISs added at an early stage of sample handling to correct for variability in RNA recovery and RT-PCR efficiency, thereby enabling true quantification of the RNA copies in the samples.

The results of the present study suggest that the dissemination of PC cells in the blood is an early event in PC progression and are in agreement with the results from previous studies (27)(32). We found that 70% of the organ-confined PCs (pT1–pT2) were positive for PSA and/or hK2, with 60% being positive for both markers. Although 3 of 10 patients with organ-confined PC were negative for both PSA and hK2 mRNA and 1 of 19 patients with benign disease was positive for PSA and hK2 mRNA, both PSA and hK2 mRNA discriminated patients with benign disease from those with PC with high statistical significance. Of PC patients with organ-confined disease, the patients with undetectable PSA or hK2 mRNA had significantly lower Gleason scores than those with detectable PSA and hK2 mRNA. The BPH patient positive for PSA and hK2 mRNA had repeatedly low percentages of free serum PSA (3.9% and 5.5%) with total PSA concentrations of 14 and 13 µg/L, respectively, at the two sampling time points. However, no cancer was found in the initial TURP performed or a subsequent biopsy. Further follow-up of this patient is needed.

PSA or hK2 mRNA was detected in all the patients with non-organ-confined PC, lymph node-positive PC, metastatic PC, and hormone-independent metastatic PC. There remains considerable overlap of both PSA and hK2 mRNA values between the different patient groups, and more extensive future studies are needed to evaluate any quantitative differences between the different patient groups.

In a previous study with 25 PC samples, we found that PSA and hK2 mRNA was present only in 3 of 8 (38%) and 2 of 8 (25%) patients with hormone-independent metastatic PC, whereas 13 of 13 patients with hormone-dependent metastatic PC and 4 of 4 patients with clinically staged, biochemically progressive PC had detectable PSA and hK2 mRNA (44). In addition, PSA and hK2 mRNA expression was significantly lower in patients with hormone-independent metastatic disease than in patients with hormone-dependent metastatic or biochemically progressive PC. In the present study, we found that 100% of patients with hormone-independent metastatic (11 of 11) and with hormone-dependent metastatic (6 of 6) disease were positive for either PSA or hK2. Four of 11 (36%) patients with hormone-independent PC had nondetectable PSA or hK2. One of six (17%) patients with hormone-dependent PC had undetectable PSA mRNA but detectable hK2 mRNA. Contrary to the previous findings, the results of the present study do not show differences in the copy numbers of PSA or hK2 mRNA between the patients with hormone-dependent and hormone-independent metastatic PC or with other stages of PC.

In principle, there are three major expectations regarding the clinical use of RT-PCR assays for the detection of circulating cancer cells. The first expectation is that one expects no detectable signal in verified benign cases. Only 1 BPH case of 19 benign cases tested positive for both PSA and hK2 mRNA. Although the serum-based indicators (total and percentage of free PSA) strongly suggested the presence of cancer, no malignant lesions were found in the biopsy. However, it is a well-documented fact that the first biopsy may miss the cancer lesion in up to 20% of the cases, as evidenced from the number of cancers found on repeat biopsy (45).

The second expectation is that the test would be ideal if it provides a reliable preoperative estimation of whether a biopsy-confirmed lesion is or is not confined to the prostate gland and thus is eligible or not eligible for curative therapy. A surprisingly high degree of positivity (70%) of the two markers was obtained for the 10 cancers that on subsequent radical prostatectomy were classified as being organ-confined. In light of the high success rate of radical prostatectomy in pathologically organ-confined cancers (46), the significance of demonstrating circulating prostate cells in these cancer patients is currently unresolved. It may be reasonable to expect that some additional qualitative variable(s) will be needed to provide a truer estimation of the pathologic potential of the circulating cells. It should also be kept in mind that the exact number of kallikrein transcripts does not provide information on the number of circulating cells or on the number of transcripts per cell. In fact, recent results by Stege et al. (47) have shown that the concentration of PSA in primary tumor tissues constitutes the best prognostic index for hormonally treated patients regarding progression of the cancer. It is therefore tempting to speculate whether the number of kallikrein transcripts per circulating cell would provide a more accurate estimation of the likelihood that a primary tumor will eventually progress.

The third expectation relates to whether RT-PCR-based quantification of mRNA will provide a clinically useful indicator for the degree of aggressiveness of the cancer. In biopsy-confirmed PC patients who tested negative for both kallikrein transcripts, interpretation of the results and the selection of treatment regimens remain open. Lack of circulating cells may be a sign of an early cancer of unknown aggressiveness, which has simply not reached the point at which dissemination of cells occurs. On the other hand, it may indicate that the cancer lesion is of an indolent nature, not demanding aggressive therapy, and thus is a candidate for watchful waiting. In both cases, the answer has to be sought from studies with extended follow-up times. In cases where a suspicion of cancer remains despite repeatedly negative biopsies, RT-PCR assays may provide more definitive or reassuring information regarding the presence or absence of disease demanding immediate attention.

To resolve the numerous questions regarding the exact positioning of RT-PCR assays in the clinical work-up of PC, it is mandatory that truly quantitative and standardized assay technologies are used that are devoid of the flaws often seen with conventional RT-PCR methods. This study is the first report describing the application of a truly quantitative assay for the detection of the two major prostatic kallikrein transcripts, PSA and hK2, in patients with benign disease and PC. Our results indicate that both PSA and hK2 mRNA measurements can be used to differentiate patients with PC from patients with benign disease. The main limitations of this preliminary clinical study are the limited size of the patient cohorts and the lack of extended clinical follow-up data. Establishing the full clinical potential of these assays is critically dependent on expanded studies with extensive follow-up times. Such studies have been initiated.


   Acknowledgments
 
This work was supported in part by grants from Biomed 2 Program, area 4.1.7. (Contract BMH4-CT96-0453); the Swedish Cancer Society (Project 3555); the Faculty of Medicine at Lund University Hospital, Malmö; the Crafoord Foundation; the Gunnar, Arvid and Elisabeth Nilsson Foundation; Fundacion Frederico S.A; and the Academy of Finland (Project 45252).


   Footnotes
 
1 Nonstandard abbreviations: PSA, prostate-specific antigen; hK2, human glandular kallikrein 2; PC, prostate cancer; BPH, benign prostatic hyperplasia; RT-PCR, reverse transcription-PCR; TURP, transurethral resection of the prostate; and IS, internal standard.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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