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Tumor Biology |
Matsunaga-Conte Prostate Cancer Research Center, Scott Department of Urology [K. M. S., S. F. S., C. N., A. K. L., W. S.] and Department of Pathology [T. M. W.], Baylor College of Medicine, and The Methodist Hospital, Houston, Texas 77030; Departments of Urology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [M. W. K.], and the Departments of Urology, Biochemistry, and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905 [C. Y. F. Y., D. J. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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643 bp in length and corresponds to the amplified product
of the native hK2 mRNA. Whereas the RT-PCR-hK2-L assay was positive in
71% of our patients with metastatic prostate cancer, 14% of healthy
control men also tested positive. By univariate
(P = 0.028) and multivariate
(P = 0.0269) analysis, which controlled
for preoperative PSA, clinical stage, and biopsy Gleason score,
RT-PCR-hK2-L status added prognostic information to the prediction of
lymph node-positive disease. We have developed a new RT-PCR assay which
demonstrates a high sensitivity for detecting hK2 mRNA. Preoperative
RT-PCR-hK2-L status helps predict pathological lymph node positivity in
patients with clinically localized prostate cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30% of these patients will fail
treatment, as evidenced by a detectable or rising PSA that often is
attributable to early dissemination of microscopic metastatic disease
(3)
. Conventional staging modalities such as bone
scan and CT scan have a limited role in staging patients with
clinically localized prostate cancer because of their poor performance
in detecting early, low-volume metastases (4
, 5)
.
Preoperative nomograms that consider established markers like PSA,
clinical stage, and biopsy Gleason Score can provide an estimate of the
risk of nodal metastases or disease recurrence, but are still imperfect
for determining the pathological stage or prognosis in individual
patients (6
, 7)
. Preoperative identification of patients
with metastatic disease would be helpful in sparing men from the
morbidity of a radical prostatectomy that would be ineffective or for
selecting patients best suited for clinical trials of neoadjuvant or
adjuvant therapy. The RT-PCR is a powerful and highly sensitive tool that has the ability to detect small numbers of prostatic cells disseminated within the peripheral blood or within other body fluids or tissues. Whereas some studies have demonstrated a significant predictive value of RT-PCR for PSA or RT-PCR for prostate-specific membrane antigen for both pathological stage (8, 9, 10, 11) and progression-free survival (12 , 13) with these assays, the majority have failed to demonstrate any clinically significant role (Refs. 14, 15, 16 ; most recent review, Ref. 17 ). In addition to the formidable technical problems regarding sample processing and handling that may underlie these conflicting results (18) , questions regarding the specificity of these markers for prostate cancer cells and the potentially variable biological potential of the cells detected by these assays have also limited the clinical utility of these assays (19, 20, 21, 22) . Whereas foci of metastatic prostate cancer detectable by conventional modalities (e.g., bone scan or computed tomography scan) are almost always associated with biologically significant advanced disease, the development of ultrasensitive techniques that can identify minute numbers of cells, like RT-PCR, places the burden on investigators to demonstrate the clinical and biological significance of these cells.
hK2 is an androgen-regulated protein that has an 80% amino acid
sequence identity with PSA (23)
and is expressed almost
exclusively in the prostatic epithelial cells. Immunohistochemical
studies have shown an incremental increase in hK2 expression from
benign epithelium, to prostatic intraepithelial neoplasia, to prostate
cancer (24)
. Furthermore, hK2 expression, in contrast with
PSA (24
, 25) , is directly associated with the Gleason
grade of the primary tumor with foci of prostate cancer metastatic to
the lymph nodes demonstrating the highest level of expression
(26)
. Finally, circulating levels of hK2 have been shown
to enhance the biochemical detection of prostate cancer when combined
with free and total PSA (27
, 28)
. These properties of hK2
suggest that it may represent a better RT-PCR target for the detection
of circulating, more biologically aggressive prostate cancer cells,
which in turn may better correlate with both occult micrometastatic
disease and the risk of disease progression.
We designed a novel, highly sensitive, and specific RT-PCR assay for the detection of prostate cancer cells expressing hK2 mRNA. First, we assessed the performance characteristics of this assay on peripheral blood specimens obtained from men without prostate cancer as well as from patients with documented metastatic disease. Subsequently, we assessed the ability of this RT-PCR-hK2 assay, performed on preoperatively obtained peripheral blood specimens, to predict advanced pathological stage in patients undergoing radical prostatectomy for the treatment of clinically localized prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hK2 and PSA cDNA.
A full-length human hK2 cDNA clone was used as a PCR template for hK2.
Previous analysis of this clone demonstrated a 70-bp deletion beginning
at position 150 and an additional 40 bp of intron 4 associated with a
splice variant of the hK2 gene (29)
reported
previously. Plasmid DNA was purified using the Qiagen-Plasmid Miniprep
Kit (Quiagen Inc., Valencia, CA). The cloned full-length human PSA
cDNA, which was used as a PCR template for PSA, was obtained as a gift
from Dr. Robert L. Vessella (Department of Urology, University of
Washington, Seattle, Washington).
Patient Selection and Sample Acquisition.
Two hundred and twenty eight consecutive patients undergoing radical
retropubic prostatectomy for clinically localized prostate cancer
(cT1–2) at The Methodist Hospital, Houston, TX, between
November 9, 1994, and November 2, 1995, were evaluated. Institutional
Review Board approved informed consent for the collection of clinical
data as well as serum and prostatic tissue samples were obtained for
all patients. Peripheral blood specimens were collected into Vacutainer
CPT 8-ml tubes containing 1 ml of 0.1 M sodium citrate
anticoagulant (Becton Dickinson, Franklin Lakes, NJ), typically
preoperatively in the morning on the day of surgery; and at least 4
weeks after prostate biopsy or other urological procedure
(e.g., cystoscopy). No patient was treated with either
neoadjuvant hormonal or radiation therapy before radical prostatectomy.
Serum PSA was measured by the Hybritech Tandem-R assay (Hybritech,
Inc., San Diego, CA). Clinical stage was assigned by the surgeon
according to the 1992 Tumor-Node-Metastasis system. The mean patient
age in this study was 61.9 ± 7.6 years (median, 62.9
years; range 37.7–75.5 years).
Control Patients.
Controls included patients with documented metastatic prostate cancer
and healthy male subjects without prostate cancer. Blood specimens from
seven patients with untreated, bone scan-proven metastatic prostate
cancer were used. The negative control group comprised 14 subjects
attending the Baylor Prostate Center’s prostate cancer screening
program who had a normal DRE and a serum PSA level of <0.5 ng/ml (mean
0.38 ± 0.09 ng/ml). These patients are unlikely to have
clinically detectable prostate cancer or to develop it over the
subsequent 10 years (30)
.
Pathological Examination.
All prostate biopsy and radical prostatectomy specimens were examined
at our institution by a single pathologist (T. M. W.), who was
blinded to clinical outcome. Standard pelvic lymph node dissection was
performed in each patient, and frozen sections of the lymph nodes were
microscopically examined for the presence of micrometastases. The
radical prostatectomy specimens were processed by whole-mount
technique, and pathological stage was assigned as described previously
(31)
. The location of each tumor focus was assessed, and
the presence of extraprostatic extension, seminal vesicle involvement,
and lymph node metastases was recorded. For the purposes of analysis,
pathological stage was categorized into four mutually exclusive groups
as follows: (a) organ-confined; (b) extracapsular
extension; (c) seminal vesicle involvement; and
(d) lymph node involvement. The Gleason grading
system was used to assign primary and secondary cancer grades.
RNA Preparation.
Mononuclear cells were separated from peripheral blood specimens by
centrifugation at room temperature for 20 min at 1500 g, and
the buffy coat fraction, containing nucleated cells, was recovered
using sterile transfer pipettes. The cells were washed once in PBS,
lysed with an acid-pH guanidine lysis buffer, and stored at -80°C
until processing. Initially RNA was extracted using the acid
phenol-chloroform method. Later, RNA was extracted using a modified
protocol, which replaced the initial ethanol precipitation by
application to Qiagen-RNeasy spin columns (Quiagen Inc.). RNA
was ethanol-precipitated and stored at -80°C in 70% ethanol and 85
mM NaOAC (pH 4.2) until assayed.
RT Reaction and cDNA Synthesis.
An aliquot containing 1 µg of RNA from each specimen was used for the
synthesis of the first strand of cDNA. In each of the reactions, a
50-µl solution containing 3 µM random hexamers, 25
mM Tris-HCl, 37.5 mM KCl, 1.5 mM
MgCl, 10 mM dithiothreitol, 0.25 mM each
deoxynucleotide triphosphate, 40 units of RNAsin RNase inhibitor
(Promega, Madison, WI), and 200 units of SuperScript II RNase
Transcriptase (Life Technologies, Inc., Rockville, MD) were processed
as described previously (32)
. Briefly, the annealing
mixture was incubated at room temperature for 15 min then incubated in
a thermocycler (Perkin-Elmer, Foster City, CA) at 37°C for 75 min.
The reverse transcriptase enzymes were inactivated by heating the
solution to 95°C for 15 s.
Oligonucleotide Primers.
Two sets of PCR primers were designed spanning intron 4 and including a
significant portion of the 3' untranslated region of the hK2
gene with maximum nonhomology to both hK1 and hK3 (PSA) at their 3'
ends (Fig. 1)
. After preliminary studies,
the primer set calculated to yield a 643-bp PCR product was selected
for additional analysis. The 5' end and the 3' end of our primer were
located at position 507 in exon 4 (5' flanking sequence from 507 to
526), and at position 1150 in the 3' untranslated region (3' flanking
sequence from 1150 to 1131), respectively. PSA cDNA and our primer for
hK2 cDNA differ at six positions within the sequence of the 5' primer
as well as the 3' primer.
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Determination of Specificity and Limit of Detection of RT-PCR
Assay for hK2 Message.
Preliminary studies using cDNA templates for hK2 and hK3 were performed
to determine assay specificity to the target species, hK2. The
detection limit of our protocol was tested in two ways. First, we
determined the detection limit of the PCR procedure with serial
dilutions of hK2 cDNA cloned into pGEM7Zf+, yielding 100, 50, 20, 10,
5, 1, and 0 copies of the plasmid/reaction. Second, we added LNCaP
cells into ATCC RPMI 7666-immortalized human lymphoblasts at a ratio of
one LNCaP cell per 104,
105, 106,
107, 108, and
109 lymphoblasts, and assayed by RT-PCR to
determine the detection limit. RNA was extracted as described above.
The quantity and quality of each RNA specimen was verified by both
spectrophotometry and gel electrophoresis. Amplification of the cloned
hK2 cDNA yielded a 653-bp fragment, which differs from the amplified
fragment from human RNA.
Nucleotide Sequencing.
The amplified 680-bp and 643-bp fragments of hK2 cDNA, referred to as
hK2-U and hK2-L amplified from LNCaP RNA, respectively, were excised
from a 2% agarose gel and purified with Bio101 Geneclean Kit (ISC
BioExpress, Kaysville, UT). The purified DNA was directly cloned using
the TA Cloning Kit (Invitrogen Co., San Diego, CA). The cloned product
was then sequenced using a 373 DNA Sequencer (Perkin-Elmer). The
sequencing reactions and analysis were performed using the
manufacturer’s recommendations.
Statistical Analysis.
The Pearson 
2
test was used to evaluate the
association between hK2 RT-PCR results and pathological stage.
Univariate and multivariate logistic regression analyses were used for
the prediction of final pathological lymph node status. Variables in
the model included typical predictors (e.g., preoperative
serum PSA, preoperative biopsy Gleason score, and clinical stage) in
addition to RT-PCR-hK2 results. Preoperative biopsy Gleason score and
the natural logarithm of serum PSA level were examined as continuous
variables. For the statistical analysis, RT-PCR-hK2 results were
examined as hK2-U (positive or negative) alone, as hK2-L (positive or
negative) alone, as combined hK2-U and hK2, as well as either hK2-U or
hk2-L. A P 
0.05 was considered statistically significant.
All analyses were performed with SAS software (version 6.12).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The
PCR assay amplified hK2 cDNA appropriately, but not PSA cDNA,
demonstrating the absence of cross-reactivity with PSA using this
assay. To determine the detection limit of the assay, we performed
serial dilutions of the plasmid hK2 cDNA, yielding 0 to 100
copies/reaction. Our PCR assay reliably detected as low as five copies
of the plasmid containing hK2 cDNA. However, lower copy numbers
occasionally yielded positive results (Fig. 3
and data not shown). Serial dilution of
LNCaP cells into immortalized human lymphoblasts at ratios of
1:104 to 1:109 demonstrated
that the lower limit of detection for this assay was at least one LNCaP
cell diluted in 109 lymphoblasts (Fig. 4)
.
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. DNA sequencing
analysis demonstrated that the larger fragment, hK2-U, corresponded to
the amplified product of a previously reported expressed, but not
translated, splice variant in the splice donor site of intron IV in the
hGK-1 gene (33)
. The smaller fragment, hK2-L,
was found to correspond to the amplified product of the native hK2
mRNA. This fragment has the same sequence as positions 507 to 1150 of
hK2 cDNA (GenBank accession no. S39329).
Detection of hK2 mRNA in Normal Control Subjects and Metastatic
Prostate Cancer Patients.
The specificity of the assay was tested on the peripheral blood
specimens of 14 healthy control subjects. Five of these specimens
(35.7%) tested positive for hK2-U and two (14.3%) tested positive for
hK2-L. We also performed RT-PCR assays on peripheral samples from seven
patients with metastatic disease. Six (85.7%) of seven specimens
tested positive, five for both hK2-U and hK2-L (71%), and one for
hK2-U only (Fig. 5)
.
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). We evaluated the relationship
between pathological stage and the results of the RT-PCR-hK2 assay,
scored separately for amplification of either hK2-L, hK2-U, or
combinations of both fragments. There was no correlation between
pathological stage and RT-PCR-hK2 results of any of the examined
fragments, although there was a trend of association between a positive
RT-PCR-hK2-L result and a more advanced pathological stage that
approached statistical significance (P = 0.056; Table 1
). RT-PCR-hK2-L (OR, 3.95;
95% CI, 1.96–8.49; P = 0.028) and the
combination of RT-PCR of hK2-L or hK2-U (OR, 4.24; 95% CI, 1.90–8.11;
P = 0.025) were found to be significant
predictors of final lymph node status (Table 2)
. Multivariate logistic regression
analysis was performed for the prediction of final pathological lymph
node status. In a model that included preoperative PSA, clinical stage,
and biopsy Gleason score, along with RT-PCR-hK2-L, preoperative serum
PSA level and clinical stage failed to predict lymph node status,
whereas preoperative biopsy Gleason score (OR, 3.28; 95% CI,
1.31–6.23; P = 0.0112) and RT-PCR-hK2-L (OR,
4.95; 95% CI 1.20–9.39; P = 0.0269) were
found to predict the presence of pelvic lymph node metastases
(Table 3)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We designed our assay to provide maximum sensitivity and specificity for hK2 without any cross-reactivity to the closely related kallikreins, hK1 and hK3. Furthermore, we designed our primers to differentiate between amplification of the native hk2 transcript (hK2-L), which encodes for the full-length hK2 protein, and an alternate spliced transcript (hK2-U), which contains an additional 37 nucleotides downstream from the native splice donor site in intron IV (33) . This larger, alternatively spliced mRNA is predicted to encode a truncated version of the hK2 protein, although no such species of hK2 protein has been identified in vivo. Recently, three additional species of hK2 transcripts have been described, including two 3.0 kb hK2 transcripts corresponding to the two known 1.5 kb major isoforms, but each with an additional 1.5 kb of 3' untranslated region attributable to transcription through the first polyadenlyation signal to a second signal located downstream. A third form, with a deletion of 13 nucleotides between exons 3 and 4 of the hKLK2 transcript that would result in a truncated hK2 protein missing 67 amino acids at the COOH-terminus of the protein, was also identified (34) . This second predicted truncated hK2 protein has also not been identified in vivo. Because of the overlapping position of our 5' primer with the 13-bp deletion in intron 3, our assay would not be predicted to amplify this third species of hK2 mRNA. However, our primers were predicted to amplify a 643-bp fragment from both the native 1.5 kb and the related 3.0-Kb transcript with the longer 3' UTR. Similarly, our primers were predicted to amplify the larger 680-bp fragment from both the 1.5 kb, and the related 3.0-kb, alternate intron IV splice variants.
By differentially amplifying these splice variants, we were able to associate a more biologically aggressive phenotype with the amplified native hK2-L fragment but not to the fragment encoding a predicted truncated and, presumably, nonfunctional hK2 protein. hK2 encodes a highly active protease that has multiple biological activities and that may contribute functionally to prostate cancer invasion and metastasis. Other active proteases such as uPA and plasmin are known to be involved in cancer invasion and metastasis. hK2 has been shown to activate single-chain uPA to the active a two-chain form of uPA (35 , 36) , which is highly correlated with prostate cancer metastasis (37) . More recently, hK2 has been shown to inactivate the major tissue inhibitor of uPA, plasminogen activator inhibitor-1 (38) . Thus, hK2 may influence the progression of prostate cancer by the activation of uPA and by the inactivation of the primary inhibitor of uPA, plasminogen activator inhibitor-1.
Patients with organ-confined, extracapsular disease and seminal vesicle invasion had similar, low rates of positive results (13–24%), in contrast to patients with lymph node involvement, 55% of whom had a positive result. Interestingly, overall 7-year, progression-free survival rates after radical prostatectomy in patients with organ-confined, extracapsular disease and seminal vesicle invasion, in whom the surgeon was able to achieve a negative surgical margin, are approximately 95%, 80%, and 50%, respectively, indicating that the majority of patients with specimen-confined prostate cancer adequately treated for their local disease will not fail distantly (39, 40, 41) . Conversely, most, if not all patients, with lymph node involvement will eventually fail local therapy with distant metastases regardless of the success of eradicating local disease (42, 43, 44) . One reason for the lack of association found between hk2 and pathological stage, despite the association between hk2 and LN+, could be that the ordinal logistic regression analysis of pathological stage makes additional assumptions about the relationship between hk2 and each level of pathological stage. In particular, it assumes that the effects of hk2 on each pathological stage are equal among adjacent pathological stages.
In studies published previously, Corey et al. (45) used primers that amplified a fragment spanning exons 2 to 4 of hK2, and Kawakami et al. (46) used primers that amplified a fragment spanning exons 3 to 4; neither assay was thus designed to differentially amplify these alternate mRNA species, and neither found any association between RT-PCR-hK2 status and pathological stage. Although we also did not find an association between RT-PCR-hK2 status and extracapsular disease or seminal vesicle involvement, RT-PCR for hK2-L, but not hK2-U, was a significant predictor of the final pathological lymph node status in a multivariate analysis that included clinical parameters such as PSA, Gleason score, and clinical stage. Neither Corey et al. (45) nor Kawakami et al. (46) detected an association between RT-PCR-hK2 positivity and lymph node metastasis, although Corey et al. (45) included only one patient with lymph nodes metastasis in their study.
Our assay was highly specific for hK2 without any detectable cross-reactivity for PSA. In addition, our assay was highly sensitive, as demonstrated by a reliable detection of five copies of hK2 cDNA and at least one LNCaP cell in 109 cultured lymphoblasts. Corey et al. (45) reported results using an assay that could detect 10 copies of hK2 cDNA and one LNCaP cell diluted in 107 peripheral blood mononuclear cells. Kawakami et al. (46) used an assay that only detected one LNCaP cell in 106 lymphocytes. These assays were thus 100- to 1000-fold less sensitive than our assay. We evaluated our RT-PCR assay on peripheral blood specimens obtained from healthy male subjects, patients with prostate cancer, and patients with untreated metastatic prostate cancer, and scored specifically for both hK2-U and hK2-L. Our assay was more sensitive (71% positive for hK2-L) for detecting hK2-expressing cells in patients with metastatic disease than the assays used in previous studies by Corey et al. (31%; Ref. 45 ) and Kawakami et al. (41%; Ref. 46 ).
Fourteen percent of the control specimens obtained from men at low risk for harboring prostate cancer tested positive for hK2-L, whereas 36% tested positive for hK2-U. In this same group of patients, 7% demonstrated a positive result with a highly sensitive RT-PCR for PSA (data not shown), which has been reported previously to detect at least one LNCaP cell diluted in 106 lymphoblasts (47) . The single control patient (1/14) who tested positive for RT-PCR-PSA was found to be negative for RT-PCR for hK2-L and hK2-U. Whereas the low rate of false positives for PSA and hK2-L were similar, the false positive rate for hK2-U was higher. However, RT-PCR-hK2-U was not a predictor of lymph node status in patients with clinically localized prostate cancer, questioning the biological relevance of this marker. The consistent results with positive and negative controls in each assayed cohort, and the low number of positive RT-PCR results for PSA, which are consistent with results from other groups, argue against a technical problem in our RT-PCR assay. More likely, our assay detected basal levels of hK2 expression, which may have arisen from nonprostatic sources of hK2 (48) , because of the high sensitivity of our PCR primer. Previously, other groups have reported a loss of diagnostic specificity of their assay because of overly sensitive PCR primers (49 , 50) . Corey et al. (45) and Kawakami et al. (46) reported no false-positive results among negative controls using assays less sensitive than ours. Whereas our study clearly demonstrates a biologically and statistically significant association between RT-PCR-hK2-L positivity and lymph node involvement, improvements in assay calibration, perhaps using semi-quantitative formats such as direct real-time fluorescence monitoring of PCR amplification, could help increase the specificity of RT-PCR while maintaining a high sensitivity for detecting biologically and clinically relevant micrometastatic disease. Ideally, after additional confirmation of these results, RT-PCR-hK2-L could be included in appropriate nomograms that use other predictive variables (e.g., PSA, clinical stage, and Gleason score) to maximize their clinical utility in predicting advanced disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the National
Cancer Institute Specialized Program of Research Excellence (SPORE
CA58203), from the NIH (CA70892), from the Frost Foundation, Inc., and
from the Max Kade Foundation, Inc. 
2 To whom requests for reprints should be
addressed, at Scott Department of Urology, Baylor College of Medicine,
Director, The Baylor Prostate Center, 6560 Fannin St., Suite
2100, Houston, TX 77030. Phone: (713) 798-8670; Fax: (713) 798-8030;
E-mail: kslawin{at}www.urol.bcm.tmc.edu
3 The abbreviations used are: PSA, prostate
specific antigen; RT-PCR, reverse transcription-PCR; hK2, human
glandular kallikrein; hK2-L, hK2 smaller fragment; hK2-U, hK2 larger
fragment; OR, odds ratio; CI, confidence interval; uPA, urokinase-type
plasminogen activator.
Received 4/28/00. Accepted 10/17/00.
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