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Detection of Clinically Significant, Occult Prostate Cancer Metastases in Lymph Nodes Using a Splice Variant-Specific RT-PCR Assay for Human Glandular Kallikrein

Shahrokh F. Shariat, Michael W. Kattan, Sibel Erdamar, Cuong Nguyen, Peter T. Scardino, David M. Spencer, Thomas M. Wheeler, Kevin M. Slawin

From the Baylor Prostate Center, the Scott Department of Urology, and the Departments of Pathology and Immunology, Baylor College of Medicine, Houston, TX; and the Departments of Urology and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY.

Address reprint requests to Kevin Mark Slawin, MD, Associate Professor, Scott Department of Urology, Baylor College of Medicine, The Baylor Prostate Center, 6560 Fannin St., STE 2100, Houston, TX 77030; email: kslawin@www.urol.bcm.tmc.edu.

	ABSTRACT

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Purpose: To compare the detection of human glandular kallikrein 2 (hK2) mRNA expression in archival lymph nodes with disease progression, the development of prostate cancer metastases, and mortality in patients undergoing radical prostatectomy for locally advanced nonmetastatic prostate cancer.

Patients and Methods: We evaluated total RNA extracted from fixed, paraffin-embedded, histopathologically normal pelvic lymph nodes, removed at radical prostatectomy, from 199 pT3N0 prostate cancer patients (150 extraprostatic extension only; 49 seminal vesicle involvement) for hK2-expressing cells using a novel reverse transcriptase polymerase chain reaction (RT-PCR)/hK2 assay. Cumulative incidence functions and Cox proportional hazards analyses were performed.

Results: Forty patients (20%) had positive results, 80 patients (40%) had negative results, and 79 patients (40%) had equivocal results. RT-PCR/hK2 status was not associated with any pathologic characteristics (P > .05). In postoperative multivariable models, the RT-PCR/hK2 result was associated with prostate cancer progression (P = .001), development of distant metastases (P = .001), and prostate cancer–specific survival (P = .005). In patients experiencing biochemical progression (n = 33), RT-PCR/hK2 status was a predictor of failure to respond to salvage radiotherapy (P = .002).

Conclusion: RT-PCR/hK2 can detect biologically and clinically significant occult prostate cancer metastases in histopathologically normal lymph nodes. In patients with locally advanced prostate cancer, RT-PCR/hK2 is strongly associated with prostate cancer progression, failure following salvage radiation therapy, development of clinically evident metastases, and prostate cancer–specific mortality after surgery.

	INTRODUCTION

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ALTHOUGH MOST patients with organ-confined prostate cancer have long-term freedom from biochemical progression after radical prostatectomy, those with locally advanced prostate cancer, in the form of extraprostatic extension (EPE) and/or seminal vesicle involvement, are at increased risk for disease progression.^1–3 In these patients, disease progression is often the result of early dissemination of microscopic metastatic disease that remains undetectable before primary therapy.⁴ Furthermore, most, if not all, patients with histopathologically evident pelvic lymph node involvement will experience failure of local therapy and eventually develop distant metastases and clinical disease progression, regardless of apparent success in eradication of local disease.^3,5–7 Conventional staging modalities such as imaging techniques (ie, bone scan, computed tomography scan, ultrasound, magnetic resonance imaging, positron emission tomography scanning, and ProstaScint [Cytogen, Princeton, NJ] scanning) and histopathologic procedures have a limited role in staging these patients because of their poor performance in detecting early, low-volume occult prostate cancer metastases.^8–11 Pre- and postoperative nomograms that consider established markers such as prostate-specific antigen (PSA), stage, and Gleason grade can provide an estimate of the risk of nodal metastasis or disease progression, but still are imperfect for determining prognosis in individual patients.^12–15 Therefore, new markers that can detect clinically relevant occult metastatic disease, which is associated with a high likelihood of eventual disease progression, may be helpful for selecting patients best suited for early systemic intervention, and for sparing men who have undergone prostatectomy from the morbidity associated with local adjuvant or salvage radiation therapy.

Immunohistochemical staining of paraffin-embedded sections of surgically removed lymph nodes using PSA and cytokeratin antibodies has been shown to be superior to routine hematoxylin and eosin staining for the detection of prostate cancer metastases. These techniques were used in several studies and have shown that up to 16% of patients with pT3N0 disease have occult prostate cancer metastases.^16–18 Reverse transcriptase polymerase chain reaction (RT-PCR) is, however, a more sensitive method than histology, flow cytometry, and immunohistochemistry for detecting small numbers of disseminated cells.¹⁹ In various cancers, including prostate cancer, RT-PCR has been shown to be superior to standard histologic and immunohistochemical approaches in sensitivity and specificity for detecting cells in regional lymph nodes.^20–22 Although the assays used in these studies identified disseminated prostatic cells, the biologic and clinical significance of these cells remain uncertain.²³ Foci of metastatic prostate cancer detectable by conventional modalities (eg, histology, bone scan, or computed tomography scan) are almost always associated with biologically significant advanced disease. New ultrasensitive assays, such as RT-PCR, that can identify very small numbers of cells, place the burden on investigators to demonstrate the clinical and biologic significance of a positive result.

We have developed a highly sensitive and specific RT-PCR assay for human glandular kallikrein 2 (hK2) mRNA²³ that was designed to differentially amplify two previously described splice variants of the hKLK2 gene.²⁴ When the assay was performed on peripheral blood of patients with clinically localized prostate cancer before radical prostatectomy, the native hK2-amplified fragment was significantly associated with an increased risk of metastasis to pelvic lymph nodes.²³ In this study, to further investigate the relationship between the native hK2 transcript and lymph node metastases, we evaluated the presence of occult micrometastatic disease in pelvic lymph nodes that remained undetectable by conventional pathologic methods from 199 consecutive patients with locally advanced prostate cancer. We modified our previous RT-PCR assay by designing a primer set that amplifies a smaller region of hK2 than our previous primer set, and we assessed the biologic and clinical significance of hK2-expressing cells in archival lymph nodes of prostate cancer patients. We found that RT-PCR detection of hK2-expressing cells in pelvic lymph nodes was associated with an increased risk for prostate cancer progression after primary and salvage local therapy, with the development of clinically detectable distant prostate cancer metastases, and with prostate cancer-specific risk of mortality.

	PATIENTS AND METHODS

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hK2 and PSA cDNA
A full-length human hK2 cDNA clone was used as PCR template for hK2. Previous analysis of this clone demonstrated a 70 base pair (bp) deletion beginning at position 150 and an additional 40 bp of intron 4 associated with a previously reported splice variant of the hK2 gene.²⁵ Plasmid DNA was purified using the Qiagen-Plasmid Miniprep Kit (Quiagen, Santa Clarita, CA). The cloned full-length human PSA cDNA, which was used as a PCR template for PSA, was obtained as a gift from Robert L. Vessella, PhD (Department of Urology, University of Washington, Seattle, WA).

Patient Selection and Sample Acquisition
We evaluated formalin-fixed, paraffin-embedded, histopathologically uninvolved pelvic lymph nodes from 217 consecutive pathologic stage pT3N0 patients who underwent radical prostatectomy for the treatment of prostate cancer by a single surgeon (P.T.S.) at The Methodist Hospital (Houston, TX) between December 1983 and November 1996. All patients underwent an extended lymph node dissection, which routinely included level I, II, and III lymph nodes.²⁶ Usable RNA was isolated from lymphadenectomy specimens from 199 patients. RNA integrity was defined by successful amplification of appropriate size fragments of glyceraldehyde-3-phosphate dehydrogenase–positive controls. Patients were identified from the database of the Baylor Specialized Program of Research Excellence in prostate cancer and selected on the basis of pathologically identified locally advanced prostate cancer and negative lymph nodes (stage pT3N0). Institutional review board–approved informed consent for the collection of clinical data, as well as serum and tissue samples, was obtained for all patients. No patient was treated with either neoadjuvant hormonal or radiation therapy before radical prostatectomy, or adjuvant radiation therapy before elevated PSA levels were observed. Serum PSA was measured by the Tandem-R assay (Hybritech, San Diego, CA). The mean patient age in this study was 62.7 ± 6.8 years (median, 63.4; range, 49.6 to 75.1 years).

Pathologic Examination
All lymph node and radical prostatectomy specimens were examined at our institution by a single pathologist (T.M.W.) who was blinded to RT-PCR/hK2 results and to clinical outcome. The radical prostatectomy specimens were processed by whole-mount technique, and pathologic parameters were evaluated as previously described.²⁷ Total tumor volume was computed by computerized planimetry from the whole-mount sections.²⁸ One hundred and fifty patients had EPE only, and 49 patients had seminal vesicle involvement (cancer within the muscular coat of the seminal vesicle, not simply tumor in the fat adjacent to the seminal vesicle).² The level of EPE, with respect to the stroma of the prostate, prostatic capsule, and periprostatic soft tissue, was classified as described previously.²⁹ Seventy-eight patients (39%) had focal EPE (tumor outside the prostate to a depth of less than one high-power field on no more than two separate sections), and 121 (61%) had established EPE (any amount of extraprostatic tumor more than focal EPE). Standard pelvic lymph node dissection was performed in each patient, with a total of 1,846 pelvic lymph nodes removed at the time of radical prostatectomy (mean, 9.6 ± 4.2 lymph nodes per patient). Frozen sections of all lymph nodes were prepared and examined at the time of surgery for the presence of micrometastases. Remaining frozen lymph node tissue was then fixed in formalin and embedded in paraffin. Paraffin sections from each lymph node were stained with hematoxylin and eosin and microscopically examined for the presence of micrometastases.

Postoperative Follow-up
Each patient was scheduled to have a digital rectal examination and serum PSA evaluation every 3 months for the first postoperative year, semiannually from the second through the fifth year, and annually thereafter. Biochemical progression was defined as a sustained elevation, on two or more occasions, of PSA more than 0.2 ng/mL. The date of progression was assigned to the date of the first value more than 0.2 ng/mL. A staging evaluation, including bone scan, ProstaScint scan, or PSA doubling time calculation was performed in 63 of the 68 patients who had PSA progression before the administration of salvage therapy. For patients who had biochemical progression, postprogression serum PSA doubling time was calculated using the following formula: DT = log² x T/[log(final PSA) - log(initial PSA)],³⁰ where DT is the serum PSA doubling time, T is the time interval between the initial and final PSA level, final PSA is the preradiation PSA level, and initial PSA is the PSA level noted at the time of the postoperative biochemical progression. All patients had at least three PSA measurements available postprogression. The natural logarithm was used in all logarithmic transformations.

Survival data were obtained from the cancer registry at The Methodist Hospital (Houston, TX) and the patients’ medical records. Death certificates were retrieved on all dead patients from the archived death certificates and reviewed for cause of death. Attribution of cause of death on the death certificate is in two parts. Part I lists death caused by immediate cause of death (final disease or condition resulting in death) or by underlying cause of death, and part II lists other significant conditions contributing to death but not resulting in the underlying cause given in part I. Information abstracted from each death certificate included the date of death and whether prostate cancer was noted in part I or II. To reduce bias in attribution of cause of death, only men who had prostate cancer listed in part I of the death certificate were considered to have died of prostate cancer for this study.

Salvage Radiation Therapy
Of the 68 patients who had cancer progression, 33 were treated with salvage radiation therapy. Twenty-one of these 33 patients (64%) were treated with external beam therapy at the Methodist Hospital, and the remainder were treated at other institutions. Radiation therapy was limited to the prostatic fossa in 32 patients (97%) and one patient received pelvic radiation with an additional boost to the prostatic fossa. Radiation was delivered with 10- to 23-MV photons. The four-fields technique (anteroposterior/posteroanterior and opposing laterals) with customized field sizes was used. Total radiation therapy dose ranged from 60 to 75.5 Gy (median, 66 Gy), delivered in daily fractions of 1.8 to 2.0 Gy. After radiation, the patients were evaluated by physical examination and serum PSA measurements approximately every 3 to 6 months. Serum PSA measurements of patients who received radiation treatment in other institutions were available through regular follow-up reports. A complete response to salvage radiation therapy was defined as the achievement and maintenance of an undetectable serum PSA level (0.2 ng/mL). Radiation therapy was considered to have failed in a patient if the postradiation serum PSA levels did not decrease to and remain at an undetectable level.

RNA Preparation
All tissue blocks from archival, formalin-fixed, paraffin-embedded pelvic lymph nodes removed at pelvic lymph node dissection during radical prostatectomy were cut into approximately 50-µm ribbons. These were transferred to a sterile 2.0-mL microcentrifuge tube (Fischer Scientific, Fair Lawn, NJ). Paraffin was removed from the lymph node tissue by two 5-minute incubations in xylene at 55°C, and thereafter in 95% ethanol. Total cellular RNA was isolated by homogenizing in 1 mL of Ultraspect RNA isolation system (Biotex Laboratories, Houston, TX). To prevent sample carryover, the homogenizer probe was cleaned by multiple washes with 75% ethanol. After the addition of 1:5 volume chloroform, the sample was centrifuged at 12,000 x g for 15 minutes. The aqueous phase was transferred to a sterile 1.5-mL centrifuge tube, and an equal volume of isopropanol was added. The solution was then incubated at 4°C for 10 minutes. RNA was pelleted by centrifugation at 12,000 x g at 4°C for 15 minutes, washed with 75% ethanol, and dried. The RNA pellet was dissolved by incubation in 30 µL diethylpyrocarbonate-treated water at 55°C for 5 to 10 minutes. The RNA yield was measured by ultraviolet absorbance at 260-nm wavelength.

Oligonucleotide Primers
We previously developed a primer set (spanning intron 4 and including a significant portion of the 3' untranslated region of the hKLK2 gene),²³ which was shown to differentially amplify the native hk2 transcript, which encodes for the full-length hK2 protein, and an alternate spliced transcript, which contains an additional 37 nucleotides downstream from the native splice donor site in intron 4.²⁴ Given the high rate of RNA degradation in archival paraffin-embedded tissue, we designed our primer set for this study to amplify a smaller fragment within the sequence amplified by our previously described primer set. The upstream primer anchored, respectively, in exon 4, nt 563 to 582: 5'-ATGTGTGCTAGAGCTTACTC-3', and the downstream primer anchored in exon 5, nt 648 to 667: 5'-AAGTGGACCCCCAGAATCAC-3'. The primer was calculated to yield two distinct amplified DNA fragments: the alternate spliced transcript of approximately 142 bp and the native hk2 transcript of approximately 105 bp.

Reverse Transcription Reaction, cDNA Synthesis, and PCR
Reverse transcription was performed as described previously, but contained 1.25 µg of RNA.²³ The PCR conditions were similar to those reported previously²³ with the exception of cycling, which was performed for 33 cycles with an annealing temperature of 63°C for 1 minute, an extension at 72°C for 2 minutes, and a final extension step at 72°C for 7 minutes. After 12 µL of each PCR product was loaded on the 2% NuSieve agarose gel (FMC BioProducts, Rockland, ME) in tris-acetate-EDTA (TAE) buffer, ethidium bromide staining was performed followed by gel documentation. After electrophoresis, PCR products were transferred onto a charged nylon membrane (Boehringer Mannheim, Indianapolis, IN), which was probed using the Genius System (Boehringer Mannheim). Both gel analysis and a second PCR reaction amplifying the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene were used to assess mRNA integrity. Internal negative control reactions for the RT-PCR were performed using all of the reagents, but without added RNA in each of the assays. None of the assays exhibited a signal from the internal negative control. Internal positive control reactions for the RT-PCR were performed using 100 and 1,000 copies of hK2 cDNA, as well as at least two sets of formalin-fixed, paraffin-embedded prostate tissue.

Determination of Specificity and Limit of Detection of RT-PCR Assay for hK2 Message
Preliminary studies using cDNA templates for hK2 and hK3 (PSA) were performed to determine assay specificity to the target species, hK2. The detection limit of the PCR procedure was tested with serial dilutions of hK2 cDNA cloned into pGEM7Zf(+), yielding 100, 50, 20, 10, 5, 1, and zero copies of the plasmid per reaction.

RT-PCR Interpretation and Scoring
We chose to score RT-PCR results solely for detection of the native hK2 fragment, which we have previously found to be associated with a more biologically aggressive prostate cancer phenotype.²³ The test results were categorized as positive if a clear 105-bp product was visible either after ethidium bromide staining of the gel before blotting or if a signal was clearly evident after probing; as equivocal if no 105-bp product band was evident on ethidium bromide staining and a barely discernible signal was present on the probed membrane; and as negative if no hK2 product was seen either during gel documentation or after membrane probing, but a band was present in each of the positive controls, indicating that the RNA was able to be reverse transcribed and subsequently amplified. All RT-PCR assay results were scored by the same investigator who was blinded to clinical and pathologic data, providing internal consistency to the application of these acknowledged subjective criteria.

Statistical Analysis
The ² test was used to evaluate the association of RT-PCR/hK2 results with clinical and pathologic features. Differences in variables with a continuous distribution across RT-PCR/hK2 categories were assessed using Kruskal-Wallis nonparametric analysis of variance. Cumulative incidence functions were generated and compared using the method of Gray.³¹ Multivariable survival analysis was performed with the Cox proportional hazards regression model. Preoperative PSA level had a skewed distribution and therefore was modeled with a log transformation in the Cox models. Restricted cubic splines were used for continuous variables in the Cox models. Statistical significance in this study was set as P < .05. All reported P values are two-sided. All analyses were performed with either the SPSS statistical package (SPSS version 10.0 for Windows, SPSS Inc., Chicago, IL) or S-Plus 2000 Professional software (Seattle, WA).

	RESULTS

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Assay Performance
To determine the specificity of our primer set for hK2, PCR assays were run on samples containing 100, 50, and 10 copies of hK2 cDNA and PSA cDNA. The PCR assay amplified hK2 cDNA appropriately, but not PSA cDNA, demonstrating the absence of cross-reactivity with PSA using this assay (Fig 1). To determine the detection limit of the assay, we performed serial dilutions of the plasmid hK2 cDNA, yielding zero to 100 copies per reaction. Our PCR assay consistently detected as few as 20 copies of the plasmid containing hK2 cDNA, with fewer copies yielding equivocal results (Fig 2).

View larger version (46K): [in this window] [in a new window]   Fig 1. Determination of the specificity of the primer for hK2 cDNA. No cross-reactivity with prostate-specific antigen (PSA) was seen. The amplified product from full-length human hK2 cDNA was 37 base pairs (bp) longer than the amplified product of the native hK2 from the prostate paraffin specimen.

View larger version (23K): [in this window] [in a new window]   Fig 2. Determination of detection limit of polymerase chain reaction (PCR) for hK2 cDNA. Reverse transcriptase PCR assay results on samples containing 100, 50, 20, 10, 5, 1, and zero copies of the plasmid hK2 cDNA. Abbreviation: bp, base pair.

View larger version (27K): [in this window] [in a new window]   Fig 3. Detection of hK2 RNA in patients with locally advanced and lymph node-negative (pT3N0) prostate cancer. Lanes marked 0 represent negative, lanes marked 1 represent equivocal, and lanes marked 2 represent positive reverse transcriptase polymerase chain reaction results.

View larger version (14K): [in this window] [in a new window]   Fig 4. Cumulative incidence functions of the probability of prostate-specific antigen progression for 199 patients with pathologic locally advanced prostate cancer and histopathologic normal pelvic lymph nodes (pT3N0) who underwent radical prostatectomy. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

Association of Lymph Node RT-PCR/hK2 Results With Development of Clinically Evident Distant Prostate Cancer Metastases
Thirty patients developed bone metastases as evidenced by imaging studies. The median follow-up period for patients who did not develop metastases was 101.8 months (range, 42.5 to 189.3 months). There were 53 patients with at least 10 years of metastasis-free follow-up. A positive RT-PCR/hK2 assay result was associated with the risk for developing distant metastases (P = .001; Fig 5), but not death from causes other than prostate cancer (P = .091). Prostatectomy Gleason sum (P = .008) and RT-PCR/hK2 results (overall, P < .001; negative versus equivocal, P = .087; HR, 0.451; 95% CI, 0.265 to 1.162; negative versus positive, P = .003; HR, 3.856; 95% CI, 1.594 to 9.331) were predictors of distant prostate cancer metastases in a multivariable postoperative model that adjusted for the effects of preoperative PSA (P = .115), seminal vesicle involvement (P = .277), level of prostatic capsular invasion (P = .405), and surgical margin status (P = .585).

View larger version (14K): [in this window] [in a new window]   Fig 5. Cumulative incidence functions of the probability of developing distant prostate cancer metastasis for 199 patients with pathologic locally advanced prostate cancer and histopathologic normal pelvic lymph nodes (pT3N0) who underwent radical prostatectomy. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

View larger version (14K): [in this window] [in a new window]   Fig 6. Cumulative incidence functions of the probability of prostate cancer-specific mortality for 199 patients with pathologic locally advanced prostate cancer and histopathologic normal pelvic lymph nodes (pT3N0) who underwent radical prostatectomy. Abbreviation: RT-PCR, reverse transcriptase polymerase chain reaction.

	DISCUSSION

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Although conventional histopathologic examination failed to reveal any evidence of lymph node metastases in these high-risk patients with pathologic locally advanced prostate cancer, RT-PCR for hK2 clearly detected disseminated prostate cells in the lymph nodes of at least 21% of the patients. Previous studies used RT-PCR targeting PSA or prostate-specific membrane antigen (PSMA) mRNA to detect prostate cancer cells in pelvic lymph nodes. Deguchi et al²⁰ reported that two of 18 patients (11%) with negative lymph nodes on conventional histology were found to be positive by RT-PCR for PSA mRNA. Takahashi et al³² demonstrated the presence of PSA mRNA in two of 12 (17%) negative lymph nodes by using RT-PCR on fine-needle aspirates of pelvic lymph nodes. Edelstein et al²² found that as many as 44% of histopathologically metastases-free, paraffin-embedded lymph nodes (16 of 36 cases) had detectable expression of PSA mRNA by RT-PCR. In a prospective study involving 33 high-risk patients, Ferrari et al²¹ found that as many as 44% of patients with histopathologically metastasis-free lymph nodes were positive by RT-PCR. More than half of the RT-PCR–positive patients (57%) were positive for both PSMA- and PSA-expressing cells. In a recent study, Potter et al¹⁸ assessed archival histopathologically normal paraffin-embedded lymph nodes removed from 102 men who had undergone radical prostatectomy; the specimens demonstrated prostate cancer involvement of the seminal vesicles. Careful histologic reevaluation by hematoxylin and eosin staining identified metastases in an additional 3% of patients. Immunohistochemical analyses for prostatic acid phosphatase, cytokeratin, and PSA (assessed in 35 patients) identified unsuspected micrometastases in an additional 6% of the patients. RT-PCR assays for PSA and PSMA was attempted but RNA was degraded in 95 of the 102 cases.

Despite these primary pathologic studies, the clinical correlation of RT-PCR–positive lymph nodes with disease potential and progression has been uncertain, placing the burden on investigators to establish a link between positive assay results and clinically and biologically meaningful end points. Illegitimate basal expression of the targeted marker mRNA in nonprostatic cells and downregulation of PSA mRNA in high-grade tumor cells^33–41 limit the utility of RT-PCR/PSA.^42–44 Although the expression of PSA has been found to be reduced in higher grade and presumably more biologically active disease,^45,46 the expression of hK2 has been found to increase gradually in conjunction with change from benign epithelium, to prostatic intraepithelial neoplasia, to prostate cancer.⁴⁶ hK2 expression was directly associated with the Gleason grade of the primary tumor, and foci of prostate cancer metastatic to the lymph nodes have been found to demonstrate the highest level of expression.⁴⁷ We have previously shown that preoperative peripheral blood RT-PCR/hK2 predicts metastases to pelvic lymph nodes²³ and overall to aggressive disease progression⁴⁸ in patients undergoing radical prostatectomy for clinically localized prostate cancer. These properties of hK2 indicate that it may represent a better RT-PCR target for the detection of biologically and clinically aggressive prostate cancer cells.

We found no association between RT-PCR/hK2 status and standard markers of biologically aggressive prostate cancer in a large consecutive cohort of patients with pathologic locally advanced prostate cancer and histopathologic cancer-free lymph nodes. However, this cohort of patients was restricted to those with a high likelihood of demonstrating aggressive parameters, possibly obscuring such an association that would be evident in a broader population of patients. Thirty-six percent of our patients had a preoperative PSA level greater than 10 ng/mL (data not shown), 25% had seminal vesicle invasion, 61% had established prostatic capsular invasion, and 69% had a final pathological Gleason sum of 7 and higher. Finally, although the association between RT-PCR/hK2 status and pathologic characteristics is important, an association with more significant end points such as biochemical progression, development of metastases, and, most importantly, survival in patients treated effectively for clinically localized disease would be more useful for managing patients with prostate cancer.^14,49

A positive RT-PCR/hK2 assay was an independent predictor of biochemical disease progression after surgery, of response to local salvage radiation therapy, and of development of clinically evident distant prostate cancer metastases. We found that although 53% of patients with positive RT-PCR/hK2 result experienced disease progression, as many as 26% of patients with negative assay result and 33% of patients with equivocal assay result also experienced disease progression. In addition, the actuarial 12-year progression-free probability of patients who had a positive RT-PCR/hK2 assay was calculated at 46%, indicating that despite the increased risk of progression, these patients had a relatively long disease-free interval. In addition, although 30% of patients with positive RT-PCR/hK2 result developed overt distant metastases at 7.5 years after surgery, 14% of patients with negative RT-PCR/hK2 result and 3% of patients with equivocal RT-PCR/hK2 result also developed overt distant metastases. In concordance with these findings, Edelstein et al²² reported that 88% of the 16 patients with a positive RT-PCR/PSA assay and no histopathologic evidence of lymph node involvement eventually experienced disease recurrence, whereas 30% of 20 patients with histopathologic and RT-PCR/PSA negative lymph nodes also had recurrent disease. These data suggest that other mechanisms of disease dissemination (via the peripheral blood and bone marrow) that bypass local lymph nodes most likely also play an important part in disease progression.^50–52

Most remarkably, we found that RT-PCR/hK2 assayed on lymph nodes was a predictor of prostate cancer-specific survival after prostatectomy. This is the first study to show an association of an RT-PCR–detected molecular target with disease-specific survival in patients with solid tumors without clinical evidence of metastases. Because of the long natural history of prostate cancer, cancer-specific mortality is rarely an evaluated end point in prostate cancer studies. Because of this long natural history, and the considerable potential for patients to die of a cause other than prostate cancer, competing risk analysis was employed. Cause-specific survival analyses depend on accurate assignment of the underlying cause of death. We relied on retrospective analysis of death certificates to assign the underlying cause of death determination. We scored as dead of prostate cancer only those patients who had prostate cancer listed in part I of their death certificate. Our study may be limited by the death certificate ascertainment of the cause of death.^53,54 Feuer et al,⁵⁵ for example, hypothesized that a shift in the likelihood of classifying prostate cancer as the underlying cause of death was the cause of an increase in prostate cancer mortality in the United States because of the rising pool of prevalent cases. However, others have found a high level of agreement between the information in hospital medical records and part I of death certificates when cause of death is viewed as a dichotomous variable.^56–58 The International Classification of Disease-9 coding rules information, which allows the use of information in part II of the death certificate to assign prostate cancer as the underlying cause of death when a review of the medical records would have suggested an alternative cause, has been shown to overestimate the frequency of death from prostate cancer compared with cause of death determination on the basis of information in part I of the death certificate alone.⁵⁶ In addition, because all patients were coded according to the death certificate data, any attribution bias toward prostate cancer-specific mortality should have been equally distributed among RT-PCR–positive, –equivocal, and –negative patients.

Our study clearly demonstrates a biologic and clinically significant association between a positive RT-PCR/hK2 assay result in histopathologic normal lymph nodes and prostate cancer progression after local therapy, development of overt distant prostate cancer metastases, and decreased survival. To our knowledge, this is the first association of a molecular assay for micrometastatic prostate cancer with profound clinical end points measured over a decade of follow-up. Prospective studies using a more objective assay (eg, real-time quantitative PCR format) are planned to confirm these results, to better predict clinical outcome after radical prostatectomy, and to establish recommendations for adjuvant and salvage therapies.

	ACKNOWLEDGMENTS

 
We thank Carolyn Schum for her excellent editorial help. We thank Veronica L. Shrode at the Methodist Hospital cancer registry for providing patient survival data.

	NOTES

 
Supported in part by grants from the National Cancer Institute Specialized Program of Research Excellence (SPORE CA58203) and from the Austrian Science Fund.

	REFERENCES

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1. Epstein JI, Partin AW, Potter SR, et al: Adenocarcinoma of the prostate invading the seminal vesicle: Prognostic stratification based on pathologic parameters. Urology 56:283–288, 2000[CrossRef][Medline]

2. Ohori M, Scardino PT, Lapin SL, et al: The mechanisms and prognostic significance of seminal vesicle involvement by prostate cancer. Am J Surg Pathol 17:1252–1261, 1993[CrossRef][Medline]

3. Catalona WJ, Smith DS: Cancer recurrence and survival rates after anatomic radical retropubic prostatectomy for prostate cancer: Intermediate-term results. J Urol 160:2428–2434, 1998[CrossRef][Medline]

4. Pound CR, Partin AW, Epstein JI, et al: Prostate-specific antigen after anatomic radical retropubic prostatectomy. Patterns of recurrence and cancer control. Urol Clin North Am 24:395–406, 1997[CrossRef][Medline]

5. Eastham J, Scardino P: Radical prostatectomy for clinical stage T1 and T2 prostate cancer, in Vogelzang NJ, Scardino PT, Shipley WU, et al (eds): Comprehensive Textbook of Genitourinary Oncology. Baltimore, MD, Williams and Wilkins, 2000, pp 722–738.

6. Walsh PC, Partin AW, Epstein JI: Cancer control and quality of life following anatomical radical retropubic prostatectomy: Results at 10 years. J Urol 152:1831–1836, 1994[Medline]

7. Gervasi LA, Mata J, Easley JD, et al: Prognostic significance of lymph nodal metastases in prostate cancer. J Urol 142:332–336, 1989[Medline]

8. Heicappell R, Muller-Mattheis V, Reinhardt M, et al: Staging of pelvic lymph nodes in neoplasms of the bladder and prostate by positron emission tomography with 2-[(18)F]-2-deoxy-D-glucose. Eur Urol 36:582–587, 1999[CrossRef][Medline]

9. Engelbrecht MR, Barentsz JO, Jager GJ, et al: Prostate cancer staging using imaging. BJU Int 86:123–134, 2000 (suppl 1)

10. Rorvik J, Haukaas S: Magnetic resonance imaging of the prostate. Curr Opin Urol 11:181–188, 2001[CrossRef][Medline]

11. Lange PH: ProstaScint scan for staging prostate cancer. Urology 57:402–406, 2001[CrossRef][Medline]

12. Partin AW, Kattan MW, Subong EN, et al: Combination of prostate-specific antigen, clinical stage, and Gleason score to predict pathological stage of localized prostate cancer: A multi-institutional update. J Am Med Assoc 277:1445–1451, 1997[Abstract/Free Full Text]

13. Kattan MW, Stapleton AM, Wheeler TM, et al: Evaluation of a nomogram used to predict the pathologic stage of clinically localized prostate carcinoma. Cancer 79:528–537, 1997[CrossRef][Medline]

14. Kattan MW, Eastham JA, Stapleton AM, et al: A preoperative nomogram for disease recurrence following radical prostatectomy for prostate cancer. J Natl Cancer Inst 90:766–771, 1998[Abstract/Free Full Text]

15. Kattan MW, Wheeler TM, Scardino PT: Postoperative nomogram for disease recurrence after radical prostatectomy for prostate cancer. J Clin Oncol 17:1499–1507, 1999[Abstract/Free Full Text]

16. Freeman JA, Esrig D, Grossfeld GD, et al: Incidence of occult lymph node metastases in pathological stage C (pT3N0) prostate cancer. J Urol 154:474–478, 1995[CrossRef][Medline]

17. Brands F, Hawes D, Stein J, et al: Occult lymph node metastases in pathological stage pT3N0 prostate cancer: Impact on survival. J Urol 163:182, 2000 (abstr A807)

18. Potter SR, Mangold LA, Shue MJ, et al: Molecular and immunohistochemical staging of men with seminal vesicle invasion and negative pelvic lymph nodes at radical prostatectomy. Cancer 89:2577–2586, 2000[CrossRef][Medline]

19. Pelkey TJ, Frierson HF Jr, Bruns DE: Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 42:1369–1381, 1996[Abstract/Free Full Text]

20. Deguchi T, Doi T, Ehara H, et al: Detection of micrometastatic prostate cancer cells in lymph nodes by reverse transcriptase-polymerase chain reaction. Cancer Res 53:5350–5354, 1993[Abstract/Free Full Text]

21. Ferrari AC, Stone NN, Eyler JN, et al: Prospective analysis of prostate-specific markers in pelvic lymph nodes of patients with high-risk prostate cancer. J Natl Cancer Inst 89:1498–1504, 1997[Abstract/Free Full Text]

22. Edelstein RA, Zietman AL, de las Morenas A, et al: Implications of prostate micrometastases in pelvic lymph nodes: An archival tissue study. Urology 47:370–375, 1996[CrossRef][Medline]

23. Slawin KM, Shariat SF, Nguyen C, et al: Detection of metastatic prostate cancer using a splice variant-specific reverse transcriptase-polymerase chain reaction assay for human glandular kallikrein. Cancer Res 60:7142–7148, 2000[Abstract/Free Full Text]

24. Riegman PH, Vlietstra RJ, van der Korput HA, et al: Identification and androgen-regulated expression of two major human glandular kallikrein-1 (hGK-1) mRNA species. Mol Cell Endocrinol 76:181–190, 1991[CrossRef][Medline]

25. Young CY, Andrews PE, Montgomery BT, et al: Tissue-specific and hormonal regulation of human prostate-specific glandular kallikrein. Biochemistry 31:818–824, 1992[CrossRef][Medline]

26. Bader P, Burkhard FC, Markwalder R, et al: Is a limited lymph node dissection an adequate staging procedure for prostate cancer? J Urol 168:514–518, 2002[CrossRef][Medline]

27. Wheeler TM, Lebovitz RM: Fresh tissue harvest for research from prostatectomy specimens. Prostate 25:274–279, 1994[Medline]

28. Greene DR, Wheeler TM, Egawa S, et al: A comparison of the morphological features of cancer arising in the transition zone and in the peripheral zone of the prostate. J Urol 146:1069–1076, 1991[Medline]

29. Wheeler TM, Dillioglugil O, Kattan MW, et al: Clinical and pathological significance of the level and extent of capsular invasion in clinical stage T1-2 prostate cancer. Hum Pathol 29:856–862, 1998[CrossRef][Medline]

30. Schmid HP, McNeal JE, Stamey TA: Observations on the doubling time of prostate cancer. The use of serial prostate-specific antigen in patients with untreated disease as a measure of increasing cancer volume. Cancer 71:2031–2040, 1993[CrossRef][Medline]

31. Gray R: A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat 16:1141–1154, 1988[CrossRef]

32. Takahashi T, Hoshi S, Kaneda T, et al: Genetic diagnosis of pelvic lymph node metastasis in prostate cancer using aspiration biopsy samples. Rinsho Byori 44:1183–1188, 1996[Medline]

33. Dumas F, Gala JL, Berteau P, et al: Molecular expression of PSMA mRNA and protein in primary renal tumors. Int J Cancer 80:799–803, 1999[CrossRef][Medline]

34. Israeli RS, Powell CT, Corr JG, et al: Expression of the prostate-specific membrane antigen. Cancer Res 54:1807–1811, 1994[Abstract/Free Full Text]

35. Uria JA, Velasco G, Santamaria I, et al: Prostate-specific membrane antigen in breast carcinoma. Lancet 349:1601, 1997 (letter)[CrossRef][Medline]

36. Renneberg H, Friedetzky A, Konrad L, et al: Prostate specific membrane antigen (PSM) is expressed in various human tissues: Implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread. Urol Res 27:23–27, 1999[CrossRef][Medline]

37. Chang SS, Reuter VE, Heston WD, et al: Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res 59:3192–3198, 1999[Abstract/Free Full Text]

38. Zarghami N, Levesque M, D’Costa M, et al: Frequency of expression of prostate-specific antigen mRNA in lung tumors. Am J Clin Pathol 108:184–190, 1997[Medline]

39. Levesque M, Hu H, D’Costa M, et al: Prostate-specific antigen expression by various tumors. J Clin Lab Anal 9:123–128, 1995[Medline]

40. Lehrer S, Terk M, Piccoli SP, et al: Reverse transcriptase-polymerase chain reaction for prostate-specific antigen may be a prognostic indicator in breast cancer. Br J Cancer 74:871–873, 1996[Medline]

41. Clements J, Mukhtar A: Glandular kallikreins and prostate-specific antigen are expressed in the human endometrium. J Clin Endocrinol Metab 78:1536–1539, 1994[Abstract]

42. Zippelius A, Kufer P, Honold G, et al: Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 15:2701–2708, 1997[Abstract/Free Full Text]

43. Smith MR, Biggar S, Hussain M: Prostate-specific antigen messenger RNA is expressed in non-prostate cells: Implications for detection of micrometastases. Cancer Res 55:2640–2644, 1995[Abstract/Free Full Text]

44. Henke W, Jung M, Jung K, et al: Detection of PSA mRNA in blood by RT-PCR does not exclusively indicate prostatic tumor cells. Clin Chem 42:1499–1500, 1996 (letter)[Free Full Text]

45. Aihara M, Lebovitz RM, Wheeler TM, et al: Prostate specific antigen and Gleason grade: An immunohistochemical study of prostate cancer. J Urol 151:1558–1564, 1994[Medline]

46. Darson MF, Pacelli A, Roche P, et al: Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: A novel prostate cancer marker. Urology 49:857–862, 1997[CrossRef][Medline]

47. Darson MF, Pacelli A, Roche P, et al: Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases. Urology 53:939–944, 1999[CrossRef][Medline]

48. Shariat S, Gottenger E, Nguyen C, et al: Preoperative blood reverse transcriptase-PCR assays for prostate-specific antigen and human glandular kallikrein for prediction of prostate cancer progression after radical prostatectomy. Cancer Res 62:5974–5979, 2002[Abstract/Free Full Text]

49. Pound CR, Partin AW, Eisenberger MA, et al: Natural history of progression after PSA elevation following radical prostatectomy. J Am Med Assoc 281:1591–1597, 1999[Abstract/Free Full Text]

50. Ellis WJ, Vessella RL, Corey E, et al: The value of a reverse transcriptase polymerase chain reaction assay in preoperative staging and followup of patients with prostate cancer. J Urol 159:1134–1138, 1998[CrossRef][Medline]

51. Melchior SW, Corey E, Ellis WJ, et al: Early tumor cell dissemination in patients with clinically localized carcinoma of the prostate. Clin Cancer Res 3:249–256, 1997[Abstract]

52. Lange PH, Vessella RL: Mechanisms, hypotheses and questions regarding prostate cancer micrometastases to bone. Cancer Metastasis Rev 17:331–336, 1998[CrossRef][Medline]

53. Carter JR: The problematic death certificate. N Engl J Med 313:1285–1286, 1985[Medline]

54. Kircher T, Anderson RE: Cause of death: Proper completion of the death certificate. J Am Med Assoc 258:349–352, 1987[Abstract/Free Full Text]

55. Feuer EJ, Merrill RM, Hankey BF: Cancer surveillance series: Interpreting trends in prostate cancer: Part II. Cause of death misclassification and the recent rise and fall in prostate cancer mortality. J Natl Cancer Inst 91:1025–1032, 1999[Abstract/Free Full Text]

56. Albertsen PC, Walters S, Hanley JA: A comparison of cause of death determination in men previously diagnosed with prostate cancer who died in 1985 or 1995. J Urol 163:519–523, 2000[CrossRef][Medline]

57. Roberts RO, Bergstralh EJ, Katusic SK, et al: Decline in prostate cancer mortality from 1980 to 1997, and an update on incidence trends in Olmsted County, Minnesota. J Urol 161:529–533, 1999[CrossRef][Medline]

58. Penson DF, Albertsen PC, Nelson PS, et al: Determining cause of death in prostate cancer: Are death certificates valid? J Natl Cancer Inst 93:1822–1823, 2001[Free Full Text]

Submitted August 22, 2002; accepted December 18, 2002.

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