Biochemical staging of prostate cancer

Urol Clin N Am 30 (2003) 263–277

Biochemical staging of prostate cancer Eduardo I. Canto, MDa, Shahrokh F. Shariat, MDa, Kevin M. Slawin, MDb,* a

Scott Department of Urology, 6535 Fannin Street, Fondren 401, Baylor College of Medicine, Houston, TX 77030, USA b The Baylor Prostate Center, Scott Department of Urology, 6560 Fannin, Suite 2100, Baylor College of Medicine, Houston, TX, USA

The widespread use of prostate-specific antigen (PSA) in the screening for prostate cancer has dramatically changed the epidemiology of this disease. In conjunction with the virtual disappearance of metastatic prostate cancer as a de novo presentation, there has been a marked stage migration toward earlier detection [1]. For example, the median PSA of patients diagnosed with clinically localized disease undergoing radical prostatectomy at The Methodist Hospital in Houston decreased from greater than 10 ng/mL prior to 1990, to 5.5 ng/mL over the last year. Seventy-five percent of these patients had a PSA of less than or equal to 7.6 ng/mL. These epidemiologic changes have improved the overall surgical cure rate of clinically localized prostate cancer to over 85% when patients are followed for more than 5 years [2]. Despite this marked stage migration, a small but significant proportion of patients continue to present with microscopic metastases already present at the time of diagnosis and are, therefore, likely to fail local therapy such as prostatectomy or radiation therapy. Although serum PSA concentration is an excellent tool when applied to patient cohorts with a wide range of PSA values, it is a poor marker for the prediction of prostate cancer stage and prognosis when applied to patients with PSA concentrations below 10 ng/ mL [3]. Because serum PSA is likely to remain one of the primary modes of diagnosis and staging of

* Corresponding author. E-mail address: [email protected] (K.M. Slawin).

prostate cancer in the near term, unrecognized microscopic metastases, in the setting of clinically localized prostate cancer, will continue to be a source of frustration for both urologists and their patients. New therapies being developed for metastatic prostate cancer are likely to work best against low-volume disease; therefore, the early identification of patients with microscopic metastases will become an even more important task in the near future. Of equal importance, and accentuated by the now widespread use of PSA-based screening, is the inability of PSA to distinguish men with significant prostate cancer from those with insignificant prostate cancer when staging patients with PSA levels below 10 ng/mL [3]. Traditionally, urologists have used the digital rectal examination (DRE), transrectal ultrasonography, biopsy Gleason score, and the serum PSA level to stage prostate cancer. For those patients with high PSA levels and high Gleason score, imaging with bone scan and CT may provide additional staging information with regard to disseminated disease [4]. Recently, by analyzing large cohorts of patients, nomograms and tables that use traditional parameters to predict pathologic stage and response to therapy have been developed. Nevertheless, even the best nomograms and staging tables that take all of the above variables into account have concordance indices between 0.73 and 0.84 [5,6]. Clearly, there is a need for additional markers that are capable of predicting pathologic stage, the presence of microscopic metastases, and the aggressiveness of a given patient’s cancer—independently or in conjunction with commonly used parameters. The availability of new validated markers would

0094-0143/03/$ - see front matter Ó 2003, Elsevier Science (USA). All rights reserved. doi:10.1016/S0094-0143(02)00183-0

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allow for the creation of more accurate nomograms that could distinguish, on an individual basis, between insignificant prostate cancer, significant but curable localized disease, and metastatic disease. This, in turn, would enhance the ability to provide optimal care for each prostate cancer patient. This article reviews various established and new biochemical markers for prostate cancer staging and prediction of response to therapy. Biopsy gleason grade In the early 1970s, clinical staging of prostate cancer was based only on DRE; the presence of cancer on biopsy; serum prostatic acid phosphatase (PAP) measurement; and available imaging modalities, including plain radiograph, radioisotopic bone scan, and lymphangiography. PAP measurement as a marker for staging suffers from a low sensitivity. Nevertheless, because of its high specificity in the absence of other causes for PAP elevation, and the high incidence of metastatic disease in the 1970s, it provided useful staging information [7]. Of the imaging modalities, only bone scan continues to have a widespread role, demonstrating a high sensitivity and acceptable specificity for the detection of metastatic disease [4]. In contrast, pelvic lymphangiography was abandoned because of low sensitivity and a high false-positive rate [8,9]. Skeletal radiography (bone survey films) suffers from low sensitivity and rarely proved to be informative compared with PAP [4]. More recently, data demonstrating the clinical utility of MRI and positron emission tomography have slowly led to more widespread use of these newer staging modalities [4]. The grading system introduced by Donald Gleason, MD, PhD, in 1974, therefore, was a significant improvement that, remarkably, has withstood the test of time and the marked changes in prostate cancer epidemiology. To this day, it remains one of the best predictors of prostate cancer stage and prognosis. Unlike traditional nuclear grading systems, the Gleason grade is based solely on the glandular architecture, ignoring the cytological characteristics of individual cells. For patients with more than one glandular pattern, survival was noted to fall somewhere between that dictated by the predominant and the secondary pattern [10]. Thus, the Gleason score (the sum of the two predominant patterns) was developed and continues to be used for the staging of prostate cancer.

A review of various representative prostatectomy series from the 1970s [9,11] revealed that as many as 40% of patients who were clinically staged as having localized cancer were found to have nodal disease at the time of prostatectomy. Because the only screening tool at the time was the DRE, essentially all men with a Gleason score of 9 or 10 presented with metastatic disease. In the larger of the prostatectomy series reported in the 1970s [9], patients with a Gleason score of 9 or 10 represented only 10% of those with patient population, but comprised 50% of those with positive lymphadenectomies [9]. Use of the Gleason score as a staging tool, therefore, had the potential to reduce unnecessary surgery by half. In these early series, it also was noted that patients with Gleason scores of 2 to 4 rarely, if ever, were found to have lymph node metastasis, even when they presented with palpable disease [9]. Currently, the most widely accepted measure of prostate cancer aggressiveness is the Gleason grade. The Gleason score on prostate biopsy is highly predictive of pathologic stage. Even in recent prostatectomy series [2,12–14], multivariate analyses consistently showed that prostate biopsy Gleason score is an independent predictor of both final pathologic stage and disease progression. Prostate cancer is organ confined in 77% of Gleason score 2 to 4 patients who undergo prostatectomy, whereas only 13% of Gleason score 8 to 10 patients have organ-confined disease [15]. The concordance index for the prediction of organconfined disease of Gleason score alone is comparable with that of PSA alone—approximately 0.6 in modern series [16]. Biopsy Gleason score has a high negative predictive value in the low range (Gleason score of 2 to 4) and a high positive predictive value in the high range (Gleason score of 8 to 9) for the presence of metastasis or extracapsular extension (ECE). Nevertheless, for moderately differentiated tumors (Gleason score of 6 to 7)—which comprise the majority of cases— Gleason score alone is a relatively poor marker for the prediction of pathologic stage or the presence of metastasis [15]. Prostatic acid phosphatase The first biochemical marker to be routinely used in the staging of prostate cancer was PAP. PAP hydrolyzes esters under acidic conditions (pH of 5) to yield inorganic phosphate. The greatest concentration of PAP is found in the prostate, although it has been detected in liver,

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brain, lung, testicle, heart, muscle, spleen, skin, and hematopoietic cells. PAP expression in the prostate is limited to epithelial cells, which secrete the enzyme into the glandular lumen. Inside these cells, PAP is normally found in lysosomes and secreting vesicles. PAP can be measured by either immunoassay or by enzymatic assay. Enzymatic assays were the first to be developed, and, until recently, were popular staging tools [7,17]. In 1938, PAP was shown to be present in high concentrations in the prostate and in bony metastasis from patients with prostate cancer [18]. Concentrations of PAP in prostate cancer tissue are only 7% to 20% that of benign prostatic hyperplasia (BPH), however [7]. Paradoxically, elevations in serum PAP correlate with advanced stages of prostate cancer. PAP elevations measured by enzymatic assay or immunoassay were shown in a number of studies to be specific for ECE or metastatic disease. Depending on how the normal range was defined, the probability of having surgically curable disease in patients with elevated PAP was shown in various studies to be as low as 5% [17]. In a study published in 1987 [19] that involved 102 patients, 84% of patients with elevated PAP levels (greater than one half of the normal range by Roy enzymatic assay, or >0.35 IU/L) had either ECE or metastatic disease. Although specific for the presence of metastatic disease, the routine use of PAP for staging has fallen out of favor with the introduction of serum PSA testing. Most patients with elevated PAP have PSA measurements of greater than 20 ng/mL [20]. Because of the shift toward earlier diagnosis of prostate cancer as a result of routine PSA-based prostate cancer screening, however, PAP measurements now are rarely informative. In a report from Johns Hopkins of 460 consecutive men with prostate cancer referred between 1990 and 1991 [20], PAP was elevated in only 4.6% and provided unique staging information that was not available from PSA measurement in only 0.9%. Furthermore, the use of PSA in prostate cancer staging is more practical than is the routine use of PAP. Not only is there no need for special handling of blood samples to preserve enzymatic activity as is the case with PAP, but PSA measurements are used for screening and are, therefore, already available at the time of diagnosis. PSA PSA is a 33-kDa serine-protease of the kallikrein family. Werle [21], who found high

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levels of these enzymes in the pancreas, introduced the term ‘‘kallikrein’’ in the 1930s. ‘‘Kallikreas’’ is the Greek word for pancreas. Most authors divide the kallikrein enzymes into two major categories: plasma kallikreins and tissue kallikreins. There are 15 genes in the kallikrein family. They share similarities at the DNA and protein level, and their genes are found in the same chromosome 19 locus as the human kallikrein 1 gene [22]. Human kallikrein 3, or PSA, is a tissue kallikrein initially identified in prostatic extracts in 1970 [23]. Its cDNA was cloned in 1987 [24]. It is made primarily by the prostatic epithelium of the periurethral glands, but it also has been detected in endometrium, breast tissue, breast cancer, breast milk, and female serum [25]. In the normal prostate, the prostatic epithelium secretes PSA into the seminal fluid, where it reaches mg/mL concentrations. Its physiologic function is to degrade seminal plasma motility inhibitor precursor/semenogelin I, the predominant protein in human ejaculate [26]. In the normal male, PSA enters the circulation through an unknown mechanism and reaches ng/mL concentrations. PSA is organ specific but is not cancer specific. Normal, hyperplastic, and neoplastic prostate epithelial cells all produce PSA, with the highest levels found in the prostatic transition zone of patients with BPH [27]. Testosterone levels and the natural variation in the epithelium-to-stroma ratio of the prostate influence PSA production per gram of prostate tissue [28]. The epithelium-to-stroma ratio has been shown to vary by as much as threefold between individuals [29]. Because PSA is produced exclusively by the epithelial component of the prostate, the amount of serum PSA per gram of prostate tissue varies significantly from one individual to another. PSA expression also has been shown to vary with Gleason grade. Based on immunohistochemical studies, PSA expression decreases with increasing Gleason grade [30]. This finding was confirmed in a large study [31] that corrected for prostate cancer volume of the pathologic specimen after prostatectomy and showed that the serum PSA level per gram of tumor decreased with increasing Gleason score. Despite the above limitations, serum PSA concentration has been shown to be proportional to the volume, Gleason score, and stage of prostate cancer [27]. This paradox may be explained by the increased release of PSA into the serum that occurs with the increased disorganization of epithelium associated with higher-grade cancers,

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leading to a greater entry of PSA into the circulation despite the lower PSA production per gram of tissue. Alternatively, the paradox could be explained by a larger tumor volume at the time of presentation in patients with highergrade cancers [31]. In a multi-institutional study involving more than 4000 patients [13], there was a linear relationship between PSA levels and tumor stage. Only 9% of patients with a PSA level of greater than 50 ng/mL had organ-confined disease. On the other hand, 64% of patients with a PSA level of less than 4 ng/mL had organconfined disease. Similarly, less than 1% of patients in the latter group had positive lymph nodes, whereas 27% of patients in the former group had positive lymph nodes [13]. As this and many other studies [3,27,31] confirm, PSA is an excellent predictor of pathologic stage when very high or low values are considered, yet greater than 50% of patients have a PSA below 10 ng/mL—a range for which PSA by itself is uninformative. PSA has a concordance index of 0.55 to 0.66 when used alone to discriminate preoperatively between pathologic T2 and T3 disease [16,30]. This means that for every randomly selected patient pair—one with T2 and the other with T3 disease—only 55% to 66% of the time will the pathologic stage of each patient be ranked correctly by consideration of the PSA level alone. Furthermore, when analyzed on an individual basis, PSA is able to predict ECE, seminal vesicle invasion, or lymph node involvement only 50% to 55% of the time [3,33]. The recent realization that the utility of PSA as a staging tool for prostate cancer diminishes as the average PSA of patients diagnosed with prostate cancer decreases has heightened the urgency to identify alternative biochemical markers that are capable of predicting both pathologic stage and risk of disease progression. PSA-based nomograms In 1987, Oesterling et al [12] demonstrated that logistic regression analysis of multiple preoperative variables could be used to predict final pathologic stage in patients undergoing radical prostatectomy. Since then, other investigators have developed similar algorithms using different variables to predict pathologic stage and lymph node involvement [34]. In 1993, Partin et al [13] introduced a nomogram in the form of an easy-touse table that predicts pathologic stage based on variables available to all urologists. The ‘‘Partin

Tables’’ predict the probability of organ-confined disease, ECE, seminal vesicle involvement, and pelvic lymph node status based on Gleason score, clinical stage, and serum PSA [13]. This nomogram was independently validated in 2000 using a large cohort of patients that underwent prostatectomy at the Mayo Clinic [5], and was found to have a concordance index of 0.84 for the prediction of node-positive disease and 0.76 for the prediction of organ-confined disease [5]. In 2001, the tables were updated to reflect the shift toward lower grade, stage, and PSA at the time of presentation [35]. The Partin Tables and other nomograms that predict pathologic stage at the time of prostatectomy facilitate the decision-making process regarding therapy for prostate cancer. The majority of patients with pT3a and even pT3b remain curable with local therapy alone, however, limiting the clinical value of nomograms that simply predict prostate cancer stage. Nomograms based on the same readily available parameters recently have been developed that predict PSA recurrencefree survival after prostatectomy, which is possibly a more meaningful endpoint. The Baylor College of Medicine group [6,34] developed a preoperative nomogram that allows patients who are considering prostatectomy as definitive therapy to make more informed decisions based on the prediction of 5-year biochemical recurrence, rather than on the prediction of pathologic stage. This preoperative nomogram has been shown to predict PSA recurrence with a concordance index of 0.79. Because of the improved ability to predict pathologic stage and disease progression using the various currently available nomograms, new prostate cancer markers must be independent predictors of either disease progression or prostate cancer stage in order to be useful. In other words, they must add unique predictive information not otherwise derivable from nomograms based on serum PSA, prostate biopsy Gleason score, and clinical stage. Molecular forms of PSA Approximately three quarters of the PSA found in serum is irreversibly bound to the protease inhibitor a1-antichymotrypsin (PSAACT). A lesser fraction of serum PSA is bound to either a2-macroglobulin (PSA-AMG) or a1protease inhibitor (PSA-API). Five percent to fifty percent of measured serum PSA is unbound, free

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PSA (fPSA) [36]. PSA-AMG may retain part of its enzymatic activity, whereas fPSA and PSA-ACT are enzymatically inactive [37]. PSA has five epitopes. Two of these are hidden when it is bound to PSA-ACT, allowing for commercial immunoassays to differentiate between PSA-ACT and fPSA. All five epitopes are hidden in PSA-AMG [34].

Percent-free PSA It has been well documented that in patients with a PSA level between 4 and 10 ng/mL, a higher fPSA fraction lowers the probability of finding prostate cancer on biopsy and raises the probability that the elevation in total PSA is due to the presence of BPH [38]. Although not completely understood, it is thought that the loss of tissue architecture that results from disorganized cancer growth leads to the release of enzymatically active forms of PSA directly into the bloodstream. This, in turn, is thought to facilitate the binding of protease inhibitors such as PSA-ACT and PSAAMG to PSA. PSA that is produced by BPH, on the other hand, is secreted into the seminal spaces from where it must leak back through the intercellular space to reach the circulation. This exposes BPH-produced PSA to proteases. Cleaved forms of PSA are not able to bind to protease inhibitors such as PSA-ACT or PSA-AMG [39]. Data on the utility of fPSA as a fraction of total PSA (%fPSA) for the staging of prostate cancer are inconclusive. Three large studies [16,40, 41] have analyzed the potential role of %fPSA in the staging of prostate cancer. The first study [40]—a large, multicenter study involving 268 men with palpably benign glands and total PSA between 4 and 10 ng/mL who underwent radical prostatectomy—found that %fPSA was a stronger predictor of postoperative pathologic outcome than was Gleason score. A value of 15% fPSA was found to discriminate between favorable and unfavorable pathologic outcome. Seventyfive percent of patients with greater than 15% fPSA had organ-confined cancer, Gleason score less than 7, and small tumors. These favorable pathologic characteristics were found in only 34% of men with a fPSA level of 15% or less [40]. Nevertheless, two later studies [16,41] have failed to validate these findings. In those studies, %fPSA had a predictive value in univariate analyses that was similar to that of total PSA and Gleason score. In multivariate analyses using PSA, Gleason score, and clinical stage, however, %fPSA failed to provide additional staging or prognostic

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data [16,41]. Although these divergent results remain unexplained, one possible explanation is that the staging utility of %fPSA—like its utility in discriminating between benign and malignant disease—is highly dependent on multiple parameters of the study population including age, race, and distribution of total PSA levels. Bound PSA Greater than half of the PSA in the serum is bound to either PSA-ACT, API, or PSA-AMG [36]. PSA-ACT is the predominant form of complex PSA in patients with prostate cancer [42], and can be measured by an assay that is available from Roche Diagnostics (Mannheim, Germany). The Bayer Diagnostics complexed PSA (cPSA) assay (Tarrytown, NY) measures PSA-ACT and PSAAPI [43]. Both markers have been studied in the setting of prostate cancer screening and the results are similar to those of %fPSA when either of the following ratios are used: cPSA/total PSA or PSAACT/total PSA [44,45]. Methods for analyzing PSA-API and PSAAMG have been reported [46,47]. The PSA-AMG/ (PSA+ PSA-AMG) ratio is higher in patients with BPH (12%) than in patients with prostate cancer (8%) [47]. The PSA-API/PSA ratio is also higher in patients with BPH (1.6%) as compared with patients with prostate cancer (0.9%) [46]. Data on the use of these bound PSA assays in the staging of prostate cancer remain scant, however. Molecular forms of free PSA fPSA is a mixture of different molecular forms of PSA. Recent studies [48] have found that the PSA produced by the transitional zone epithelium of prostates with nodular BPH has a high percentage of PSA molecules that have been clipped at amino acid residues Lys145–146 and Lys182–183 (see Fig. 1). This enzymatically inactive form of PSA has been named BPSA. A dual monoclonal antibody assay for BPSA (detection limit of 0.06 ng/mL) has been tested in men with symptomatic BPH and without clinical BPH and in healthy subjects. The median BPSA values in the patients with symptomatic BPH were significantly higher than those in the patients without BPH symptoms. In the healthy male control group, BPSA was almost undetectable. BPSA performed better than did fPSA in this setting [49]. Although it has yet to be studied in the staging of prostate cancer, ratios of BPSA to fPSA or BPSA to total PSA could prove to be useful prostate cancer staging tools.

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patients with very high concentrations of total PSA have detected -1, -5 and -7pPSA in addition to -2 and -4pPSA. The relevance of -1, -5 and -7pPSA for patients with total PSA in the more clinically applicable range of 4 to 20 ng/mL is unknown. Furthermore, studies have yet to be performed to test the ability of any of the fPSA molecular forms to aid in the staging of prostate cancer. Nevertheless, measurement of the various molecular forms of fPSA and their respective ratios hold great promise as biochemical markers for prostate cancer staging. Human kallikrein-2

Fig. 1. High-performance hydrophobic interaction chromatography profile of immunoaffinity-purified PSA isolated from prostatic tissues. Three matched tissue specimens were analyzed: transition zone tissue (TZ), peripheral zone cancer (PZ-C, containing 80% to 100% tumor), and peripheral zone benign tissue (PZ-N). BPSA elutes at 8 minutes. Other forms of PSA elute at 10 minutes. (From Mikolajczyk SD, Millar LS, Wang TJ, et al. ‘‘BPSA,’’ a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia. Urology 2000;55(1):43; with permission.)

Prostate cancer has been found to produce more than one molecular form of fPSA. Mikolajczyk et al [50] found that prostate cancer produces PSA with either two (-2pPSA) or four (-4pPSA) unclipped amino acids from its leader sequence. These molecular forms of PSA are, for the most part, not present in transitional zone epithelium, where BPH occurs [50]. In a study of eight patients with total PSA levels between 6 and 24 ng/mL (five patients with biopsy-proven cancer and three biopsy-negative men) [51], the fraction of -2pPSA in prostate cancer patients ranged from 25% to 95%, whereas that of the biopsy-negative patients was between 6% and 19%. In this study [51], -2pPSA also was shown to be a stable (not cleaved by either hK2 or trypsin), enzymatically inactive form of fPSA. Recent studies [52] analyzing the fPSA content of prostate cancer

Human kallikrein-2 (hK2) shows a 78% amino acid sequence identity to PSA but differs in its enzymatic specificity. Like PSA, hK2 is expressed in various tissues, but the highest level of expression is found in the prostate [22]. hK2 manifests trypsinlike substrate specificity. It can activate the zymogen form of urokinase and can generate enzymatically active PSA from fulllength PSA [53]. In seminal plasma, hK2 can cleave the gel-forming proteins semenogelin I, semenogelin II, and fibronectin [54]. hK2 protein levels in both seminal plasma and serum are less than 3% that of PSA, although at the mRNA level, hK2 expression is only approximately half that of PSA [55]. Like PSA, hK2 forms complexes with various plasma protease inhibitors such as ACT, a2-antiplasmin, antithrombin III, plasminogen activator inhibitor 1, AMG, and protease inhibitor 6 (PI-6) [56,57]. Unlike PSA, however, most of the hK2 in serum is found in the free, unbound form. hK2 bound to ACT represents only 4% to 19% of the total hK2 [58]. hK2 bound to PI6 is associated with prostate tumor tissue and may be formed as the result of tumor necrosis [57]. The ratio of hK2 to fPSA has been shown to enhance prostate cancer detection in patients with serum PSA concentrations of 2 to 4 ng/mL and 4 to 10 ng/mL [59]. Three published studies [32,60,61] compared hK2 to PSA in the preoperative staging of clinically localized prostate cancer. The best data were reported in the most recent of these studies, which was a multi-institutional study [32]. One hundred and sixty-one serum samples (48 patients with stage pT3a cancer, and 113 with stage
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pT3a cancer) that were collected prior to surgery were used to compare preoperative hK2, PSA, and fPSA serum levels. Calculation of concordance indices revealed that the area under the curve (AUC) for hK2 (AUC=0.64) and the algorithm of [hK2]  [total PSA/fPSA] (AUC=0.68) were both larger than that of total PSA (AUC= 0.55). Only the difference between the AUC for PSA and the AUC for the algorithm [hK2]  [total PSA/fPSA] achieved statistical significance (P= 0.007), however. There was no statistical difference in mean PSA and fPSA levels between patients with stage pT2a/b cancer and patients with greater than or equal to stage pT3a cancer. On the other hand, the mean of the hK2 levels and the mean of the results of the algorithm [hK2]  [total PSA/fPSA] both achieved statistically significant differences when men with greater than or equal to stage pT3a cancer were compared with men with stage pT2a/b cancer (P=0.004 and P= 0.0004, respectively). When the analysis was limited to men with total PSA between 10 and 20 ng/mL, only the difference in the mean of the algorithm [hK2]  [total PSA/fPSA] remained statistically significant when men with greater than or equal to stage pT3a disease were compared with men with stage pT2a/b disease [32].

Insulin-like growth factor binding protein 2 and 3 The local expression of insulin-like growth factor 1 (IGF-1), IGF binding proteins (IGFBPs), and IGF receptors has been associated with tumor prognosis, progression, pathologic stage, and tumor grade in patients with colon, breast, lung, and prostate cancers [62]. Six IGFBPs have been identified. These proteins modulate the levels of free IGF-1 and IGF-2, and also can mediate cell growth and induction of apoptosis independently of IGFs. IGFBP-2 is the main IGFBP produced by prostate epithelial cells. IGFBP-3 is the primary carrier for IGF in the blood [63,64]. Although epidemiologic studies have found a positive association between IGF-1 and the risk of developing various cancers including lung, breast, endometrial, colorectal, and prostate cancer, others have failed to find an association between the presence of prostate cancer and IGF1 levels [62,65–67]. We have shown that systemic levels of IGF-1 are not associated with metastasis, established markers of biologically aggressive disease, or disease progression in patients with clinically localized prostate cancer [67].

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Immunohistochemical studies [68] have found that IGFBP-2 immunoreactivity is higher and that of IGFBP-3 is lower in prostate cancer tissue than in benign prostate tissue. Preoperative and postoperative measurement of plasma IGF-1, IGFBP2, and IGFBP-3 revealed that IGF-1 was not associated with disease stage or progression. IGFBP-2 and IGFBP-3 levels, on the other hand, proved to be independent predictors of disease progression in multivariate models that included PSA, biopsy Gleason score, and clinical stage. IGFBP-3 levels were lowest in patients with bony metastasis, followed by patients with lymph node metastasis and patients with nonmetastatic prostate cancer. Healthy subjects had the highest plasma IGFBP-3 levels [69]. The relationship between plasma IGFBP-2 and prostate cancer is more complex. IGFBP-2 plasma concentration was higher in patients with prostate cancer as compared with healthy subjects, but among patients with clinically localized prostate cancer, IGFBP-2 was inversely associated with tumor volume and with features of advanced disease (eg, higher final Gleason score, ECE, and seminal vesicle involvement). A higher IGFBP-2 level was a significant predictor of organ-confined disease. A lower preoperative IGFBP-2 was significantly associated with an increased probability of ECE and disease progression when adjusted for preoperative PSA, biopsy Gleason score, and clinical stage in 120 patients undergoing radical prostatectomy [69].

Transforming growth factor b1 Transforming growth factor b1 (TGF-b1) is one of more than 30 structurally related polypeptides including activins, bone morphogenic proteins, and other TGF-bs [70]. Signaling by TGF-b1 is mediated by ligand-induced heteromeric complex formation of distinct type I and type II receptors. The type I receptor is phosphorylated by the constitutively active type II receptor upon dimerization. The now-activated type I receptor propagates the signal within the cell by the phosphorylation of mothers against decapentaplegic-related protein (Smad) [71]. The TGF-b1 family of cytokines regulates cellular proliferation, chemotaxis, differentiation, extracellular matrix production, immune response, and angiogenesis; however, TGF-b1 can play opposing roles in tumor development. Although in the initial phases of tumorigenesis it may act as

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a tumor suppressor, during later phases, it may function as a tumor promoter [72]. Elevated TGF-b1 in the blood has been reported in patients with various tumor types [73]. TGF-b1 has been shown to promote cell motility and metastasis in experimental prostate cancer models [74,75]. Immunohistochemical studies have shown increased expression of TGF-b1 in neoplastic prostatic epithelium when compared with normal prostate [76]. Although the relationship between TGF-b1 and prostate cancer invasion and metastasis remains controversial, a number of studies have documented a positive correlation between prostate cancer stage and TGF-b1 [77,78]. For example, by measuring the plasma levels of TGF-b1 in 10 patients with bone scan-proven metastasis, 19 men with regional lymphatic metastasis, and 44

healthy men without prostate cancer, we showed that TGF-b1 levels were dramatically elevated in patients with metastatic disease (see Fig. 2) [14]. The differences between studies on TGF-b1 could be explained, in part, by the differences in sample collection. TGF-b1 levels in serum are three to six times higher than in plasma. TGFb1 is stored in platelet granules and is released upon platelet activation [14]. Measurement of TGF-b1 in serum is, therefore, affected by the release from damaged platelets and may not correlate with the TGF-b1 level in the patient’s blood prior to specimen collection and handling. Two studies [79,80] have failed to find a correlation between TGF-b1 and prostate cancer stage. In one of these studies [80], serum rather than plasma was used. In the second study [79], although there was no correlation between

Fig. 2. Box plot of the TGF-b1 levels in radical prostatectomy patients stratified by progression status at 48 months (OC, organ confined; ECE, extracapsular extension; SVI, seminal vesicle involvement; LN Mets, lymph node metastases), healthy men, patients with prostate cancer metastatic to lymph nodes (LN Mets), or patients with prostate cancer metastatic to bones (Bone Mets). (From Shariat SF, Shalev M, Menesses-Diaz A, et al. Preoperative plasma levels of transforming growth factor b1 strongly predict progression in patients undergoing radical prostatectomy. J Clin Oncol 2001;19(11):2861; with permission.)

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plasma TGF-b1 and prostate cancer stage, there was, nevertheless, a direct correlation between stage and urinary levels of TGF-b1. In a large study that evaluated the relationship between TGF-b1 and prostate cancer stage and prognosis [14], we found a significant correlation between preoperative TGF-b1 and established markers of biologically aggressive prostate cancer. By evaluating plasma TGF-b1 levels in 120 consecutive prostatectomy patients at Baylor, we showed that TGF-b1 levels correlated with preoperative PSA, ECE, seminal vesicle involvement, and lymph node involvement. TGF-b1 was an independent predictor of final pathologic stage and disease progression. For each pathologic stage, patients who progressed had higher preoperative TGF-b1 levels than did patients who remained disease free. Patients with aggressive recurrences, which were presumed to be due to micrometastatic disease, had higher TGF-b1 levels than did those with less aggressive recurrences, which were presumed to be local recurrences. Interestingly, TGF-b1 levels did not correlate with surgical margin status. Indeed, in a postoperative multivariate model, both TGF-b1 and surgical margin status were independent predictors of disease progression [14]. In a separate study evaluating preoperative and 6-weeks postoperative levels of TGF-b1 (S.F. Shariat, M.W. Kattan, B. Andrews, et al, unpublished data), plasma levels of TGF-b1 decreased the most in patients who did not progress. In patients who did not experience cancer progression, the decrease in TGF-b1 levels was significantly greater than that of all patients combined (33% versus 18%). In contrast, in patients who did experience disease progression, postoperative TGF-b1 levels fell only minimally (9%) and were not significantly different from preoperative TGF-b1 levels (S.F. Shariat, M.W. Kattan, B. Andrews, et al, unpublished data). Although immunohistochemical studies have shown that both normal and neoplastic prostate tissue produce TGF-b1, these clinical data support the hypothesis that elevated plasma levels of TGF-b1 in patients with metastatic prostate cancer are the result of direct production by foci of metastatic tumor or by normal cells in response to metastasis, and not necessarily the result of production by the primary tumor. Taken together, these data suggest that preoperative and postoperative plasma TGF-b1 measurements could be used to improve the accuracy of preoperative and postoperative nomograms

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designed to predict pathologic stage and disease progression. Interleukin 6 and interleukin 6 soluble receptor IL-6 is a pleiotropic cytokine that acts on a variety of cell types. It has important regulatory functions in the immune system, is a mediator of the acute-phase response, and is involved in the regulation of differentiation, proliferation, and survival of target cells. IL-6 has been shown to inhibit tumor immunity and induce natural killer cell dysfunction, allowing tumor cells to escape immune surveillance [81]. IL-6 also regulates bone turnover by promoting osteoclastic activity and mediates tumor-associated cachexia [82,83]. IL-6 exerts its biological activities through interaction with the IL-6 receptor complex. The IL-6 receptor complex is composed of IL-6R-a and gp130. To exert its biological activity, IL-6 first binds to the low-affinity IL-6R-a. The IL-6/ IL-6R-a complex recruits the signal-transducing gp130 subunit. Soluble forms of both gp130 and IL-6R-a (IL-6sR) have been described. IL-6sR is generated by alternative RNA splicing or limited proteolytic cleavage of its membrane-bound form. Interestingly, when bound to IL-6sR, IL-6 is capable of eliciting a biological response in cells that express only gp130. This type of activation renders virtually all cells capable of responding to IL-6 in the presence of IL-6sR [84]. Immunohistochemical and biochemical studies [85] have shown that prostate cancer tissue expresses IL-6 and IL-6 receptor, allowing for an autocrine/paracrine loop to be established. Furthermore, IL-6 protein concentrations are approximately 18 times higher in clinically localized prostate cancer than in normal prostate tissue. The concentration of IL-6R-a is also higher in prostate cancer than in normal prostate tissue [85]. Elevated circulating levels of IL-6 have been associated with advanced prostate cancer, and these have been shown to correlate with both decreased survival and extent of disease on bone scans [86]. We hypothesized that men with clinically localized prostate cancer harboring occult metastases also would have elevated levels of plasma IL-6 and IL6sR. Therefore, to determine the relationship among plasma IL-6, IL-6sR, and prostate cancer stage, we measured the preoperative plasma levels of these two proteins in a cohort of 120 consecutive patients with clinically localized prostate cancer who underwent prostatectomy.

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We confirmed that plasma IL-6 levels were dramatically elevated in patients with metastatic prostate cancer. We also found that IL-6sR levels were highest in patients with bone metastasis, followed by patients with metastasis to regional lymph nodes. Patients with nonmetastatic prostate cancer and healthy subjects had the lowest plasma levels of IL-6sR. Preoperative plasma IL-6 and IL-6sR levels correlated with PSA, Gleason score, and tumor volume, but did not predict stage [87]. IL-6 and IL-6sR levels did predict PSA progression-free survival, even after adjusting for the effects of PSA, Gleason score, and clinical stage. When both cytokines were considered in the same model, however, only IL-6sR remained an independent predictor of disease progression. In patients whose disease did progress, preoperative plasma IL-6 and IL-6sR levels were significantly higher in those with characteristics of aggressive failure (eg, PSA doubling time <10 months, failure to respond to salvage radiation therapy, and metastases detectable by imaging studies) than in those with characteristics of nonaggressive PSA failures, suggestive of a correlation between these two proteins and metastatic rather than locally recurrent prostate cancer [87]. The stronger predictive value of preoperative IL-6sR over that of IL-6 for prostate cancer progression supports the role of IL-6sR as an agonistic regulator of IL-6 functions, and suggests an underlying biological mechanism for its superiority to IL-6 for prognostic purposes in patients with prostate cancer. To investigate the relationship between early postoperative plasma levels of IL-6 and IL-6sR and disease progression, and to gain insight into the origin of these proteins in prostate cancer patients, we studied preoperative and postoperative levels of IL-6 and IL-6sR in a cohort of 302 consecutive patients who underwent radical prostatectomy for clinically localized disease (S.F. Shariat, M.W. Kattan, B. Andrews, et al, unpublished data). We found that IL-6 and IL-6sR levels decreased postoperatively, independent of whether cure was achieved. Circulating levels of IL-6 have been reported to significantly decrease after surgery for colon cancer, regardless of whether cure was surgically achieved [88]. Together with the fact that the plasma levels of these two markers correlated with tumor volume in our studies, this suggests that the plasma levels of IL-6 and IL-6sR are a consequence of cytokine production by cells within the prostate, most likely

the tumor cells (S.F. Shariat, M.W. Kattan, B. Andrews, et al, unpublished data). Furthermore, circulating levels of IL-6 and its soluble receptor appear to be associated with the potential of prostate cancer to metastasize, rather than with the metastases themselves.

Polymerase chain reaction The polymerase chain reaction (PCR) is a clever method of amplifying DNA using sequence-specific primers and heat-stable bacterial DNA polymerase. When messenger ribonucleic acid (mRNA) rather than DNA is the starting material, an additional step is performed to reverse transcribe the mRNA into complementary DNA prior to starting the reaction (RT-PCR). Taking advantage of the ability of RT-PCR to amplify minute amounts of RNA, various investigators have tested its ability to predict clinical stage and recurrence after prostatectomy based on amplification of tissue-specific mRNA. RT-PCR for PSA, prostate-specific membrane antigen, and hK2 has been tested for this purpose. In addition, to improve the specificity of the assay, some investigators have used ‘‘nested PCR.’’ This technique uses two sets of PCR primers with the second set of primers (the nested primers) designed to bind within the segment of DNA amplified by the first set of primers. The logic behind the technique is that if the incorrect gene is amplified, the second set of primers will not work, thereby allowing the researcher to exclude falsepositive tests that result from a nonspecific amplification. RT-PCR is extremely sensitive. It can detect one LNCaP cancer cell in 107 to 109 lymphocytes. Nevertheless, depending on how it is performed, there can be a large variation in the assay’s sensitivity and specificity. Furthermore, in newly diagnosed patients, there is shedding of prostate cancer cells into the bloodstream despite the absence of true metastatic or micrometastatic disease, as shown by the disappearance of these cells after a curative prostatectomy [89]. More than a dozen studies have tested the association between preoperative PSA, RT-PCR, and final pathologic stage, but only two groups [90,91] have found a statistically significant correlation. The results are similar for prediction of recurrence [90]. In our comparison of RT-PCR for PSA and RT-PCR for a native hK2 transcript mRNA (hK2-L) (S.F. Shariat, E. Gottenger, C. Nguyen,

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et al, unpublished data), we found that only RTPCR for hK2-L correlated with both a higher Gleason score and the presence of metastasis. Nevertheless, hK2-L RT-PCR was not an independent predictor of recurrence after prostatectomy in our dataset when adjusting for preoperative PSA, biopsy Gleason score, and clinical stage. RT-PCR of bone marrow in patients with prostate cancer has been found to correlate with disease recurrence after prostatectomy in two studies [92,93]. Others have found no correlation between PSA, RT-PCR from bone marrow, and pathologic stage, however. In these studies, there was a high proportion of patients with a positive bone marrow RT-PCR for PSA, suggesting that circulating prostate cancer cells may be nonspecifically sequestered in the bone marrow. In the postoperative setting, once the prostate is removed and there is no ‘‘false’’ shedding of cells from the prostate, RT-PCR can be a highly sensitive and specific marker for metastatic disease. It can be performed using mRNA obtained from lymph nodes, bone marrow, or peripheral blood (S.F. Shariat, M.W. Kattan, S. Erdamar, et al, unpublished data; S.F. Shariat, M.W. Kattan, W. Song, et al, unpublished data). A positive PSA RTPCR 6 weeks after prostatectomy was highly predictive of eventual disease recurrence, even in patients with an undetectable PSA by the ultrasensitive, Immulite PSA assay (Diagnostics Products Corporation, Los Angeles, CA). In this study (S.F. Shariat, M.W. Kattan, W. Song, et al, unpublished data), only postoperative positive PSA RT-PCR was associated with pathologic stage, including ECE and seminal vesicle involvement. A positive postoperative RT-PCR for PSA was an independent predictor of recurrence. Furthermore, postoperative RT-PCR for PSA was associated with aggressive failures that were likely due to the presence of metastatic rather than locally recurrent disease (S.F. Shariat, M.W. Kattan, W. Song, et al, unpublished data). In our series (S.F. Shariat, M.W. Kattan, S. Erdamar, et al, unpublished data), RT-PCR of lymph node tissue for hK2 was an independent predictor of biochemical recurrence after surgery, response to salvage radiation therapy, and development of clinically evident distant prostate cancer metastasis. Nevertheless, 26% of patients with a negative hK2 RT-PCR experienced disease progression, suggesting that there are means of disseminating prostate cancer other than the pelvic lymph nodes assayed (S.F. Shariat, M.W. Kattan, S. Erdamar, et al, unpublished data).

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Summary PSA continues to be one of the most effective and widely used cancer screening tools available. Its popularity in prostate cancer screening, however, has eroded its usefulness in the staging of this disease. As more men are screened every year on a routine basis with DRE and PSA, the average PSA at diagnosis has drifted down to well below 10 ng/mL in many centers, including ours. This trend is likely to accelerate, as a PSA cut off for prompting biopsy of the prostate of 2.5 ng/mL gains more widespread acceptance. The recent realization that, at these levels, serum PSA is more reflective of the presence of BPH than of the extent of cancer and, therefore, does not provide additional staging information, has renewed the search for new biochemical markers that are capable of predicting prostate cancer stage and prognosis. Because of the heterogeneity of this disease, it is unlikely that a single biochemical marker that is capable of accurately staging all prostate cancer patients will be found. For this reason, nomograms that are capable of integrating various parameters to predict stage and prognosis will remain indispensable. As new biochemical markers that provide independent predictive information about stage or prognosis are identified, they can be incorporated into currently available nomograms. Of the biochemical markers discussed in this article, IL-6sR and TGF-b1 are the most promising. By incorporating them into a preoperative nomogram designed to predict PSA recurrence, we found that they improved the ability to predict biochemical recurrence by a statistically and clinically significant margin [94]. The ability to stage prostate cancer and predict response to therapy has improved dramatically over the last 3 decades. Nevertheless, there is still a need for new biochemical markers that will improve the ability to predict an individual patient’s stage and response to therapy. Incorporating these new markers into nomograms will enhance the ability to provide optimal care for each prostate cancer patient.

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