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Molecular Markers and Prostate Cancer Prognosis
Comprehensive Review
Molecular Markers and Prostate Cancer Prognosis Jonathan L. Chin Robert E. Reiter David Geffen School of Medicine University of California Los Angeles
Abstract Prostate cancer is the most common malignancy among American men and is the second-leading cause of cancer-related mortality. Although radical prostatectomy and radiation therapy offer hope for cure for the majority of men with localized tumors, we continue to lack the tools to definitively determine which cancers need to be treated, which cancers will recur after treatment, and which cancers will behave aggressively when they have metastasized. Recent breakthroughs in molecular biology have led to the identification of a number of potential biomarkers for prostate cancer, many of which have been suggested to have prognostic significance. Eventually, combinations of these markers will hopefully enable us to more rationally facilitate counseling and direct management for men with prostate cancer.
Introduction
Clinical Prostate Cancer, Vol. 3, No. 3, 157-164, 2004 Key words: Biomarker, Fluorescence in situ hybridization, Immunohistochemistry, Proteasome, Proteomics Submitted: Aug 18, 2004; Revised: Nov 9, 2004 Accepted: Nov 10, 2004 Address for correspondence: Robert E. Reiter, MD David Geffen School of Medicine University of California Los Angeles 650 Charles East Young Dr Room 66-134 CHS Los Angeles, CA 90095 Fax: 310-206-5343 e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Cancer Information Group, ISSN #1540-0352, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
Adenocarcinoma of the prostate ranks as the most common malignancy among American men and is second only to lung cancer in terms of cancer-related mortality in men, with approximately 200,000 new cases and almost 30,000 deaths per year.1 Since the widespread adoption of prostatespecific antigen (PSA) screening for prostate cancer, we have witnessed impressive stage migration with more and more cancers being detected at a potentially curable juncture. Still, a plethora of dilemmas persists regarding the management of prostate cancer. Which cancers need to be treated? Which cancers will progress regardless of treatment? Which cancers can be cured? Is one treatment modality more appropriate than another for a particular patient? As a case in point, Thompson et al recently noted that as many as 15% of asymptomatic men > 62 years of age with PSA levels < 4.0 ng/mL (the upper limit of normal) harbor prostate cancer, with a significant percentage of these having a Gleason score of ≥ 7.2 However, we currently have no means of assessing whether these tumors are clinically significant. Clearly, we cannot rely on PSA alone to dictate management. Moreover, the natural history of a given tumor must be balanced against life expectancy, and given the significant morbidities of the various treatments for prostate cancer, quality of life issues must be taken into serious consideration as well. Although parameters such as Gleason score, stage, and PSA can offer rough estimates as to eventual outcome, additional prognostic markers are urgently needed in order to accurately risk-stratify patients and dictate therapy. Prognostic markers for prostate cancer can arise from an assortment of techniques and sources (Table 1).3-67 Tissue-based markers can be assessed by immunohistochemistry (IHC). Fluorescence in situ hybridization (FISH) can analyze chromosomal aberrations arising in malignant tissue. Various serum markers can be detected at the protein level or at the RNA level. The field of proteomics explores the protein “signature” of biologic samples such as serum, tissue, or urine and can delineate characteristic differences between malignant and benign specimens. Although nothing to
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Table 1 Potential Biomarkers in Prostate Cancer3-67 Marker
Source
Technique
Comments
Ki-67
Tissue
IHC
Proliferative antigen3-6
Bcl-2
Tissue
IHC
Apoptosis inhibitor3,7-12
Tissue, serum
IHC, ELISA
Proliferation, chemotaxis, angiogenesis, and cellular differentiation13-16
E-cadherin
Tissue
IHC
Adhesion molecule10,17-23
CD44
Tissue
IHC
Adhesion molecule24,25
HER2/neu
Tissue
IHC, FISH
Growth factor receptor26-33
p53
Tissue
IHC
Tumor suppressor9,11,34-40
PTEN
Tissue
IHC
Tumor suppressor41-45
p27
Tissue
IHC
Tumor suppressor46-49
skp2
Tissue
IHC
Positive cell cycle regulator50
MYC
Tissue
IHC, FISH
Protooncogene51-54
PSCA
Tissue
IHC
Coamplified with MYC55-57
EZH2
Tissue
RT-PCR
Transcriptional repressor58,59
Caveolin-1
Tissue, serum
IHC, sandwich immunoassay
Molecular transport, cell adhesion, and signal transduction60-62
Proteomics
Tissue, serum
Mass spectrometry
Protein signature63-67
TGF-β1
Abbreviations: ELISA = enzyme-linked immunosorbent assay; RT-PCR = reverse-transcriptase polymerase chain reaction
date has definitively improved upon our usual risk-stratifying parameters for prostate cancer, much work has been done on a variety of molecular and genetic biomarkers. The prognostic significance of several biomarkers that may be important in the management of prostate cancer is reviewed in this article.
Proliferative Antigens Ki-67 Ki-67 is a nuclear antigen present throughout the cell cycle, but not in noncycling or quiescent cells, and its expression is associated with cellular proliferation. Multiple studies have demonstrated that elevated expression of Ki-67 is associated with an adverse prognosis in prostate cancer. Biopsy specimens from 106 patients with stage T1-T3 prostate cancer treated with external-beam radiation were stained for Ki-67.3 With a median follow-up of 62 months, elevated Ki-67 staining was negatively correlated with progression-free survival (PFS). In a separate study involving 104 patients treated by radical prostatectomy, the surgical specimens were stained for Ki-67 in the area of highest tumor grade.4 In a univariate analysis, Ki-67 expression was significantly associated with time to biochemical failure, as were age, grade, stage, margin status, tumor size, and preoperative PSA. In a multivariate analysis, only stage, PSA, and Ki-67 expression remained as significant independent predictors of time to biochemical recurrence. A similar study of 180 patients treated with radical prostatectomy again demonstrated a correlation between Ki-67 expression and diseasefree survival (DFS).5 Finally, Ki-67 expression has also been shown to predict disease progression after initial hormonal therapy in patients with advanced prostate cancer.6 Thus, Ki-67 might eventually serve as a significant biomarker in patients managed with a variety of treatment modalities.
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Apoptosis-Related Proteins Bcl-2 Bcl-2 is an integral membrane protein that plays a role in the inhibition of apoptosis. Overexpression of Bcl-2 has been found to play a role in a number of human malignancies.7 The evidence for the role of Bcl-2 as a biomarker in prostate cancer has been mixed. Expression of Bcl-2 in prostate needle biopsy specimens predicted biochemical failure in those treated with radiation.3,8,9 Similarly, multiple studies have confirmed that in patients with localized prostate cancer treated with radical prostatectomy, Bcl-2 expression in the prostatectomy specimen correlated with biochemical recurrence.10-11 However, in contrast to the radiation therapy (RT) studies, staining in the preoperative biopsy specimen did not correlate with outcome.11 Finally, in a cohort of 221 elderly patients (median age, 75 years) with prostate cancer who were followed expectantly, Bcl-2 expression was significantly associated with disease-specific and overall survival (OS).12 However, in a multivariate analysis, this correlation did not maintain its significance.
Growth Factors Transforming Growth Factor–β1 Transforming growth factor–β1 (TGF-β1) plays a role in the control of cellular proliferation, chemotaxis, angiogenesis, and cellular differentiation. Loss of the inhibitory effects of TGF-β1 has been associated with the progression of cancer, and elevated local and circulating levels have correlated with prostate cancer invasion and metastasis.13 In a cohort of 120 consecutive patients who underwent radical prostatectomy for clinically localized prostate cancer, preoperative TGF-β1 levels were measured and correlated with clinical outcome.14 For comparison,
Jonathan L. Chin, Robert E. Reiter TGF-β1 serum levels were also measured in 44 healthy men, 19 men with lymph node metastases, and 10 men with bone metastases. TGF-β1 levels in patients with metastatic lesions (lymph node or bone) were significantly higher than those in the radical prostatectomy group or healthy subjects. Moreover, preoperative TGF-β1 levels strongly predicted organ-confined disease and PSA progression, suggesting that this growth factor might be useful in identifying patients with occult metastatic disease and therefore those who are at higher risk of recurrence after radical prostatectomy. In a related finding, Wikstrom et al determined that increased TGF-β1 staining was associated with tumor grade and metastasis.15 Other groups have investigated a relationship between the loss of TGF-β receptors and clinical outcome. In a study of 52 patients treated with radical prostatectomy, loss of TGF-β1 R1 was significantly associated with Gleason score, tumor stage, 4-year survival rate, and biochemical recurrence.16 Thus, expression of the TGF-β1 receptor by IHC may be of use as a prognostic marker, as well as expression of the growth factor itself.
Adhesion Molecules E-cadherin E-cadherin, located on chromosome 16q22.1, is cell membrane protein that is involved in intercellular adhesion. It has been theorized that E-cadherin serves as suppressor of invasion. Thus, decreased or absent expression of this protein may place a patient at risk for locally invasive cancer and/or metastasis. Indeed, E-cadherin expression, as assessed by IHC, is decreased or absent in prostate cancer.17 Li et al demonstrated that hypermethylation of the E-cadherin promoter correlates with reduced or absent E-cadherin protein staining in prostate cancer specimens, and the severity of hypermethylation is associated with tumor grade.18 Demethylation restored E-cadherin messenger RNA (mRNA) transcription in prostate cancer lines. E-cadherin protein levels are inversely correlated with Gleason grade, disease stage, presence of metastasis, and survival.19 Decreased expression of E-cadherin in radical prostatectomy specimens portends an elevated risk of PSA recurrence in patients with T2 tumors.10 Finally, abnormal E-cadherin expression in prostatectomy specimens was strongly associated with the detection of prostate cancer cells in the peripheral blood.20 One particular E-cadherin polymorphism (–160 C/A) has been associated with an increased risk of prostate cancer.21 Subjects with ≥ 1 A allele had a relative risk of 3.6 of having prostate cancer compared with the CC carriers. In a study of > 1000 Swedish subjects, this E-cadherin polymorphism was found to be associated with hereditary prostate cancer but not sporadic prostate cancer.22 Conversely, in a case-control study of Japanese subjects, this E-cadherin polymorphism did not correlate with an increased risk of prostate cancer.23
CD44 CD44, a type 1 transmembrane glycoprotein involved in cell–cell and cell–matrix interactions, is another cell adhesion
protein whose prognostic role in prostate cancer has been studied. Patients with lymph node metastases are much more likely to have decreased staining for CD44.24 In addition, loss of CD44 in radical prostatectomy specimens is an independent predictor of biochemical recurrence.25
Oncogenes HER2/neu HER2/neu, located on chromosome 17p, is a transmembrane tyrosine kinase growth factor receptor with homology to the epidermal growth factor receptor (EGFR). Amplification and subsequent overexpression of HER2/neu is an adverse prognostic indicator in breast cancer.26 In prostate cancer as well, HER2/neu may play a role in advanced disease. Signoretti et al examined a panel of prostate specimens obtained from patients treated by surgery alone, by maximal androgen blockade (MAB) followed by surgery, or by MAB alone who subsequently had progression to metastatic, androgen-independent (AI) disease.27 As determined by IHC, only 25% of specimens from the surgery-alone group stained positive for HER2/neu, whereas 59% from the MAB/surgery group and 78% from the AI group were positive. Moreover, patients treated with MAB demonstrated amplification of the HER2/neu gene by FISH, whereas patients treated by surgery alone did not manifest gene amplification. These data suggest that HER2/neu expression increases with progression to AI disease. In contrast, in a study of 88 radical prostatectomy specimens (presumably androgen-dependent), HER2/neu amplification was found in only 8 of 86 tumors (9.3%), and even in these cases, amplification was only at a low to moderate level.28 Not only does it appear that HER2/neu is overexpressed in advanced prostate cancer, but several studies have also suggested that HER2/neu carries a negative prognostic significance in prostate cancer in general. In a study of 112 patients undergoing curative RT for localized prostate cancer, HER2/neu expression in the needle biopsy specimen was inversely correlated with both PFS and disease-specific survival.29 Similarly, in 70 Spanish patients diagnosed with metastatic prostate cancer and treated with MAB, HER2 overexpression on IHC decreased mean cancer-specific survival from 54 months to 33 months.30 In contrast, in a study of 113 patients treated by radical prostatectomy, HER2/neu gene amplification correlated with biochemical recurrence in a univariate analysis but did not reach significance (P = 0.125) in a multivariate analysis with grade and DNA ploidy.31 HER2/neu protein expression by IHC was not significantly associated with biochemical recurrence. As noted, HER2/neu has been established as a negative prognostic indicator in breast cancer. Moreover, targeting HER2 with the humanized monoclonal antibody trastuzumab has benefited patients with metastatic breast tumors that overexpressed HER2/neu.32 The addition of trastuzumab to chemotherapy was associated with a longer time to disease progression, a higher rate of objective response, a longer duration of response, a lower rate of death at 1 year, longer survival, and a 20% reduction in the risk of death. The efficacy of trastuzumab in breast carcinomas that overexpress HER2/neu raised the hope that it would offer similar advantages in prostate tumors that similarly overexpress
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Figure 1 Positive Surgical Margins: Impact of p27 and Stage on Survival
Disease-Free Survival (%)
100 p27+, No SVI
75
50 p27–, No SVI or p27+, SVI
25 p27–, SVI
0
12
P < 0.001
24
36 Months
48
60
72
Patients with positive surgical margins and low or absent p27 immunostaining of their radical prostatectomy specimens have a significantly worse outcome than those with normal p27 levels. Low p27 appears to have the same dire prognostic significance as SVI (Reiter et al, unpublished data). Abbreviation: SVI = seminal vesicle invasion
HER2/neu. Morris et al conducted a preliminary study evaluating the efficacy of trastuzumab in 23 patients with progressive metastatic prostate cancer.33 After preliminary screening for HER2/neu, all patients were administered weekly trastuzumab until they experienced disease progression, after which paclitaxel was added. All of the patients had disease progression within 12 weeks regardless of HER2/neu status. However, this study was hampered by the fact that, of the 130 patients screened for HER2/neu, only 6 eligible patients had HER2/neu–positive disease. Significantly, in several cases, the HER2/neu status of paired primary prostate specimens and metastatic tissue did not match, with the former staining negative and the latter staining positive. Because obtaining metastatic tissue is neither facile nor routine, the authors emphasized that improved methods of screening for HER2/neu is crucial in order to properly evaluate the efficacy of targeting HER2/neu in prostate cancer treatment.
Tumor Suppressors
chemical recurrence, whereas p53 levels in the preoperative biopsy specimen was not.11 Positive p53 staining heralded PSA failure in patients with localized tumors treated with radiation.9,37 Finally, in a study of patients with locally advanced prostate cancer treated with a combination of RT and MAB versus RT alone, abnormal p53 protein expression by IHC of pretreatment prostate specimens (needle biopsy or transurethral resection) was significantly associated with an increased incidence of distant metastasis and shortened PFS and OS.38 Interestingly, time to metastasis was unrelated to p53 status in patients treated with RT alone. Unfortunately, the data for p53 have been far from uniformly positive. Multiple studies have failed to demonstrate a relationship between p53 and outcome, including disease-specific survival in advanced prostate cancer treated with hormone ablation,39 PSA recurrence following radical prostatectomy,10 and PSA recurrence in radical prostatectomy patients subsequently treated with neoadjuvant androgen ablation and salvage RT.40
PTEN PTEN is a phospholipid phosphatase that acts as a negative regulator in the phosphatidylinositol-3,4,5-triphosphate (PIP3)/Akt signaling pathway. The end result of this pathway is to inhibit apoptosis and promote proliferation.41 PTEN dephosphorylates and thereby inactivates the second messenger PIP3. Thus, inactivating mutations of PTEN can lead to persistent levels of PIP3 and constitutive activation of PIP3 kinase (PI3K) with subsequent uncontrolled cellular proliferation. Loss of PTEN expression has been correlated with elevated Gleason score and advanced stage in 109 primary prostate tumors,42 and has been shown to be an independent predictor of biochemical failure in men treated by radical prostatectomy.43 One of the downstream kinases activated in the PIP3/Aktpathway is mTOR (mammalian target of rapamycin). PTEN loss or inactivation leads to overactive mTOR and unchecked cellular proliferation.44,45 Blocking mTOR should, in essence, take over the inhibitory role of the defective or deficient PTEN. Thus, in another example of rational and targeted therapy, the mTOR inhibitor rapamycin is currently being studied in phase II clinical trials as a neoadjuvant therapy in patients whose needle biopsies stain negative for PTEN by IHC.
p53
p27
The p53 tumor suppressor plays a role in cell cycle arrest and apoptosis, and loss of function mutations in p53 is present in a wide range of human malignancies. A number of studies have suggested that p53 expression might have prognostic value in patients with prostate cancer. In a study of 221 patients with prostate cancer treated with watchful waiting (median follow-up, 15 years), the degree of p53 nuclear accumulation in the biopsy specimen was significantly associated with disease-specific survival in both the overall study population as well as in the subgroup of 125 patients with clinically localized disease.34 Similarly, nuclear accumulation of p53 in radical prostatectomy specimens correlated with increased risk of PSA recurrence and reduced disease-specific survival in multiple studies.11,35,36 Of note, in one study, expression of p53 in the radical prostatectomy specimen was significantly associated with bio-
Protein p27 (Kip1) is a cyclin-dependent kinase inhibitor that plays a role in cell cycle arrest and apoptosis. Protein p27 expression in needle biopsies is inversely correlated with tumor stage and grade.46 Moreover, in a multivariate analysis of a cohort of 52 Spanish patients who had been treated with radical prostatectomy, a low level of IHC staining for p27 in the surgical specimen was the lone independent predictor of biochemical recurrence.47 At 3 years, 59% of the patients with low p27 had experienced a PSA recurrence, whereas only 18% of the patients with normal p27 expression had a relapse. Similarly, in a separate study of 86 patients also treated with radical prostatectomy, Yang et al demonstrated that p27 was a strong independent predictor of DFS, second only to pathologic stage in a multivariate analysis that included age, preoperative PSA, Gleason score, and
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Jonathan L. Chin, Robert E. Reiter stage.48 Even more strikingly, multivariate analysis demonstrated that p27 was the strongest predictor of biochemical recurrence among factors studied in patients with pathologic T2a-T3b disease. Thus, absent or low levels of p27 protein expression appear to be an adverse prognostic factor in patients with clinically organ-confined disease treated by radical prostatectomy. Another interesting finding is that patients who have positive surgical margins have a relative risk of 3.4 of having recurrence if they have low expression of p27 (P < 0.04; Reiter et al, unpublished data). Moreover, in patients with positive margins, low or absent p27 staining carries as dire a prognostic significance as seminal vesicle invasion (Figure 1). Thus, p27 status might help determine whether a patient with a positive margin would benefit from adjuvant radiation. Importantly, p27 status at the time of the initial prostate biopsy also seems to predict biochemical recurrence. In a study of 161 men with subsequent surgical management, p27 immunostaining in < 45% of the cells resulted in significant preoperative risk stratification for time to PSA failure (hazard ratio, 2.41; P = 0.010).49 This relationship maintained its significance in a multivariate analysis. Therefore, p27 may be an important prognostic factor at the time of diagnosis.
skp2 The F-box protein skp2 is a positive regulator of G1-S transition and promotes ubiquitin-mediated proteolysis of the cyclindependent kinase inhibitor p27. Thus, upregulation of skp2 should result in decreased p27 and subsequent loss of cell cycle control. In an immunohistochemical analysis, skp2 levels were dramatically increased in prostatic intraepithelial neoplasia (PIN) and radical prostatectomy specimens.50 In addition, skp2 levels were positively correlated with preoperative PSA, Gleason score, and stage. Finally, a higher skp2 labeling index negatively impacted biochemical recurrence–free survival after radical prostatectomy.
Proteasome Targeting in Prostate Cancer The proteasome is a multifunctional protease present in all eukaryotic cells that is primarily responsible for intracellular protein destruction.67 Proteins that have been selected for degradation are tagged with chains of ubiquitin monomers, which are subsequently recognized by the proteasome complex. The proteasome plays a critical role in the regulation of many cellular processes, including activation and deactivation of transcription factors, cell cycle progression, and apoptosis. Many tumor suppressors have been shown to be regulated by the ubiquitin-proteasome pathway, including p5367 and p27.68 Bortezomib (Velcade®, PS-341) is a selective proteasome inhibitor that is currently being evaluated in clinical trials for the treatment of a variety of human malignancies, including prostate cancer. Williams et al demonstrated that bortezomib can strongly inhibit proliferation and induce apoptosis of prostate cancer xenograft cell lines in vitro and suppress tumor growth in vivo.69 Similarly, Ikezoe et al showed that bortezomib triggered growth arrest and apoptosis of androgen-dependent human prostate cancer LNCaP cells, possibly by interrupting the androgen receptor sig-
naling pathway.70 A recent phase I clinical trial investigating bortezomib in advanced prostate cancer exhibited a modest PSA response in patients with androgen-independent disease.71 Thus, preliminary data suggest that proteasome inhibition may be an enticing novel therapeutic strategy. Indeed, for prostate cancers deficient in tumor suppressors regulated by the ubiquitin-proteasome pathway, inhibition of the proteasome might restore the tumor suppressor to its normal function and thereby impede tumor growth.
Other Biomarkers Chromosome 8q, MYC, and PSCA Gain of chromosome 8q is one of the most frequent changes in prostate cancer. The 8q gain was seen in 89% of hormone refractory prostate specimens,72 85% of lymph node metastases,73 and 50% of bone metastases,74 but in only 6% of primary, localized tumors,72 suggesting that this is a late event in prostate carcinogenesis. The MYC gene, mapped to chromosome 8q24, is an intriguing candidate for this locus. MYC is amplified in a significant number of advanced prostate cancers.51 Overexpression of MYC in transgenic mice leads to the formation of lesions similar to PIN followed by overt adenocarcinoma.52 In a study of tumor samples from 144 patients with high grade prostate cancer, Sato et al demonstrated that MYC amplification assessed by FISH predicts systemic progression and prostate cancer–specific death in patients with highgrade advanced tumors.53 Conversely, FISH analysis of 195 organ-confined radical prostatectomy specimens demonstrated a correlation between MYC amplification and Gleason grade, but not between MYC amplification and biochemical recurrence.54 PSCA is a cell surface antigen overexpressed in prostate cancer that maps to chromosome 8q24.2, just distal to MYC.55 PSCA has been shown to be coamplified with MYC in locally advanced prostate tumors,56 and PSCA expression has been shown to increase with Gleason grade, tumor stage, and metastasis.57 Monoclonal antibodies against PSCA inhibit prostate cancer tumor growth and metastasis in animal models, offering hope that PSCA might be an excellent therapeutic target as well as a prognostic indicator in aggressive, MYC-amplified tumors.
EZH2 EZH2 (polycomb group protein enhancer of zeste homolog 2) is a transcriptional repressor that, using complementary DNA microarray analysis, has been shown to be overexpressed at the mRNA level in hormone refractory metastatic prostate cancer relative to localized prostate cancer.58 In a separate study, the same group assessed a panel of 14 candidate prognostic biomarkers identified with use of the tissue microarrays in a cohort of 259 patients who had undergone radical prostatectomy for localized prostate cancer. They determined that patients whose tumors exhibited moderate or strong immunostaining for EZH2 coupled with at most moderate expression of E-cadherin had a relative risk of approximately 3.0 for biochemical recurrence, even after adjusting for stage, grade, and preoperative PSA.59 Thus, EZH2:ECAD status might prove useful in identifying a high-risk cohort of patients following radical prostatectomy.
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Molecular Prognostic Markers in Prostate Cancer Caveolin-1 Caveolin-1 is a major structural protein of caveolae, specialized plasma membrane invaginations that are abundant in smooth muscle cells, adipocytes, and endothelium. Caveolae appear to mediate molecular transport, cell adhesion, and signal transduction activities in a cell- and context-specific fashion. Positive caveolin-1 immunostaining correlates positively with Gleason score, positive surgical margins, and lymph node metastasis, and is an independent predictor for time to disease progression in patients treated by radical prostatectomy.60 In a similar study involving a cohort of Japanese patients with T2 disease, caveolin1 staining again predicted biochemical recurrence.61 Caveolin-1 is particularly intriguing because unlike many of the other markers discussed herein, this protein is secreted into the serum. In fact, Tahir et al recently reported the development of a serum assay for caveolin-1 that was able to differentiate between patients with and without prostate cancer, suggesting that this protein may eventually serve as important serum biomarker.62
Proteomics Much interest has recently been directed towards evaluating the diagnostic potential of patterns of protein expression in various disease states using surface enhanced laser desorption/ionization time of flight mass spectrometry. Li et al compared the serum proteomics mass spectra of 246 men who had undergone radical prostatectomy with 99 contemporary men with negative prostate biopsy results.63 The cases and controls were randomly split into training and testing groups. The training set identified a panel of 3 proteins that optimally separated cases from controls, and the sensitivity and specificity of this protein panel was compared with that of PSA. At a selected specificity of 45%, the sensitivity of the selected protein panel was 76%, a clear improvement of the PSA sensitivity of 57%. Other studies similarly were able to differentiate malignant from benign prostates with high specificity and sensitivity using proteomic analysis of serum64-66 and tissue.67-75 Although to date attention has focused on the diagnostic capabilities of proteomics in prostate cancer, it is certainly quite likely that tumors of differing aggressiveness and metastatic potential will differ in their proteomic signatures as well.
Discussion The ideal prognostic marker would have as close to 100% sensitivity and 100% specificity as possible. Obviously, a novel marker would have to have independent prognostic value above and beyond that obtainable by existing parameters (eg, Gleason grade, disease stage, PSA). Optimally, the marker would not only predict outcome but would also suggest the best form of treatment for a given patient. With the remarkable advances in molecular biology, a wide variety of markers have become available. Tissue-based markers are typically detected by immunohistochemical staining. Specimens consist of prostate needle biopsies or the entire surgical specimen obtained after radical prostatectomy. The predictive value of biopsy specimens is hampered by the multifocal and heterogeneous nature of prostate tumors, thereby leading to sam-
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pling error. Analysis of the entire organ avoids this problem. However, in many cases one would like to scrutinize the prognostic marker before definitive treatment. Fluorescence in situ hybridization uses fluorescently labeled DNA probes to identify chromosomal abnormalities. Centromeric probes attach to the centromeres of chromosomes and are useful in determining the number of copies of a given chromosome within a cell. Normal cells harbor 2 copies of a chromosome, whereas malignant cells will often have additional copies. Locus-specific probes provide information about whether a given gene (or more specifically, an area on a chromosome) is amplified or deleted. Some markers are detectable in the serum at the protein level (eg, by enzyme-linked immunosorbent assay), or at the RNA level (eg, by reverse-transcriptase polymerase chain reaction). The burgeoning field of proteomics has allowed us to analyze the protein “signature” of a given disease process. Surfaceenhanced laser desorption/ionization time of flight mass spectrometry is an affinity-based mass spectrometry method in which proteins are selectively adsorbed to a chemically modified chip. Weakly bound proteins are washed away and the remaining samples are analyzed by mass spectrometry. By this technique, disease-associated proteins from specimens such as tissue, urine, and serum can be identified. Finally, although beyond the scope of this article, genetic markers might also eventually be useful in predicting outcome. Much work has been done in elucidating genetic mutations and/or polymorphisms that may play a role in prostate cancer and is reviewed by Chin and Reiter.76 A fascinating example of a genetic mutation leading to a tailored therapy comes from the realm of lung cancer.77 Gefitinib is an inhibitor of the EGFR tyrosine kinase and has been approved for the treatment of non–small-cell lung carcinoma. Response to this agent has varied greatly, with women, patients with adenocarcinoma, and Japanese patients most likely to benefit. Interestingly, Paez et al noticed that mutations in the EGFR correlated with precisely these demographic characteristics.77 They went on to demonstrate that 5 of 5 patients who exhibited radiographic and/or symptomatic improvement following gefitinib treatment in fact carried mutations in the EGFR kinase domain, whereas all 4 nonresponders whom they tested carried the wild-type EGFR. Thus, there seems to be an exquisite correlation between EGFR somatic mutation and gefitinib sensitivity, suggesting that screening for this marker may identify those patients most likely to respond to this specific chemotherapeutic agent. As noted, none of the discussed biomarkers have yet achieved clinical utility. Most of the studies to date are retrospective in nature and many present inconsistent data. Clearly, to be useful, one would need to design a study that demonstrates that a given marker can provide not only statistically significant prognostic information, but clinically significant information as well. Such a marker would have to improve upon or add to parameters such as Gleason score, tumor stage, PSA doubling time, and margin status. Moreover, an optimal marker would help the urologist decide which tumors need to be treated, which treatment modality might be most efficacious, which tumors should be
Jonathan L. Chin, Robert E. Reiter considered cured after primary definitive therapy, and which tumors might benefit from aggressive adjuvant therapy. One can envision that combinations of markers might be even more effective at directing therapy. Another key question that must be addressed is which patients would benefit from testing for biomarkers. Should every patient with prostate cancer be tested or merely a subset of patients? The answer undoubtedly will depend on the nature of the biomarker itself. A marker that can be isolated in a biopsy specimen or in peripheral blood at the time of diagnosis and can predict outcome after various treatment modalities with high sensitivity and specificity might be useful for routine screening in virtually all patients with prostate cancer. Conversely, for example, a marker that is obtained from a surgical specimen or that specializes in predicting response to adjuvant therapy after primary treatment would have a more limited target population. Finally, the costeffectiveness of biomarker testing must be rigorously established. In summary, prospective randomized controlled studies must ascertain that a given marker or markers can accurately predict outcome in a clinically meaningful and cost-effective way before they enter everyday clinical practice.
Conclusion The heterogeneity of prostate cancer continues to confound clinicians entrusted with the responsibility of helping patients choose the most sensible course of treatment. The current clinical and pathologic features (Gleason score, stage, PSA) that are used to risk-stratify patients fall far short in terms of accurately forecasting outcome for any particular patient. Indeed, in the PSA era, more and more patients are presenting within a very narrow range of these parameters, rendering their predictive utility even less helpful. Thus, additional biomarkers that can relieve some of the mystery and rationally direct prostate cancer treatment are urgently needed. The exponential growth of molecular biology techniques and the progressive elucidation of the biologic pathways of prostate cancer offers hope that this demand will soon be met.
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10. Wu TT, Hsu YS, Wang JS, et al. The role of p53, bcl-2 and E-cadherin expression in predicting biochemical relapse for organ confined prostate cancer in Taiwan. J Urol 2003; 170:78-81. 11. Stackhouse GB, Sesterhenn IA, Bauer JJ, et al. p53 and bcl-2 immunohistochemistry in pretreatment prostate needle biopsies to predict recurrence of prostate cancer after radical prostatectomy. J Urol 1999; 162:2040-2045. 12. Borre M, Stausbol-Gron B, Nerstrom B, et al. Immunohistochemical BCL2 and Ki-67 expression predict survival in prostate cancer patients followed expectantly. Prostate Cancer Prostatic Dis 1998; 1:268-275. 13. Ivanovic V, Melman A, Davis-Joseph B, et al. Elevated plasma levels of TGFbeta 1 in patients with invasive prostate cancer. Nat Med 1995; 1:282-284. 14. Shariat SF, Shalev M, Menesses-Diaz A, et al. Preoperative plasma levels of transforming growth factor beta(1) (TGF-beta(1)) strongly predict progression in patients undergoing radical prostatectomy. J Clin Oncol 2001; 19:2856-2864. 15. Wikstrom P, Stattin P, Franck-Lissbrant I, et al. Transforming growth factor beta1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer. Prostate 1998; 37:19-29. 16. Kim IY, Ahn HJ, Lang S, et al. Loss of expression of transforming growth factor-beta receptors is associated with poor prognosis in prostate cancer patients. Clin Cancer Res 1998; 4:1625-1630. 17. Umbas R, Schalken JA, Aalders TW, et al. Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer. Cancer Res 1992; 52:5104-5109. 18. Li LC, Zhao H, Nakajima K, et al. Methylation of the E-cadherin gene promoter correlates with progression of prostate cancer. J Urol 2001; 166:705-709. 19. Umbas R, Isaacs WB, Bringuier PP, et al. Decreased E-cadherin expression is associated with poor prognosis in patients with prostate cancer. Cancer Res 1994; 54:3929-3933. 20. Loric S, Paradis V, Gala JL, et al. Abnormal E-cadherin expression and prostate cell blood dissemination as markers of biological recurrence in cancer. Eur J Cancer 2001; 37:1475-1481. 21. Verhage BA, van Houwelingen K, Ruijter TE, et al. Single-nucleotide polymorphism in the E-cadherin gene promoter modifies the risk of prostate cancer. Int J Cancer 2002; 100:683-685. 22. Jonsson BA, Adami HO, Hagglund M, et al. -160C/A polymorphism in the E-cadherin gene promoter and risk of hereditary, familial and sporadic prostate cancer. Int J Cancer 2004; 109:348-352. 23. Tsukino H, Kuroda Y, Imai H, et al. Lack of evidence for the association of E-cadherin gene polymorphism with increased risk or progression of prostate cancer. Urol Int 2004; 72:203-207. 24. Noordzij MA, van Steenbrugge GJ, Schroder FH, et al. Decreased expression of CD44 in metastatic prostate cancer. Int J Cancer 1999; 84:478-483. 25. Vis AN, Noordzij MA, Fitoz K, et al. Prognostic value of cell cycle proteins p27(kip1) and MIB-1, and the cell adhesion protein CD44s in surgically treated patients with prostate cancer. J Urol 2000; 164:2156-2161. 26. Coradini D, Daidone MG. Biomolecular prognostic factors in breast cancer. Curr Opin Obstet Gynecol 2004; 16:49-55. 27. Signoretti S, Montironi R, Manola J, et al. HER2-neu expression and progression toward androgen independence in human prostate cancer. J Natl Cancer Inst 2000; 92:1918-1925. 28. Mark HF, Feldman D, Das S, et al. Fluorescence in situ hybridization study of HER2/neu oncogene amplification in prostate cancer. Exp Mol Pathol 1999; 66:170-178. 29. Fossa A, Lilleby W, Fossa SD, et al. Independent prognostic significance of HER2 oncoprotein expression in pN0 prostate cancer undergoing curative radiotherapy. Int J Cancer 2002; 99:100-105. 30. Morote J, de Torres I, Caceres C, et al. Prognostic value of immunohistochemical expression of the c-erbB-2 oncoprotein in metastasic prostate cancer. Int J Cancer 1999; 84:421-425. 31. Ross JS, Sheehan CE, Hayner-Buchan AM, et al. Prognostic significance of HER2/neu gene amplification status by fluorescence in situ hybridization of prostate carcinoma. Cancer 1997; 79:2162-2170. 32. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344:783-792. 33. Morris MJ, Reuter VE, Kelly WK, et al. HER2 profiling and targeting in prostate carcinoma. Cancer 2002; 94:980-986. 34. Borre M, Stausbol-Gron B, Overgaard J. p53 accumulation associated with bcl-2, the proliferation marker MIB-1 and survival in patients with prostate cancer subjected to watchful waiting. J Urol 2000; 164:716-721. 35. Deliveliotis C, Skolarikos A, Karayannis A, et al. The prognostic value of p53 and DNA ploidy following radical prostatectomy. World J Urol 2003; 21:171-176.
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56. Reiter RE, Sato I, Thomas G, et al. Coamplification of prostate stem cell antigen (PSCA) and MYC in locally advanced prostate cancer. Genes Chromosomes Cancer 2000; 27:95-103. 57. Gu Z, Thomas G, Yamashiro J, et al. Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer. Oncogene 2000; 19:1288-1296. 58. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002; 419:624-629. 59. Rhodes DR, Sanda MG, Otte AP, et al. Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 2003; 95:661-668. 60. Yang G, Truong LD, Wheeler TM, et al. Caveolin-1 expression in clinically confined human prostate cancer: a novel prognostic marker. Cancer Res 1999; 59:5719-5723. 61. Satoh T, Yang G, Egawa S, et al. Caveolin-1 expression is a predictor of recurrence-free survival in pT2N0 prostate carcinoma diagnosed in Japanese patients. Cancer 2003; 97:1225-1233. 62. Tahir SA, Ren C, Timme TL, et al. Development of an immunoassay for serum caveolin-1: a novel biomarker for prostate cancer. Clin Cancer Res 2003; 9:3653-3659. 63. Li J, White N, Zhang Z, et al. Detection of prostate cancer using serum proteomics pattern in a histologically confirmed population. J Urol 2004; 171:1782-1787. 64. Banez LL, Prasanna P, Sun L, et al. Diagnostic potential of serum proteomic patterns in prostate cancer. J Urol 2003; 170:442-446. 65. Petricoin EF III, Ornstein DK, Paweletz CP, et al. Serum proteomic patterns for detection of prostate cancer. J Natl Cancer Inst 2002; 94:1576-1578. 66. Adam BL, Qu Y, Davis JW, et al. Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res 2002; 62:3609-3614. 67. Adams J. Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer. Curr Opin Chem Biol 2002; 6:493-500. 68. Pagano M, Tam SW, Theodoras AM, et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 1995; 269:682-685. 69. Williams S, Pettaway C, Song R, et al. Differential effects of the proteasome inhibitor bortezomib on apoptosis and angiogenesis in human prostate tumor xenografts. Mol Cancer Ther 2003; 2:835-843. 70. Ikezoe T, Yang Y, Saito T, et al. Proteasome inhibitor PS-341 down-regulates prostate-specific antigen (PSA) and induces growth arrest and apoptosis of androgen-dependent human prostate cancer LNCaP cells. Cancer Sci 2004; 95:271-275. 71. Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 2004; 22:2108-2121. 72. Visakorpi T, Kallioniemi AH, Syvanen AC, et al. Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization. Cancer Res 1995; 55:342-347. 73. Cher ML, Bova GS, Moore DH, et al. Genetic alterations in untreated metastases and androgen-independent prostate cancer detected by comparative genomic hybridization and allelotyping. Cancer Res 1996; 56:3091-3102. 74. Alers JC, Krijtenburg PJ, Rosenberg C, et al. Interphase cytogenetics of prostatic tumor progression: specific chromosomal abnormalities are involved in metastasis to the bone. Lab Invest 1997; 77:437-448. 75. Zheng Y, Xu Y, Ye B, et al. Prostate carcinoma tissue proteomics for biomarker discovery. Cancer 2003; 98:2576-2582. 76. Chin JL, Reiter RE. Genetic alterations in prostate cancer. Curr Urol Rep 2004; 5:157-165. 77. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304:1497-1500.
Molecular Markers and Prostate Cancer Prognosis Jonathan L. Chin Robert E. Reiter David Geffen School of Medicine University of California Los Angeles
Abstract Prostate cancer is the most common malignancy among American men and is the second-leading cause of cancer-related mortality. Although radical prostatectomy and radiation therapy offer hope for cure for the majority of men with localized tumors, we continue to lack the tools to definitively determine which cancers need to be treated, which cancers will recur after treatment, and which cancers will behave aggressively when they have metastasized. Recent breakthroughs in molecular biology have led to the identification of a number of potential biomarkers for prostate cancer, many of which have been suggested to have prognostic significance. Eventually, combinations of these markers will hopefully enable us to more rationally facilitate counseling and direct management for men with prostate cancer.
Introduction
Clinical Prostate Cancer, Vol. 3, No. 3, 157-164, 2004 Key words: Biomarker, Fluorescence in situ hybridization, Immunohistochemistry, Proteasome, Proteomics Submitted: Aug 18, 2004; Revised: Nov 9, 2004 Accepted: Nov 10, 2004 Address for correspondence: Robert E. Reiter, MD David Geffen School of Medicine University of California Los Angeles 650 Charles East Young Dr Room 66-134 CHS Los Angeles, CA 90095 Fax: 310-206-5343 e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Cancer Information Group, ISSN #1540-0352, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
Adenocarcinoma of the prostate ranks as the most common malignancy among American men and is second only to lung cancer in terms of cancer-related mortality in men, with approximately 200,000 new cases and almost 30,000 deaths per year.1 Since the widespread adoption of prostatespecific antigen (PSA) screening for prostate cancer, we have witnessed impressive stage migration with more and more cancers being detected at a potentially curable juncture. Still, a plethora of dilemmas persists regarding the management of prostate cancer. Which cancers need to be treated? Which cancers will progress regardless of treatment? Which cancers can be cured? Is one treatment modality more appropriate than another for a particular patient? As a case in point, Thompson et al recently noted that as many as 15% of asymptomatic men > 62 years of age with PSA levels < 4.0 ng/mL (the upper limit of normal) harbor prostate cancer, with a significant percentage of these having a Gleason score of ≥ 7.2 However, we currently have no means of assessing whether these tumors are clinically significant. Clearly, we cannot rely on PSA alone to dictate management. Moreover, the natural history of a given tumor must be balanced against life expectancy, and given the significant morbidities of the various treatments for prostate cancer, quality of life issues must be taken into serious consideration as well. Although parameters such as Gleason score, stage, and PSA can offer rough estimates as to eventual outcome, additional prognostic markers are urgently needed in order to accurately risk-stratify patients and dictate therapy. Prognostic markers for prostate cancer can arise from an assortment of techniques and sources (Table 1).3-67 Tissue-based markers can be assessed by immunohistochemistry (IHC). Fluorescence in situ hybridization (FISH) can analyze chromosomal aberrations arising in malignant tissue. Various serum markers can be detected at the protein level or at the RNA level. The field of proteomics explores the protein “signature” of biologic samples such as serum, tissue, or urine and can delineate characteristic differences between malignant and benign specimens. Although nothing to
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Table 1 Potential Biomarkers in Prostate Cancer3-67 Marker
Source
Technique
Comments
Ki-67
Tissue
IHC
Proliferative antigen3-6
Bcl-2
Tissue
IHC
Apoptosis inhibitor3,7-12
Tissue, serum
IHC, ELISA
Proliferation, chemotaxis, angiogenesis, and cellular differentiation13-16
E-cadherin
Tissue
IHC
Adhesion molecule10,17-23
CD44
Tissue
IHC
Adhesion molecule24,25
HER2/neu
Tissue
IHC, FISH
Growth factor receptor26-33
p53
Tissue
IHC
Tumor suppressor9,11,34-40
PTEN
Tissue
IHC
Tumor suppressor41-45
p27
Tissue
IHC
Tumor suppressor46-49
skp2
Tissue
IHC
Positive cell cycle regulator50
MYC
Tissue
IHC, FISH
Protooncogene51-54
PSCA
Tissue
IHC
Coamplified with MYC55-57
EZH2
Tissue
RT-PCR
Transcriptional repressor58,59
Caveolin-1
Tissue, serum
IHC, sandwich immunoassay
Molecular transport, cell adhesion, and signal transduction60-62
Proteomics
Tissue, serum
Mass spectrometry
Protein signature63-67
TGF-β1
Abbreviations: ELISA = enzyme-linked immunosorbent assay; RT-PCR = reverse-transcriptase polymerase chain reaction
date has definitively improved upon our usual risk-stratifying parameters for prostate cancer, much work has been done on a variety of molecular and genetic biomarkers. The prognostic significance of several biomarkers that may be important in the management of prostate cancer is reviewed in this article.
Proliferative Antigens Ki-67 Ki-67 is a nuclear antigen present throughout the cell cycle, but not in noncycling or quiescent cells, and its expression is associated with cellular proliferation. Multiple studies have demonstrated that elevated expression of Ki-67 is associated with an adverse prognosis in prostate cancer. Biopsy specimens from 106 patients with stage T1-T3 prostate cancer treated with external-beam radiation were stained for Ki-67.3 With a median follow-up of 62 months, elevated Ki-67 staining was negatively correlated with progression-free survival (PFS). In a separate study involving 104 patients treated by radical prostatectomy, the surgical specimens were stained for Ki-67 in the area of highest tumor grade.4 In a univariate analysis, Ki-67 expression was significantly associated with time to biochemical failure, as were age, grade, stage, margin status, tumor size, and preoperative PSA. In a multivariate analysis, only stage, PSA, and Ki-67 expression remained as significant independent predictors of time to biochemical recurrence. A similar study of 180 patients treated with radical prostatectomy again demonstrated a correlation between Ki-67 expression and diseasefree survival (DFS).5 Finally, Ki-67 expression has also been shown to predict disease progression after initial hormonal therapy in patients with advanced prostate cancer.6 Thus, Ki-67 might eventually serve as a significant biomarker in patients managed with a variety of treatment modalities.
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Apoptosis-Related Proteins Bcl-2 Bcl-2 is an integral membrane protein that plays a role in the inhibition of apoptosis. Overexpression of Bcl-2 has been found to play a role in a number of human malignancies.7 The evidence for the role of Bcl-2 as a biomarker in prostate cancer has been mixed. Expression of Bcl-2 in prostate needle biopsy specimens predicted biochemical failure in those treated with radiation.3,8,9 Similarly, multiple studies have confirmed that in patients with localized prostate cancer treated with radical prostatectomy, Bcl-2 expression in the prostatectomy specimen correlated with biochemical recurrence.10-11 However, in contrast to the radiation therapy (RT) studies, staining in the preoperative biopsy specimen did not correlate with outcome.11 Finally, in a cohort of 221 elderly patients (median age, 75 years) with prostate cancer who were followed expectantly, Bcl-2 expression was significantly associated with disease-specific and overall survival (OS).12 However, in a multivariate analysis, this correlation did not maintain its significance.
Growth Factors Transforming Growth Factor–β1 Transforming growth factor–β1 (TGF-β1) plays a role in the control of cellular proliferation, chemotaxis, angiogenesis, and cellular differentiation. Loss of the inhibitory effects of TGF-β1 has been associated with the progression of cancer, and elevated local and circulating levels have correlated with prostate cancer invasion and metastasis.13 In a cohort of 120 consecutive patients who underwent radical prostatectomy for clinically localized prostate cancer, preoperative TGF-β1 levels were measured and correlated with clinical outcome.14 For comparison,
Jonathan L. Chin, Robert E. Reiter TGF-β1 serum levels were also measured in 44 healthy men, 19 men with lymph node metastases, and 10 men with bone metastases. TGF-β1 levels in patients with metastatic lesions (lymph node or bone) were significantly higher than those in the radical prostatectomy group or healthy subjects. Moreover, preoperative TGF-β1 levels strongly predicted organ-confined disease and PSA progression, suggesting that this growth factor might be useful in identifying patients with occult metastatic disease and therefore those who are at higher risk of recurrence after radical prostatectomy. In a related finding, Wikstrom et al determined that increased TGF-β1 staining was associated with tumor grade and metastasis.15 Other groups have investigated a relationship between the loss of TGF-β receptors and clinical outcome. In a study of 52 patients treated with radical prostatectomy, loss of TGF-β1 R1 was significantly associated with Gleason score, tumor stage, 4-year survival rate, and biochemical recurrence.16 Thus, expression of the TGF-β1 receptor by IHC may be of use as a prognostic marker, as well as expression of the growth factor itself.
Adhesion Molecules E-cadherin E-cadherin, located on chromosome 16q22.1, is cell membrane protein that is involved in intercellular adhesion. It has been theorized that E-cadherin serves as suppressor of invasion. Thus, decreased or absent expression of this protein may place a patient at risk for locally invasive cancer and/or metastasis. Indeed, E-cadherin expression, as assessed by IHC, is decreased or absent in prostate cancer.17 Li et al demonstrated that hypermethylation of the E-cadherin promoter correlates with reduced or absent E-cadherin protein staining in prostate cancer specimens, and the severity of hypermethylation is associated with tumor grade.18 Demethylation restored E-cadherin messenger RNA (mRNA) transcription in prostate cancer lines. E-cadherin protein levels are inversely correlated with Gleason grade, disease stage, presence of metastasis, and survival.19 Decreased expression of E-cadherin in radical prostatectomy specimens portends an elevated risk of PSA recurrence in patients with T2 tumors.10 Finally, abnormal E-cadherin expression in prostatectomy specimens was strongly associated with the detection of prostate cancer cells in the peripheral blood.20 One particular E-cadherin polymorphism (–160 C/A) has been associated with an increased risk of prostate cancer.21 Subjects with ≥ 1 A allele had a relative risk of 3.6 of having prostate cancer compared with the CC carriers. In a study of > 1000 Swedish subjects, this E-cadherin polymorphism was found to be associated with hereditary prostate cancer but not sporadic prostate cancer.22 Conversely, in a case-control study of Japanese subjects, this E-cadherin polymorphism did not correlate with an increased risk of prostate cancer.23
CD44 CD44, a type 1 transmembrane glycoprotein involved in cell–cell and cell–matrix interactions, is another cell adhesion
protein whose prognostic role in prostate cancer has been studied. Patients with lymph node metastases are much more likely to have decreased staining for CD44.24 In addition, loss of CD44 in radical prostatectomy specimens is an independent predictor of biochemical recurrence.25
Oncogenes HER2/neu HER2/neu, located on chromosome 17p, is a transmembrane tyrosine kinase growth factor receptor with homology to the epidermal growth factor receptor (EGFR). Amplification and subsequent overexpression of HER2/neu is an adverse prognostic indicator in breast cancer.26 In prostate cancer as well, HER2/neu may play a role in advanced disease. Signoretti et al examined a panel of prostate specimens obtained from patients treated by surgery alone, by maximal androgen blockade (MAB) followed by surgery, or by MAB alone who subsequently had progression to metastatic, androgen-independent (AI) disease.27 As determined by IHC, only 25% of specimens from the surgery-alone group stained positive for HER2/neu, whereas 59% from the MAB/surgery group and 78% from the AI group were positive. Moreover, patients treated with MAB demonstrated amplification of the HER2/neu gene by FISH, whereas patients treated by surgery alone did not manifest gene amplification. These data suggest that HER2/neu expression increases with progression to AI disease. In contrast, in a study of 88 radical prostatectomy specimens (presumably androgen-dependent), HER2/neu amplification was found in only 8 of 86 tumors (9.3%), and even in these cases, amplification was only at a low to moderate level.28 Not only does it appear that HER2/neu is overexpressed in advanced prostate cancer, but several studies have also suggested that HER2/neu carries a negative prognostic significance in prostate cancer in general. In a study of 112 patients undergoing curative RT for localized prostate cancer, HER2/neu expression in the needle biopsy specimen was inversely correlated with both PFS and disease-specific survival.29 Similarly, in 70 Spanish patients diagnosed with metastatic prostate cancer and treated with MAB, HER2 overexpression on IHC decreased mean cancer-specific survival from 54 months to 33 months.30 In contrast, in a study of 113 patients treated by radical prostatectomy, HER2/neu gene amplification correlated with biochemical recurrence in a univariate analysis but did not reach significance (P = 0.125) in a multivariate analysis with grade and DNA ploidy.31 HER2/neu protein expression by IHC was not significantly associated with biochemical recurrence. As noted, HER2/neu has been established as a negative prognostic indicator in breast cancer. Moreover, targeting HER2 with the humanized monoclonal antibody trastuzumab has benefited patients with metastatic breast tumors that overexpressed HER2/neu.32 The addition of trastuzumab to chemotherapy was associated with a longer time to disease progression, a higher rate of objective response, a longer duration of response, a lower rate of death at 1 year, longer survival, and a 20% reduction in the risk of death. The efficacy of trastuzumab in breast carcinomas that overexpress HER2/neu raised the hope that it would offer similar advantages in prostate tumors that similarly overexpress
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Figure 1 Positive Surgical Margins: Impact of p27 and Stage on Survival
Disease-Free Survival (%)
100 p27+, No SVI
75
50 p27–, No SVI or p27+, SVI
25 p27–, SVI
0
12
P < 0.001
24
36 Months
48
60
72
Patients with positive surgical margins and low or absent p27 immunostaining of their radical prostatectomy specimens have a significantly worse outcome than those with normal p27 levels. Low p27 appears to have the same dire prognostic significance as SVI (Reiter et al, unpublished data). Abbreviation: SVI = seminal vesicle invasion
HER2/neu. Morris et al conducted a preliminary study evaluating the efficacy of trastuzumab in 23 patients with progressive metastatic prostate cancer.33 After preliminary screening for HER2/neu, all patients were administered weekly trastuzumab until they experienced disease progression, after which paclitaxel was added. All of the patients had disease progression within 12 weeks regardless of HER2/neu status. However, this study was hampered by the fact that, of the 130 patients screened for HER2/neu, only 6 eligible patients had HER2/neu–positive disease. Significantly, in several cases, the HER2/neu status of paired primary prostate specimens and metastatic tissue did not match, with the former staining negative and the latter staining positive. Because obtaining metastatic tissue is neither facile nor routine, the authors emphasized that improved methods of screening for HER2/neu is crucial in order to properly evaluate the efficacy of targeting HER2/neu in prostate cancer treatment.
Tumor Suppressors
chemical recurrence, whereas p53 levels in the preoperative biopsy specimen was not.11 Positive p53 staining heralded PSA failure in patients with localized tumors treated with radiation.9,37 Finally, in a study of patients with locally advanced prostate cancer treated with a combination of RT and MAB versus RT alone, abnormal p53 protein expression by IHC of pretreatment prostate specimens (needle biopsy or transurethral resection) was significantly associated with an increased incidence of distant metastasis and shortened PFS and OS.38 Interestingly, time to metastasis was unrelated to p53 status in patients treated with RT alone. Unfortunately, the data for p53 have been far from uniformly positive. Multiple studies have failed to demonstrate a relationship between p53 and outcome, including disease-specific survival in advanced prostate cancer treated with hormone ablation,39 PSA recurrence following radical prostatectomy,10 and PSA recurrence in radical prostatectomy patients subsequently treated with neoadjuvant androgen ablation and salvage RT.40
PTEN PTEN is a phospholipid phosphatase that acts as a negative regulator in the phosphatidylinositol-3,4,5-triphosphate (PIP3)/Akt signaling pathway. The end result of this pathway is to inhibit apoptosis and promote proliferation.41 PTEN dephosphorylates and thereby inactivates the second messenger PIP3. Thus, inactivating mutations of PTEN can lead to persistent levels of PIP3 and constitutive activation of PIP3 kinase (PI3K) with subsequent uncontrolled cellular proliferation. Loss of PTEN expression has been correlated with elevated Gleason score and advanced stage in 109 primary prostate tumors,42 and has been shown to be an independent predictor of biochemical failure in men treated by radical prostatectomy.43 One of the downstream kinases activated in the PIP3/Aktpathway is mTOR (mammalian target of rapamycin). PTEN loss or inactivation leads to overactive mTOR and unchecked cellular proliferation.44,45 Blocking mTOR should, in essence, take over the inhibitory role of the defective or deficient PTEN. Thus, in another example of rational and targeted therapy, the mTOR inhibitor rapamycin is currently being studied in phase II clinical trials as a neoadjuvant therapy in patients whose needle biopsies stain negative for PTEN by IHC.
p53
p27
The p53 tumor suppressor plays a role in cell cycle arrest and apoptosis, and loss of function mutations in p53 is present in a wide range of human malignancies. A number of studies have suggested that p53 expression might have prognostic value in patients with prostate cancer. In a study of 221 patients with prostate cancer treated with watchful waiting (median follow-up, 15 years), the degree of p53 nuclear accumulation in the biopsy specimen was significantly associated with disease-specific survival in both the overall study population as well as in the subgroup of 125 patients with clinically localized disease.34 Similarly, nuclear accumulation of p53 in radical prostatectomy specimens correlated with increased risk of PSA recurrence and reduced disease-specific survival in multiple studies.11,35,36 Of note, in one study, expression of p53 in the radical prostatectomy specimen was significantly associated with bio-
Protein p27 (Kip1) is a cyclin-dependent kinase inhibitor that plays a role in cell cycle arrest and apoptosis. Protein p27 expression in needle biopsies is inversely correlated with tumor stage and grade.46 Moreover, in a multivariate analysis of a cohort of 52 Spanish patients who had been treated with radical prostatectomy, a low level of IHC staining for p27 in the surgical specimen was the lone independent predictor of biochemical recurrence.47 At 3 years, 59% of the patients with low p27 had experienced a PSA recurrence, whereas only 18% of the patients with normal p27 expression had a relapse. Similarly, in a separate study of 86 patients also treated with radical prostatectomy, Yang et al demonstrated that p27 was a strong independent predictor of DFS, second only to pathologic stage in a multivariate analysis that included age, preoperative PSA, Gleason score, and
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Jonathan L. Chin, Robert E. Reiter stage.48 Even more strikingly, multivariate analysis demonstrated that p27 was the strongest predictor of biochemical recurrence among factors studied in patients with pathologic T2a-T3b disease. Thus, absent or low levels of p27 protein expression appear to be an adverse prognostic factor in patients with clinically organ-confined disease treated by radical prostatectomy. Another interesting finding is that patients who have positive surgical margins have a relative risk of 3.4 of having recurrence if they have low expression of p27 (P < 0.04; Reiter et al, unpublished data). Moreover, in patients with positive margins, low or absent p27 staining carries as dire a prognostic significance as seminal vesicle invasion (Figure 1). Thus, p27 status might help determine whether a patient with a positive margin would benefit from adjuvant radiation. Importantly, p27 status at the time of the initial prostate biopsy also seems to predict biochemical recurrence. In a study of 161 men with subsequent surgical management, p27 immunostaining in < 45% of the cells resulted in significant preoperative risk stratification for time to PSA failure (hazard ratio, 2.41; P = 0.010).49 This relationship maintained its significance in a multivariate analysis. Therefore, p27 may be an important prognostic factor at the time of diagnosis.
skp2 The F-box protein skp2 is a positive regulator of G1-S transition and promotes ubiquitin-mediated proteolysis of the cyclindependent kinase inhibitor p27. Thus, upregulation of skp2 should result in decreased p27 and subsequent loss of cell cycle control. In an immunohistochemical analysis, skp2 levels were dramatically increased in prostatic intraepithelial neoplasia (PIN) and radical prostatectomy specimens.50 In addition, skp2 levels were positively correlated with preoperative PSA, Gleason score, and stage. Finally, a higher skp2 labeling index negatively impacted biochemical recurrence–free survival after radical prostatectomy.
Proteasome Targeting in Prostate Cancer The proteasome is a multifunctional protease present in all eukaryotic cells that is primarily responsible for intracellular protein destruction.67 Proteins that have been selected for degradation are tagged with chains of ubiquitin monomers, which are subsequently recognized by the proteasome complex. The proteasome plays a critical role in the regulation of many cellular processes, including activation and deactivation of transcription factors, cell cycle progression, and apoptosis. Many tumor suppressors have been shown to be regulated by the ubiquitin-proteasome pathway, including p5367 and p27.68 Bortezomib (Velcade®, PS-341) is a selective proteasome inhibitor that is currently being evaluated in clinical trials for the treatment of a variety of human malignancies, including prostate cancer. Williams et al demonstrated that bortezomib can strongly inhibit proliferation and induce apoptosis of prostate cancer xenograft cell lines in vitro and suppress tumor growth in vivo.69 Similarly, Ikezoe et al showed that bortezomib triggered growth arrest and apoptosis of androgen-dependent human prostate cancer LNCaP cells, possibly by interrupting the androgen receptor sig-
naling pathway.70 A recent phase I clinical trial investigating bortezomib in advanced prostate cancer exhibited a modest PSA response in patients with androgen-independent disease.71 Thus, preliminary data suggest that proteasome inhibition may be an enticing novel therapeutic strategy. Indeed, for prostate cancers deficient in tumor suppressors regulated by the ubiquitin-proteasome pathway, inhibition of the proteasome might restore the tumor suppressor to its normal function and thereby impede tumor growth.
Other Biomarkers Chromosome 8q, MYC, and PSCA Gain of chromosome 8q is one of the most frequent changes in prostate cancer. The 8q gain was seen in 89% of hormone refractory prostate specimens,72 85% of lymph node metastases,73 and 50% of bone metastases,74 but in only 6% of primary, localized tumors,72 suggesting that this is a late event in prostate carcinogenesis. The MYC gene, mapped to chromosome 8q24, is an intriguing candidate for this locus. MYC is amplified in a significant number of advanced prostate cancers.51 Overexpression of MYC in transgenic mice leads to the formation of lesions similar to PIN followed by overt adenocarcinoma.52 In a study of tumor samples from 144 patients with high grade prostate cancer, Sato et al demonstrated that MYC amplification assessed by FISH predicts systemic progression and prostate cancer–specific death in patients with highgrade advanced tumors.53 Conversely, FISH analysis of 195 organ-confined radical prostatectomy specimens demonstrated a correlation between MYC amplification and Gleason grade, but not between MYC amplification and biochemical recurrence.54 PSCA is a cell surface antigen overexpressed in prostate cancer that maps to chromosome 8q24.2, just distal to MYC.55 PSCA has been shown to be coamplified with MYC in locally advanced prostate tumors,56 and PSCA expression has been shown to increase with Gleason grade, tumor stage, and metastasis.57 Monoclonal antibodies against PSCA inhibit prostate cancer tumor growth and metastasis in animal models, offering hope that PSCA might be an excellent therapeutic target as well as a prognostic indicator in aggressive, MYC-amplified tumors.
EZH2 EZH2 (polycomb group protein enhancer of zeste homolog 2) is a transcriptional repressor that, using complementary DNA microarray analysis, has been shown to be overexpressed at the mRNA level in hormone refractory metastatic prostate cancer relative to localized prostate cancer.58 In a separate study, the same group assessed a panel of 14 candidate prognostic biomarkers identified with use of the tissue microarrays in a cohort of 259 patients who had undergone radical prostatectomy for localized prostate cancer. They determined that patients whose tumors exhibited moderate or strong immunostaining for EZH2 coupled with at most moderate expression of E-cadherin had a relative risk of approximately 3.0 for biochemical recurrence, even after adjusting for stage, grade, and preoperative PSA.59 Thus, EZH2:ECAD status might prove useful in identifying a high-risk cohort of patients following radical prostatectomy.
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Molecular Prognostic Markers in Prostate Cancer Caveolin-1 Caveolin-1 is a major structural protein of caveolae, specialized plasma membrane invaginations that are abundant in smooth muscle cells, adipocytes, and endothelium. Caveolae appear to mediate molecular transport, cell adhesion, and signal transduction activities in a cell- and context-specific fashion. Positive caveolin-1 immunostaining correlates positively with Gleason score, positive surgical margins, and lymph node metastasis, and is an independent predictor for time to disease progression in patients treated by radical prostatectomy.60 In a similar study involving a cohort of Japanese patients with T2 disease, caveolin1 staining again predicted biochemical recurrence.61 Caveolin-1 is particularly intriguing because unlike many of the other markers discussed herein, this protein is secreted into the serum. In fact, Tahir et al recently reported the development of a serum assay for caveolin-1 that was able to differentiate between patients with and without prostate cancer, suggesting that this protein may eventually serve as important serum biomarker.62
Proteomics Much interest has recently been directed towards evaluating the diagnostic potential of patterns of protein expression in various disease states using surface enhanced laser desorption/ionization time of flight mass spectrometry. Li et al compared the serum proteomics mass spectra of 246 men who had undergone radical prostatectomy with 99 contemporary men with negative prostate biopsy results.63 The cases and controls were randomly split into training and testing groups. The training set identified a panel of 3 proteins that optimally separated cases from controls, and the sensitivity and specificity of this protein panel was compared with that of PSA. At a selected specificity of 45%, the sensitivity of the selected protein panel was 76%, a clear improvement of the PSA sensitivity of 57%. Other studies similarly were able to differentiate malignant from benign prostates with high specificity and sensitivity using proteomic analysis of serum64-66 and tissue.67-75 Although to date attention has focused on the diagnostic capabilities of proteomics in prostate cancer, it is certainly quite likely that tumors of differing aggressiveness and metastatic potential will differ in their proteomic signatures as well.
Discussion The ideal prognostic marker would have as close to 100% sensitivity and 100% specificity as possible. Obviously, a novel marker would have to have independent prognostic value above and beyond that obtainable by existing parameters (eg, Gleason grade, disease stage, PSA). Optimally, the marker would not only predict outcome but would also suggest the best form of treatment for a given patient. With the remarkable advances in molecular biology, a wide variety of markers have become available. Tissue-based markers are typically detected by immunohistochemical staining. Specimens consist of prostate needle biopsies or the entire surgical specimen obtained after radical prostatectomy. The predictive value of biopsy specimens is hampered by the multifocal and heterogeneous nature of prostate tumors, thereby leading to sam-
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pling error. Analysis of the entire organ avoids this problem. However, in many cases one would like to scrutinize the prognostic marker before definitive treatment. Fluorescence in situ hybridization uses fluorescently labeled DNA probes to identify chromosomal abnormalities. Centromeric probes attach to the centromeres of chromosomes and are useful in determining the number of copies of a given chromosome within a cell. Normal cells harbor 2 copies of a chromosome, whereas malignant cells will often have additional copies. Locus-specific probes provide information about whether a given gene (or more specifically, an area on a chromosome) is amplified or deleted. Some markers are detectable in the serum at the protein level (eg, by enzyme-linked immunosorbent assay), or at the RNA level (eg, by reverse-transcriptase polymerase chain reaction). The burgeoning field of proteomics has allowed us to analyze the protein “signature” of a given disease process. Surfaceenhanced laser desorption/ionization time of flight mass spectrometry is an affinity-based mass spectrometry method in which proteins are selectively adsorbed to a chemically modified chip. Weakly bound proteins are washed away and the remaining samples are analyzed by mass spectrometry. By this technique, disease-associated proteins from specimens such as tissue, urine, and serum can be identified. Finally, although beyond the scope of this article, genetic markers might also eventually be useful in predicting outcome. Much work has been done in elucidating genetic mutations and/or polymorphisms that may play a role in prostate cancer and is reviewed by Chin and Reiter.76 A fascinating example of a genetic mutation leading to a tailored therapy comes from the realm of lung cancer.77 Gefitinib is an inhibitor of the EGFR tyrosine kinase and has been approved for the treatment of non–small-cell lung carcinoma. Response to this agent has varied greatly, with women, patients with adenocarcinoma, and Japanese patients most likely to benefit. Interestingly, Paez et al noticed that mutations in the EGFR correlated with precisely these demographic characteristics.77 They went on to demonstrate that 5 of 5 patients who exhibited radiographic and/or symptomatic improvement following gefitinib treatment in fact carried mutations in the EGFR kinase domain, whereas all 4 nonresponders whom they tested carried the wild-type EGFR. Thus, there seems to be an exquisite correlation between EGFR somatic mutation and gefitinib sensitivity, suggesting that screening for this marker may identify those patients most likely to respond to this specific chemotherapeutic agent. As noted, none of the discussed biomarkers have yet achieved clinical utility. Most of the studies to date are retrospective in nature and many present inconsistent data. Clearly, to be useful, one would need to design a study that demonstrates that a given marker can provide not only statistically significant prognostic information, but clinically significant information as well. Such a marker would have to improve upon or add to parameters such as Gleason score, tumor stage, PSA doubling time, and margin status. Moreover, an optimal marker would help the urologist decide which tumors need to be treated, which treatment modality might be most efficacious, which tumors should be
Jonathan L. Chin, Robert E. Reiter considered cured after primary definitive therapy, and which tumors might benefit from aggressive adjuvant therapy. One can envision that combinations of markers might be even more effective at directing therapy. Another key question that must be addressed is which patients would benefit from testing for biomarkers. Should every patient with prostate cancer be tested or merely a subset of patients? The answer undoubtedly will depend on the nature of the biomarker itself. A marker that can be isolated in a biopsy specimen or in peripheral blood at the time of diagnosis and can predict outcome after various treatment modalities with high sensitivity and specificity might be useful for routine screening in virtually all patients with prostate cancer. Conversely, for example, a marker that is obtained from a surgical specimen or that specializes in predicting response to adjuvant therapy after primary treatment would have a more limited target population. Finally, the costeffectiveness of biomarker testing must be rigorously established. In summary, prospective randomized controlled studies must ascertain that a given marker or markers can accurately predict outcome in a clinically meaningful and cost-effective way before they enter everyday clinical practice.
Conclusion The heterogeneity of prostate cancer continues to confound clinicians entrusted with the responsibility of helping patients choose the most sensible course of treatment. The current clinical and pathologic features (Gleason score, stage, PSA) that are used to risk-stratify patients fall far short in terms of accurately forecasting outcome for any particular patient. Indeed, in the PSA era, more and more patients are presenting within a very narrow range of these parameters, rendering their predictive utility even less helpful. Thus, additional biomarkers that can relieve some of the mystery and rationally direct prostate cancer treatment are urgently needed. The exponential growth of molecular biology techniques and the progressive elucidation of the biologic pathways of prostate cancer offers hope that this demand will soon be met.
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