New concepts in the pathology of prostatic epithelial carcinogenesis

NEW CONCEPTS IN THE PATHOLOGY OF PROSTATIC EPITHELIAL CARCINOGENESIS ANGELO M. DE MARZO, MATHEW J. PUTZI,

AND

WILLIAM G. NELSON

ABSTRACT The development of drugs to prevent prostate cancer is underway, yet monitoring the potential efficacy of these agents during clinical trials relies on measuring intermediate endpoints. In this review, various candidate markers are presented that are under different stages of evaluation as intermediate endpoint biomarkers. In addition, the near future will bring an unprecedented wave of new potential biomarkers. For instance, through genomics-based methods many new genes are being discovered whose altered expression may be involved in different phases of prostate cancer development and progression. In the development of rational approaches for selecting which of these untested biomarkers may be useful to measure systematically, there must be an improved understanding of the mechanisms of prostatic carcinogenesis. We submit that this improved understanding will come through new knowledge of the biology of normal prostate epithelial cells, the determination of the precise target cells of transformation, and how their growth regulation is genetically and epigenetically perturbed during the phases of initiation and progression. In this review, therefore, we also present our recent immune-mediated oxidant injury and regeneration hypothesis of why and how the prostate is targeted for carcinogenesis. UROLOGY 57 (Suppl 4A): 103–114, 2001. © 2001, Elsevier Science Inc.

BIOMARKERS IN PROSTATIC ADENOCARCINOMA The monitoring of efficacy of chemoprevention trials for prostate cancer relies on the measurement of intermediate endpoint biomarkers (IEB).1,2 Several categories of potential markers have emerged, many of which can be analyzed directly on tissue sections from patient specimens. These include morphological entities such as high-grade prostatic intraepithelial neoplasia (PIN), as well as markers that can be analyzed by immunohistochemistry, such as those involved in cell proliferation, apoptosis, and angiogenesis. Many of these, which have been under various stages of evaluation as potential prognostic markers in prostate cancer, From the Department of Pathology (AMD), Brady Urological Institute (AMD, MJP, WGN), and Johns Hopkins Oncology Center (WGN), Johns Hopkins Medical Institutions, Baltimore, Maryland, USA This research was funded in part by Grant No. P50CA58236 from the National Institutes of Health/National Cancer Institute Specialized Program in Research Excellence (SPORE) in Prostate Cancer, and by Grant No.1K08CA78588-01 to Dr. De Marzo from the National Institutes of Health/National Cancer Institute Reprint requests: Angelo M. De Marzo, MD, PhD, BuntingBlaustein Cancer Research Building, Room 153, 1650 Orleans Street, Baltimore, MD 21231. E-mail: [email protected] © 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED

are listed in the tables in this article. The development and validation of the next generation of candidate IEBs will rely on a better understanding of prostatic carcinogenesis, which in turn will rely on a deeper understanding of the biology of normal prostate epithelial cells, and how their growth regulation is genetically and epigenetically perturbed during the phases of initiation and progression. In this review, we update our hypothesis of why and how the prostate is targeted for carcinogenesis. Tables I through IV represent a partial listing of various biomarkers of histopathology, as well as those implicated either directly or indirectly in the initiation and/or progression of human prostate carcinogenesis. Table I3– 8 lists germline genetic alterations thought to be either directly or indirectly involved in modifying the risk for the development of prostatic carcinogenesis. Table II9 –34 lists somatic genomic alterations, as this type of heritable change is the likely initiating event in sporadic prostate carcinogenesis. Table III35– 44 lists serum markers for assessing prostate cancer risk and disease monitoring. Table IV45–133 lists the key histopathological features of various benign or proposed cancer precursor lesions of the prostate as well as primary carcinoma. Next, Table IV lists se0090-4295/01/$20.00 PII S0090-4295(00)00952-3 103

TABLE I. Germline alterations proposed as hereditary prostate cancer genes or genetic risk factor genes* Germline Alteration

Selected References

Familial genes HPC-1 X chromosome Risk-modifying gene polymorphisms Vitamin D receptor 3-␤-hydroxysteroid dehydrogenase type II Type II steroid 5-␣-reductase Androgen receptor polymorphisms

3 4 5 6 7 8

HPC ⫽ hereditary prostate cancer. * Essentially all of these genetic associations are still emerging.

TABLE II. Selected somatic genomic alterations and prostate carcinogenesis Somatic Alteration Type LOH 8p 6q 7q 9p and p16 10q 12p 13q 16q 17p 18q pTEN 2q, 5q, 6q, 15q, 11p, 1q, 3q, and 2p Chomosomal gain/amplification 8q gain 7 gain Xq gain 11p, 1q, 3q, 2p C-myc amplification AR amplification PSCA Her-2/Neu Cyclin D1 Ploidy CpG island methylation GSTP1 p16INK4a Endothelin receptor b Androgen receptor E-cadherin CD44

lected biomarkers, categorized by function, that show alterations or altered levels of expression in prostate cancer. Many of these markers have been covered in prior reviews50,115,134 –136 or are presented in other articles in this supplement. For those biomarkers present, there are many “holes in 104

Selected References 9 10 11,12 13 14 15 16 17 15 18 19 20 17 21 17,22 23 24 17 22,25 24 22 26 27 28 29 30 31 32 33 34

the grid,” reflecting our lack of knowledge. Although this list appears long, in fact it represents only the tip of the iceberg. The human genome project, in combination with newly emerging methods of transcriptome and proteome analysis, is already creating an exponential increase in the numUROLOGY 57 (Supplement 4A), April 2001

TABLE III. Serum markers and prostate carcinogenesis Selected References Serum markers PSA HK-2 Endothelin-1 PSAP VEGF (plasma) IGF-1 IGFBP-3 Lycopene Soy isoflavonoids Selenium Circulating tumor cells

35 36 31 37 38 39 40 41 42 43 44

HK ⫽ human kallikrein; IGF ⫽ insulinlike growth factor; IGFBP ⫽ IGF binding protein; PSA ⫽ prostate-specific antigen; PSAP ⫽ prostate-specific acid phosphatase; VEGF ⫽ vascular endothelial growth factor.

ber of genes and expressed sequenced tags (ESTs) the alterations of which have been examined in various lesions of the prostate. For instance, as of July 1999, using cDNA libraries in the prostate expression database,137 there were 719 unique unknown genes expressed in normal prostate, 111 in PIN, and 202 in carcinoma (data available at http://chroma. mbt.washington.edu/PEDB/). In addition, new methods of chemical analysis, such as improvements in liquid chromatography/mass spectrometry instrumentation and software,138 will allow the use of tissue samples obtained from patients to assay for biomarkers that reflect metabolic alterations, such as the products of oxidative damage to DNA bases. To uncover the significance of these gene expression and metabolic perturbations, they must be examined in the context of normal prostate cells and of the specific cell types involved in neoplastic transformation of the prostatic epithelium. INITIATION OF PROSTATE CARCINOGENESIS Currently, the precursor to many peripheral zone prostatic carcinomas is believed to be high-grade prostatic intraepithelial neoplasia (HGPIN).139,140 This is based in part on the following: (1) an increased frequency of HGPIN in prostates that contain adenocarcinoma (CaP) as compared with those without CaP; (2) spatial colocalization of HGPIN and CaP in the different prostate zones; (3) frequent morphological transitions occurring between HGPIN and CaP; and (4) shared phenotypic and molecular genetic alterations between HGPIN and CaP. It is believed that HGPIN arises from low-grade PIN, which in turn is thought to arise within normal prostate epithelium. The cell type of origin of UROLOGY 57 (Supplement 4A), April 2001

HGPIN, however, remains elusive. For instance, does HGPIN always arise from low-grade PIN? Does “normal” epithelium give rise to PIN, or might another intermediate lesion be involved? Finally, do all clinically significant prostatic CaPs arise from HGPIN lesions or, again, might another intermediate be involved? A widely held view of carcinogenesis indicates that the common carcinomas generally arise in renewal tissues, stem cells of which have acquired somatic mutations in growth regulatory genes.141 In normal human prostate epithelium, most cell division takes place in the basal cell compartment,51,53 where the tissue stem cells are thought to reside. The luminal secretory cells, which are thought to be derived from differentiation of basal cells, are the mature cells that perform the androgen-regulated differentiated functions of the prostate, such as prostate-specific antigen (PSA) production and secretion. Both CaP and HGPIN cells possess many phenotypic and morphological features of secretory cells, yet they also contain features of the stem cell compartment such as DNA replication competence, immortality, and telomerase expression.100,101 Thus, we have argued that in carcinoma these “stem cell–like” features have been shifted up into the secretory compartment.142 This cell shift in proliferation, or “topographic infidelity of proliferation,”142 that occurs in HGPIN51,53 is present in many other precursor lesions, including those of the large bowel and uterine cervix. Based on this, as well as patterns of cytokeratin expression,143 it has been postulated that an intermediate, or transiently proliferating, prostatic epithelial cell, with gene expression and morphologic features of both basal and secretory cells, is the target of neoplastic transformation.142,143 To determine the precise cell of origin 105

TABLE IV. Selected histologic and gene expression biomarkers in prostate carcinogenesis*

Histopathology Nuclear enlargement Nuclear hyperchromasia Nucleolar enlargement Cell crowding Nuclear pleomorphism Cytoplasmic hyperchromasia Nuclear morphometric/chromatin texture alterations MAC Gene expression Proliferation markers Ki-67 PCNA Cyclin-dependent kinase inhibitors p27Kip1 p16INK4a p21 Tumor suppressor gene products pRB p53 pTEN Apoptosis TUNEL/apoptotic bodies bcl-2 Angiogenesis Microvessels (CD31, CD34) Hif-1␣ Adhesion/cell surface molecules E-cadherin P-cadherin CD44H/S CD44v6 PSCA* PSMA ␣-Catenin C-CAM Ep-CAM Heat shock proteins HSP27 Metastasis suppressor genes KA1 Nm23 Immortalization Telomerase activity Growth factors FGF (␣, ␤, 8) TGF-␣ KGF TGF-␤1 Bone morphogenetic protein–6 Growth factor receptors EGF-R P185 c-erbB2/HER2-neu P180 c-erbB-3 c-met Prolactin receptor Growth factor binding proteins IGFBP-2 IGFBP-3

106

Selected References

Normal

PIA

HGPIN†

CaP

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹‡

⫺/⫹ ⫺/⫹ ⫺/⫹ ⫺/⫹ ⫺/⫹ ⫺/⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹/⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹/⫹⫹⫹ ⫹⫹⫹

⫹§ ⫹§

⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹㛳 ⫹⫹⫹㛳

⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹㛳 ⫺ ⫺/⫹㛳

⫹⫹⫹

⫹⫹ (V)

⫹⫹ (V) ⫹ ⫹

52,56–58 59,60 61,62

⫹⫹ ⫹/⫺ ⫹⫹⫹

⫹ (V) ⫹⫹ (M) ⫹ (V)

63–66 50,66–68 69

⫹⫹ ⫺/⫹

⫹⫹ ⫹/⫺ (M)

52,70 50,52,71,72

⫹ ⫹/⫺

⫹⫹⫹ ⫹/⫺ (V)

73–76 77

⫹⫹ (V) ⫺ ⫹⫹ (V) ⫹/⫺ ⫹⫹ ⫹⫹⫹ ⫹ (V) ⫺ ⫹⫹⫹

78–83 84,85 86,87 86 22,88 89–92 81,93,94 95 96

⫹⫹ (M)

97

45

⫹⫹ ⫺/⫹§ ⫹⫹⫹ ⫹/⫺ ⫹⫹⫹§ NA ⫺/⫹ ⫹⫹⫹ ⫹⫹⫹ (B) ⫹⫹⫹ ⫹⫹§ ⫹⫹㛳 ⫹⫹ (S) ⫹⫹⫹ ⫹⫹§ ⫹⫹㛳

⫺ ⫹⫹⫹ (BS)

⫹/⫺ ⫹⫹ (V) ⫹/⫺ ⫹⫹ ⫹ ⫹⫹⫹

⫺

46–48 49

50–52 52–55

⫹⫹ ⫹⫹§

⫹⫹

⫺/⫹ ⫹⫹

98 99

⫺

⫹ (V)

⫹⫹⫹

100,101

⫹ (B) ⫺ ⫺ ⫺/⫹ (B) ⫹

⫹⫹ ⫹⫹⫹ ⫺/⫹

⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹

102,103 104 105 106,107 108–111

⫹⫹§ ⫹⫹⫹§ ⫹⫹§ ⫹⫹ (B) ⫹⫹⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ (I⫹)

⫹⫹ (V) ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

⫺ ⫹⫹⫹ (BS)

⫹⫹⫹ (I⫺)

⫹⫹ (M) ⫹⫹⫹ (I⫺)

55,112 26,55,113,114 113,115 55,116 117 97,118,119 120

UROLOGY 57 (Supplement 4A), April 2001

TABLE IV. Continued

Nuclear receptors Androgen receptor Estrogen receptor–␣ Carcinogen detoxification GSTP1 Intermediate filaments Cytokeratins 5,14 Arachidonic acid metabolizing enzymes 15-LOX-2 Cyclooxygenases COX-2 Metabolic genes Fatty acid synthetase Apolipoprotein D Other AIPC

Normal

PIA

HGPIN†

CaP

Selected References

⫹⫹⫹㛳 ⫹ (B)

⫹⫹㛳

⫹⫹⫹ ⫺/⫹

⫹⫹⫹ ⫹

52,115,121–123 122,124,125

⫹⫹⫹ (BS)

⫺

⫺

29,52,126

⫺

⫺

52,127

⫹⫹⫹§ ⫹⫹⫹ (B)

⫹

⫹⫹⫹ (S)

⫹⫹ (V)

128

⫹

⫹⫹

129

⫺/⫹ ⫺/⫹ (S) ⫹⫹⫹ (B)

⫹⫹

⫹⫹

⫹⫹⫹

⫹⫹⫹ ⫹⫹⫹ (V) ⫹⫹⫹

130,131 132 133

KEY: AIPC ⫽ activated in prostate cancer; B ⫽ basal specific; BS ⫽ equal in basal and secretory cells; CaP ⫽ adenocarcinoma; CAM ⫽ cellular adhesion molecule; COX ⫽ cyclooxygenase; EGF ⫽ epidermal growth factor; FGF ⫽ fibroblast growth factor; GSTP1 ⫽ glutathione-S-transferase Pi; HGPIN ⫽ high-grade prostatic intraepithelial neoplasia; I⫹ ⫽ marked increase in intensity of staining compared with normal; I⫺ ⫽ marked decrease in intensity of staining compared with normal; IGFBP ⫽ insulinlike growth factor binding protein; KGF ⫽ keratinocyte growth factor; M ⫽ markedly elevated in hormone-refractory prostate cancers; PCNA ⫽ proliferating cell nuclear antigen; PIA ⫽ proliferative inflammatory atrophy; PSCA ⫽ prostate stem cell antigen; PSMA ⫽ prostate-specific membrane antigen; S ⫽ secretory specific; TGF ⫽ transforming growth factor; TUNEL ⫽ terminal deoxynucleotidyl transferase (TdT)–mediated dUTP in situ nick end-labeling; V ⫽ significant case-to-case variability with some cases positive and some negative; ⫺/⫹ ⫽ positive in few cells only; ⫹/⫺ ⫽ positive in minority of cells; ⫹ ⫽ positive in many cells; ⫹⫹ ⫽ positive in most cells; ⫹⫹⫹ ⫽ positive in the vast majority of cells. * Expression data are based on studies with human tissues almost exclusively at the protein level (or enzymatic level for telomerase) using immunohistochemistry. Markers are related to their expression in normal-appearing epithelium (stromal staining is excluded). Due to space constraints, only selected markers are included and many references could not be included. Relative expression levels are noted, but there are often discrepancies in terms of the percentage of positive cases as well as the percentage of cells staining. Blank spaces indicate that no information is currently available, or could not be found using MEDLINE search. See original reference articles for more quantitative information. † For HGPIN, data are for secretory cells only. ‡ Changes detected in normal-appearing epithelium in prostates with carcinoma versus prostates without carcinoma. § Basal greater than secretory. 㛳 Secretory greater than basal.

for prostatic carcinoma, we would like to decipher at what point the topographic fidelity of proliferation is altered during the course of neoplastic transformation, and whether there is an expansion of intermediate target cells in the preneoplastic phase of carcinogenesis. Even though we have some knowledge about a potential precursor lesion, HGPIN, we still do not know why prostatic epithelium is targeted for carcinogenesis. In many organ systems, including the liver, stomach, and large bowel, long-standing chronic inflammation has been linked to the development of carcinoma.144 –146 Carcinogenesis is thought to result in this setting from recurrent bouts of tissue damage and regeneration in the presence of highly reactive oxygen and nitrogen species. These reactive molecules, such as H202 and nitric oxide (NO), are released from the inflammatory cells and can interact with DNA in the proliferating epithelium to produce permanent genomic alterations.145 This inflammation– carcinoma sequence has been invoked as a potential mechanism of prostatic carcinogenesis.121,147–149 Interestingly, focal prostatic glandular atrophy, UROLOGY 57 (Supplement 4A), April 2001

which has been put forth previously as a precursor of prostatic CaP,150,151 occurs often in close association with chronic inflammation.152–154 In addition, prostatic inflammation may be influenced by dietary practices, as soy products have been shown to inhibit the development of prostatic inflammation in rats.155 Finally, it is possible that the potential efficacy of antioxidant treatments, such as vitamin E (␣-tocopherol) in smokers156 and selenium,43 may prevent prostate cancer by decreasing oxidative genome damage mediated by inflammatory cells. We have recently presented the hypothesis that focal atrophy of the prostate may indeed be a precursor to CaP, but that it may often progress via an intermediate transition into HGPIN.52 We based this hypothesis on the following lines of evidence52: (1) Focal atrophy, for which we have proposed the term “proliferative inflammatory atrophy” (PIA), is often associated with chronic and acute inflammation. (2) As compared with normalappearing epithelium, PIA is highly proliferative. (3) As with HGPIN and carcinoma, PIA occurs in the peripheral zone frequently but less often in the 107

FIGURE 1. New model for development of high-grade PIN and early adenocarcinoma from PIA. Mitotic figures indicated to show increased cell proliferation. Clear cells lack GSTP1 protein expression. The sequence of events of the genomic changes (GSTP1 methylation, 8p loss and 8q gain) remain unknown. In normal epithelium, the cell division takes place predominantly in the basal cell compartment. By contrast, most cell division shifts up into the secretory compartment in PIA, and PIN. Although depicted primarily in the initial phase, ongoing oxidative stress is likely occurring throughout all phases of carcinogenesis.

central zone. (4) Inflammation is extremely rare to nonexistent in the seminal vesicles, which have a relative risk of 10-6 or less of developing carcinoma compared with the adjacent prostate gland.121 (5) PIA contains many proliferating cells in the luminal layer, which is similar to PIN. (6) Many of the luminal cells in PIA have decreased expression of the cyclin-dependent kinase inhibitor, p27Kip1, which has been implicated in the initiation and progression of prostatic cancer. (7) PIA contains many cells with phenotypic features of “intermediate cells,” which have been proposed as the target cells for carcinogenesis in the prostate. (8) PIA contains very few cells undergoing apoptosis, with many cells in the luminal layer expressing bcl-2. (9) PIA shows increased expression of the carcinogen-detoxifying enzyme, glutathione-S-transferase Pi (GSTP1), in many of the cells, consistent with a stress response to an increased oxidative burden. (10) Finally, PIA shows frequent morphologic transitions to PIN.52,157 Based on these findings, we propose a new model (Figure 1) of prostate carcinogenesis whereby chronic and acute inflammation, in conjunction with dietary and other environmental factors, target prostate epithelial cells for injury and destruction. Increased proliferation occurs as a regenerative response to lost epithelial cells. The increased proliferation may be related mechanistically to decreased p27Kip1. Furthermore, and consistent with a regenerative response, epithelial apoptosis is quite low in PIA, perhaps mechanistically related to increased 108

bcl-2. This results in an expansion of intermediate cells that are in various stages of differentiation between basal and secretory cells. In this process, GSPT1 expression is elevated in many of the cells in PIA as a genome protective measure. We also hypothesize that, although elevated in many of the cells in PIA, GSTP1 expression is lost in some cells as the result of aberrant methylation of the CpG island of the GSTP1 gene promoter. This alteration would place these cells at increased risk for the accumulation of additional genetic damage, with acceleration of the neoplastic process toward PIN and carcinoma. No convincing evidence has shown PIA to be a precursor to HGPIN and/or carcinoma. Much work needs to be done to further test this hypothesis. To facilitate these studies, and to determine whether PIA might be useful as a potential IEB that is modified by dietary or drug treatments, there must be studies showing that there is reasonable interobserver agreement regarding the diagnosis of PIA. We are currently developing criteria for recognizing PIA in pathologic samples. PIA is to be distinguished from diffuse hormonal atrophy of the prostate, which is related to androgen withdrawal. In contrast to PIA, diffuse hormonal atrophy occurs relatively uniformly throughout the prostate and shows elevated levels of apoptosis. PIA represents a spectrum of more focal atrophic lesions, not related to androgen withdrawal, and not showing elevated levels of apoptosis.52,158 PIA encompasses the lesions previUROLOGY 57 (Supplement 4A), April 2001

TABLE V. Other potential new biomarkers* ● New/other markers — Six-transmembrane epithelial antigen of the prostate — Caveolin-1 — DD3 — Tissue transglutaminase — HK2 — Prostate-derived Ets factor ● Developmental regulators — Nkx3.1 — Hox ● Early growth response genes ● Nuclear matrix proteins — D-1, D-2, D-3 — AM-1, AM-2, PC-1 ● Markers of oxidative injury — 8-hydroxydeoxyguanosine — Thymidine glycol (Urinary) — Malondialdehyde — 5-hydroxy-2⬘-deoxycytidine * Space constraints do not permit references. See supplemental materials on the world wide web.

ously referred to by Franks as sclerotic atrophy, and postatrophic hyperplasia, the latter being divided into lobular hyperplasia and sclerotic atrophy with hyperplasia.150 McNeal referred to these lesions as postinflammatory atrophy.154 More recently, Ruska et al. have divided most focally atrophic epithelial lesions into two types referred to as simple atrophy and postatrophic hyperplasia.158 Although not strictly required, PIA is usually accompanied by chronic inflammation and at times by acute inflammation as well. In addition, there may be epithelial disruption, occurring frequently adjacent to corpora amylacea. We have characterized the inflammatory infiltrate in terms of mononuclear cells, including lymphocytes and macrophages. There is a spectrum of inflammatory cells, but in the typical cases there are variable numbers of lymphocytes and macrophages in the lumen as well as within the epithelial and the smooth muscle/stromal compartments. The inflammatory infiltrate is predominantly T-lymphocytes and macrophages with variable numbers of B-cells. Our more recent work suggests that the extent of epithelial disruption in PIA lesions that contain moderate to marked chronic inflammation may be related to the expression of inducible nitric oxide synthetase (iNOS) in macrophages.159 FUTURE DIRECTIONS Biomarker validation is similar to drug development, with many initial promising candidates that are proven ineffective; final acceptance is very rare. UROLOGY 57 (Supplement 4A), April 2001

So far, for prostate cancer prognostication there are few biomarkers in routine clinical practice. This fact may be attributable to our historic inability to discover potential new targets at a rapid rate and, more important, to a lack of standardization in the measurement and validation of targets that are identified. In addition, many markers are found to correlate with other known prognostic markers, such as Gleason score, preoperative PSA, and pathologic stage, which are easily obtained and routinely collected variables. In such cases, the new markers often do not add to the predictive power of the standard markers in multivariate analyses. How these markers might function in the setting of chemoprevention trials, however, is essentially untested. In our view the near future is bright. In addition to the continued practice of using time-honored approaches of clinical observations, epidemiology, and histopathologic assessment to generate new potential insights into the pathogenesis of this disease, we expect that the emerging high-throughput technologies for studying gene expression will greatly accelerate new prognostic target discoveries. Validation of these new potential biomarkers will be greatly facilitated by the ability to probe their expression using either antibodies or in situ hybridization on tissue microarrays.97,160 Because these tissue microarrays allow simultaneous assessment of hundreds of patient samples on a single slide, they are poised to become invaluable tools for analysis and validation of new biomarkers (Table V) . 109

REFERENCES 1. Crawford ED, Fair WR, Kelloff GJ, et al: Chemoprevention of prostate cancer: guidelines for possible intervention strategies. J Cell Biochem Suppl 16H: 140 –145, 1992. 2. Kelloff GJ, Lieberman R, Steele VE, et al: Chemoprevention of prostate cancer: concepts and strategies. Eur Urol 35: 342–350, 1999. 3. Smith JR, Freije D, Carpten JD, et al: Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search. Science 274: 1371–1374, 1996. 4. Xu J, Meyers D, Freije D, et al: Evidence for a prostate cancer susceptibility locus on the X chromosome. Nat Genet 20: 175–179, 1998. 5. Taylor JA, Hirvonen A, Watson M, et al: Association of prostate cancer with vitamin D receptor gene polymorphism. Cancer Res 56: 4108 – 4110, 1996. 6. Elo JP, Akinola LA, Poutanen M, et al: Characterization of 17-beta-hydroxysteroid dehydrogenase isoenzyme expression in benign and malignant human prostate. Int J Cancer 66: 37– 41, 1996. 7. Reichardt JK, Makridakis N, Henderson BE, et al: Genetic variability of the human SRD5A2 gene: implications for prostate cancer risk. Cancer Res 55: 3973–3975, 1995. 8. Kantoff P, Giovannucci E, and Brown M: The androgen receptor CAG repeat polymorphism and its relationship to prostate cancer. Biochim Biophys Acta 1378: C1–C5, 1998. 9. Isaacs WB, Bova GS, Morton RA, et al: Genetic alterations in prostate cancer. Cold Spring Harb Symp Quant Biol 59: 653– 659, 1994. 10. Suzuki H, Emi M, Komiya A, et al: Localization of a tumor suppressor gene associated with progression of human prostate cancer within a 1.2 Mb region of 8p22–p21.3. Genes Chromosomes Cancer 13: 168 –174, 1995. 11. Cooney KA, Wetzel JC, Consolino CM, et al: Identification and characterization of proximal 6q deletions in prostate cancer. Cancer Res 56: 4150 – 4153, 1996. 12. Srikantan V, Sesterhenn IA, Davis L, et al: Allelic loss on chromosome 6Q in primary prostate cancer. Int J Cancer 84: 331–335, 1999. 13. Oakahashi S, Shan AL, Ritland SR, et al: Frequent loss of heterozygosity at 7q31.1 in primary prostate cancer is associated with tumor aggressiveness and progression. Cancer Res 55: 4114 – 4119, 1995. 14. Komiya A, Suzuki H, Aida S, et al: Mutational analysis of CDKN2 (CDK4I/MTS1) gene in tissues and cell lines of human prostate cancer. Jpn J Cancer Res 86: 622– 625, 1995. 15. Carter BS, Ewing CM, Ward WS, et al: Allelic loss of chromosomes 16q and 10q in human prostate cancer. Proc Natl Acad Sci USA 87: 8751– 8755, 1990. 16. Kibel AS, Schutte M, Kern SE, et al: Identification of 12p as a region of frequent deletion in advanced prostate cancer. Cancer Res 58: 5652–5655, 1998. 17. 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 56: 3091–3102, 1996. 18. Bova GS, and Isaacs WB: Review of allelic loss and gain in prostate cancer. World J Urol 14: 338 –346, 1996. 19. Bostwick DG, Shan A, Qian J, et al: Independent origin of multiple foci of prostatic intraepithelial neoplasia: comparison with matched foci of prostate carcinoma. Cancer 83: 1995–2002, 1998. 20. Whang YE, Wu X, Suzuki H, et al: Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. Proc Natl Acad Sci USA 95: 5246 –5250, 1998. 21. Qian J, Jenkins RB, and Bostwick DG: Chromosomal 110

anomalies in atypical adenomatous hyperplasia and carcinoma of the prostate using fluorescence in situ hybridization. Urology 46: 837– 842, 1995. 22. 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 27: 95– 103, 2000. 23. Visakorpi T: Molecular genetics of prostate cancer. Ann Chir Gynaecol 88: 11–16, 1999. 24. Visakorpi T, Hyytinen E, Koivisto P, et al: In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 9: 401– 406, 1995. 25. Jenkins RB, Qian J, Lieber MM, et al: Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization. Cancer Res 57: 524 –531, 1997. 26. Zhau HE, Wan DS, Zhou J, et al: Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines. Mol Carcinog 5: 320 –327, 1992. 27. Bubendorf L, Kononen J, Koivisto P, et al: Survey of gene amplifications during prostate cancer progression by high-throughput fluorescence in situ hybridization on tissue microarrays [published erratum appears in Cancer Res 59: 1388, 1999]. Cancer Res 59: 803– 806, 1999. 28. Peters-Gee JM: An update on the role of ploidy in prostate carcinoma. Henry Ford Hosp Med J 40: 99 –102, 1992. 29. Lee WH, Morton RA, Epstein JI, et al: Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA 91: 11733–11737, 1994. 30. Jarrard DF, Bova GS, Ewing CM, et al: Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer. Genes Chromosomes Cancer 19: 90 –96, 1997. 31. Pirtskhalaishvili G, and Nelson JB: Endothelium-derived factors as paracrine mediators of prostate cancer progression. Prostate 44: 77– 87, 2000. 32. Jarrard DF, Kinoshita H, Shi Y, et al: Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells. Cancer Res 58: 5310 –5314, 1998. 33. Graff JR, Herman JG, Lapidus RG, et al: E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 55: 5195–5199, 1995. 34. Lou W, Krill D, Dhir R, et al: Methylation of the CD44 metastasis suppressor gene in human prostate cancer. Cancer Res 59: 2329 –2331, 1999. 35. Carter HB, and Pearson JD: Prostate-specific antigen testing for early diagnosis of prostate cancer: formulation of guidelines. Urology 54: 780 –786, 1999. 36. Partin AW, Catalona WJ, Finlay JA, et al: Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis. Urology 54: 839 – 845, 1999. 37. Lowe FC, and Trauzzi SJ: Prostatic acid phosphatase in 1993. Its limited clinical utility. Urol Clin North Am 20: 589 – 595, 1993. 38. Duque JL, Loughlin KR, Adam RM, et al: Plasma levels of vascular endothelial growth factor are increased in patients with metastatic prostate cancer. Urology 54: 523–527, 1999. 39. Pollak M, Beamer W, and Zhang JC: Insulin-like growth factors and prostate cancer. Cancer Metast Rev 17: 383–390, 1998. 40. Giovannucci E: Insulin-like growth factor-i and binding protein-3 and risk of cancer. Horm Res 51(suppl S3): 34 – 41, 1999. 41. Giovannucci E: Tomatoes, tomato-based products, lyUROLOGY 57 (Supplement 4A), April 2001

copene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst 91: 317–331, 1999. 42. Jacobsen BK, Knutsen SF, and Fraser GE: Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States). Cancer Causes Control 9: 553– 557, 1998. 43. Nelson MA, Porterfield BW, Jacobs ET, et al: Selenium and prostate cancer prevention. Semin Urol Oncol 17: 91–96, 1999. 44. Ts’o PO, Pannek J, Wang ZP, et al: Detection of intact prostate cancer cells in the blood of men with prostate cancer. Urology 49: 881– 885, 1997. 45. Epstein JI: Pathology of prostatic intraepithelial neoplasia and adenocarcinoma of the prostate: prognostic influences of stage, tumor volume, grade, and margins of resection. Semin Oncol 21: 527–541, 1994. 46. Mohler JL, Partin AW, Epstein JI, et al: Prediction of prognosis in untreated stage A2 prostatic carcinoma. Cancer 69: 511–519, 1992. 47. Boone CW, and Kelloff GJ: Biomarker end-points in cancer chemoprevention trials. IARC Sci Publ 142: 273–280, 1997. 48. Lopez-Beltran A, Artacho-Perula E, Roldan-Villalobos R, et al: Nuclear volume estimates in prostatic intraepithelial neoplasia. Anal Quant Cytol Histol 22: 37– 44, 2000. 49. Bartels PH, Montironi R, Hamilton PW, et al: Nuclear chromatin texture in prostatic lesions. II. PIN and malignancy associated changes. Anal Quant Cytol Histol 20: 397– 406, 1998. 50. Moul JW: Angiogenesis, p53, bcl-2 and Ki-67 in the progression of prostate cancer after radical prostatectomy. Eur Urol 35: 399 – 407, 1999. 51. Bonkhoff H, Stein U, and Remberger K: The proliferative function of basal cells in the normal and hyperplastic human prostate. Prostate 24: 114 –118, 1994. 52. De Marzo AM, Marchi VL, Epstein JI, et al: Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am J Pathol 155: 1985–1992, 1999. 53. McNeal JE, Haillot O, and Yemoto C: Cell proliferation in dysplasia of the prostate: analysis by PCNA immunostaining. Prostate 27: 258 –268, 1995. 54. Visakorpi T: Proliferative activity determined by DNA flow cytometry and proliferating cell nuclear antigen (PCNA) immunohistochemistry as a prognostic factor in prostatic carcinoma. J Pathol 168: 7–13, 1992. 55. Myers RB, and Grizzle WE: Changes in biomarker expression in the development of prostatic adenocarcinoma. Biotech Histochem 72: 86 –95, 1997. 56. Guo YP, Sklar GN, Borkowski A, et al: Loss of the cyclin-dependent kinase inhibitor P27(Kip1) protein in human prostate cancer correlates with tumor grade. Clin Cancer Res 3: 2269 –2274, 1997. 57. De Marzo AM, Meeker AM, Epstein JI, et al: Prostate stem cell compartments: expression of p27Kip1 in normal, hyperplastic and cancer cells. Am J Pathol 153: 911–919, 1998. 58. Tsihlias J, Kapusta LR, DeBoer G, et al: Loss of cyclindependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma. Cancer Res 58: 542–548, 1998. 59. Lee CT, Capodieci P, Osman I, et al: Overexpression of the cyclin-dependent kinase inhibitor p16 is associated with tumor recurrence in human prostate cancer. Clin Cancer Res 5: 977–983, 1999. 60. Halvorsen OJ, Hostmark J, Haukaas HS, et al: Prognostic significance of p16 and CDK4 proteins in localized prostate carcinoma. Cancer 88: 416 – 424, 2000. UROLOGY 57 (Supplement 4A), April 2001

61. Matsushima H, Sasaki T, Goto T, et al: Immunohistochemical study of p21WAF1 and p53 proteins in prostatic cancer and their prognostic significance. Hum Pathol 29: 778 –783, 1998. 62. Sarkar FH, Li Y, Sakr WA, et al: Relationship of p21(WAF1) expression with disease-free survival and biochemical recurrence in prostate adenocarcinomas (PCa). Prostate 40: 256 –260, 1999. 63. Phillips SM, Barton CM, Lee SJ, et al: Loss of the retinoblastoma susceptibility gene (RB1) is a frequent and early event in prostatic tumorigenesis. Br J Cancer 70: 1252–1257, 1994. 64. Theodorescu D, Broder SR, Boyd JC, et al: p53, bcl-2 and retinoblastoma proteins as long-term prognostic markers in localized carcinoma of the prostate. J Urol 158: 131–137, 1997. 65. Baldi A, Esposito V, De Luca A, et al: Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer. Clin Cancer Res 3: 1691–1697, 1997. 66. Tamboli P, Amin MB, Xu HJ, et al: Immunohistochemical expression of retinoblastoma and p53 tumor suppressor genes in prostatic intraepithelial neoplasia: comparison with prostatic adenocarcinoma and benign prostate. Mod Pathol 11: 247–252, 1998. 67. Grizzle WE, Myers RB, Arnold MM, et al: Evaluation of biomarkers in breast and prostate cancer. J Cell Biochem Suppl 19: 259 –266, 1994. 68. Brooks JD, Bova GS, Ewing CM, et al: An uncertain role for p53 gene alterations in human prostate cancers. Cancer Res 56: 3814 –3822, 1996. 69. McMenamin ME, Soung P, Perera S, et al: Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res 59: 4291– 4296, 1999. 70. Montironi R, Magi Galluzzi CM, Marina S, et al: Quantitative characterization of the frequency and location of cell proliferation and death in prostate pathology. J Cell Biochem Suppl 19: 238 –245, 1994. 71. McDonnell TJ, Troncoso P, Brisbay SM, et al: Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 52: 6940 – 6944, 1992. 72. Kyprianou N, Tu H, and Jacobs SC: Apoptotic versus proliferative activities in human benign prostatic hyperplasia. Hum Pathol 27: 668 – 675, 1996. 73. Montironi R, Diamanti L, Thompson D, et al: Analysis of the capillary architecture in the precursors of prostate cancer: recent findings and new concepts. Eur Urol 30: 191–200, 1996. 74. Silberman MA, Partin AW, Veltri RW, et al: Tumor angiogenesis correlates with progression after radical prostatectomy but not with pathologic stage in Gleason sum 5 to 7 adenocarcinoma of the prostate. Cancer 79: 772–779, 1996. 75. Bostwick DG, Wheeler TM, Blute M, et al: Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies. Urology 48: 47–57, 1996. 76. Rubin MA, Buyyounouski M, Bagiella E, et al: Microvessel density in prostate cancer: lack of correlation with tumor grade, pathologic stage, and clinical outcome. Urology 53: 542–547, 1999. 77. Zhong H, De Marzo AM, Laughner E, et al: Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases. Cancer Res 59: 5830 –5835, 1999. 78. 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 52: 5104 –5109, 1992. 111

79. Umbas R, Isaacs WB, Bringuier PP, et al: Decreased E-cadherin expression is associated with poor prognosis in patients with prostate cancer. Cancer Res 54: 3929 –3933, 1994. 80. Ross JS, Figge HL, Bui HX, et al: E-cadherin expression in prostatic carcinoma biopsies: correlation with tumor grade, DNA content, pathologic stage, and clinical outcome. Mod Pathol 7: 835– 841, 1994. 81. Umbas R, Isaacs WB, Bringuier PP, et al: Relation between aberrant alpha-catenin expression and loss of E-cadherin function in prostate cancer. Int J Cancer 74: 374 –377, 1997. 82. Bryden AA, Freemont AJ, Clarke NW, et al: Paradoxical expression of E-cadherin in prostatic bone metastases. BJU Int 84: 1032–1034, 1999. 83. De Marzo AM, Knudsen B, Chan-Tack K, et al: E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens. Urology 53: 707–713, 1999. 84. Soler AP, Harner GD, Knudsen KA, et al: Expression of P-cadherin identifies prostate-specific-antigen-negative cells in epithelial tissues of male sexual accessory organs and in prostatic carcinomas—implications for prostate cancer biology. Am J Pathol 151: 471– 478, 1997. 85. Jarrard DF, Paul R, van Bokhoven A, et al: P-Cadherin is a basal cell-specific epithelial marker that is not expressed in prostate cancer. Clin Cancer Res 3: 2121–2128, 1997. 86. De Marzo AM, Bradshaw C, Sauvageot J, et al: CD44 and CD44v6 downregulation in clinical prostatic carcinoma: relation to Gleason grade and cytoarchitecture. Prostate 34: 162–168, 1998. 87. Kallakury BV, Sheehan CE, Ambros RA, et al: Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas. J Clin Oncol 16: 1302–1309, 1998. 88. Reiter RE, Gu Z, Watabe T, et al: Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci USA 95: 1735–1740, 1998. 89. Renneberg H, Wennemuth G, Konrad L, et al: Immunohistochemistry of a prostate membrane specific protein during development and maturation of the human prostate. J Anat 190: 343–349, 1997. 90. Douglas TH, Morgan TO, McLeod DG, et al: Comparison of serum prostate specific membrane antigen, prostate specific antigen, and free prostate specific antigen levels in radical prostatectomy patients. Cancer 80: 107–114, 1997. 91. Sweat SD, Pacelli A, Murphy GP, et al: Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastases. Urology 52: 637– 640, 1998. 92. Chang SS, Reuter VE, Heston WD, et al: Short-term neoadjuvant androgen deprivation therapy does not affect prostate specific membrane antigen expression in prostate tissues. Cancer 88: 407– 415, 2000. 93. Murant SJ, Handley J, Stower M, et al: Co-ordinated changes in expression of cell adhesion molecules in prostate cancer. Eur J Cancer 33: 263–271, 1997. 94. Aaltomaa S, Lipponen P, Ala-Opas M, et al: Alpha-catenin expression has prognostic value in local and locally advanced prostate cancer. Br J Cancer 80: 477– 482, 1999. 95. Kleinerman DI, Troncoso P, Lin SH, et al: Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor. Cancer Res 55: 1215–1220, 1995. 96. Poczatek RB, Myers RB, Manne U, et al: Ep-Cam levels 112

in prostatic adenocarcinoma and prostatic intraepithelial neoplasia. J Urol 162: 1462–1466, 1999. 97. Bubendorf L, Kolmer M, Kononen J, et al: Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays. J Natl Cancer Inst 91: 1758 –1764, 1999. 98. Dong JT, Suzuki H, Pin SS, et al: Down-regulation of the KAI1 metastasis suppressor gene during the progression of human prostatic cancer infrequently involves gene mutation or allelic loss. Cancer Res 56: 4387– 4390, 1996. 99. Myers RB, Srivastava S, Oelschlager DK, et al: Expression of nm23–H1 in prostatic intraepithelial neoplasia and adenocarcinoma. Hum Pathol 27: 1021–1024, 1996. 100. Sommerfeld HJ, Meeker AK, Piatyszek MA, et al: Telomerase activity: a prevalent marker of malignant human prostate tissue. Cancer Res 56: 218 –222, 1996. 101. Koeneman KS, Pan CX, Jin JK, et al: Telomerase activity, telomere length, and DNA ploidy in prostatic intraepithelial neoplasia (PIN). J Urol 160: 1533–1539, 1998. 102. Dorkin TJ, Robinson MC, Marsh C, et al: aFGF immunoreactivity in prostate cancer and its co-localization with bFGF and FGF8. J Pathol 189: 564 –569, 1999. 103. Dorkin TJ, Robinson MC, Marsh C, et al: FGF8 overexpression in prostate cancer is associated with decreased patient survival and persists in androgen independent disease. Oncogene 18: 2755–2761, 1999. 104. Leav I, McNeal JE, Ziar J, et al: The localization of transforming growth factor alpha and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions. Hum Pathol 29: 668 – 675, 1998. 105. Planz B, Aretz HT, Wang Q, et al: Immunolocalization of the keratinocyte growth factor in benign and neoplastic human prostate and its relation to androgen receptor. Prostate 41: 233–242, 1999. 106. Truong LD, Kadmon D, McCune BK, et al: Association of transforming growth factor-beta 1 with prostate cancer: an immunohistochemical study. Hum Pathol 24: 4 –9, 1993. 107. Isaacs WB, Bova GS, Morton RA, et al: Molecular biology of prostate cancer progression. Cancer Surv 23: 19 –32, 1995. 108. Bentley H, Hamdy FC, Hart KA, et al: Expression of bone morphogenetic proteins in human prostatic adenocarcinoma and benign prostatic hyperplasia. Br J Cancer 66: 1159 – 1163, 1992. 109. Barnes J, Anthony CT, Wall N, et al: Bone morphogenetic protein-6 expression in normal and malignant prostate. World J Urol 13: 337–343, 1995. 110. Hamdy FC, Autzen P, Robinson MC, et al: Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue. Cancer Res 57: 4427– 4431, 1997. 111. Autzen P, Robson CN, Bjartell A, et al: Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies. Br J Cancer 78: 1219 –1223, 1998. 112. Myers RB, Kudlow JE, and Grizzle WE: Expression of transforming growth factor-alpha, epidermal growth factor and the epidermal growth factor receptor in adenocarcinoma of the prostate and benign prostatic hyperplasia. Mod Pathol 6: 733–737, 1993. 113. Myers RB, Srivastava S, Oelschlager DK, et al: Expression of p160erbB-3 and p185erbB-2 in prostatic intraepithelial neoplasia and prostatic adenocarcinoma [see comments]. J Natl Cancer Inst 86: 1140 –1145, 1994. 114. Myers RB, Brown D, Oelschlager DK, et al: Elevated serum levels of p105(erbB-2) in patients with advanced-stage prostatic adenocarcinoma. Int J Cancer 69: 398 – 402, 1996. UROLOGY 57 (Supplement 4A), April 2001

115. Myers RB, and Grizzle WE: Biomarker expression in prostatic intraepithelial neoplasia. Eur Urol 30: 153–166, 1996. 116. Pisters LL, Troncoso P, Zhau HE, et al: c-met protooncogene expression in benign and malignant human prostate tissues. J Urol 154: 293–298, 1995. 117. Leav I, Merk FB, Lee KF, et al: Prolactin receptor expression in the developing human prostate and in hyperplastic, dysplastic, and neoplastic lesions. Am J Pathol 154: 863– 870, 1999. 118. Thrasher JB, Tennant MK, Twomey PA, et al: Immunohistochemical localization of insulin-like growth factor binding proteins 2 and 3 in prostate tissue: clinical correlations [see comments]. J Urol 155: 999 –1003, 1996. 119. Ho PJ, and Baxter RC: Insulin-like growth factorbinding protein-2 in patients with prostate carcinoma and benign prostatic hyperplasia. Clin Endocrinol (Oxf) 46: 145– 154, 1997. 120. Hampel OZ, Kattan MW, Yang G, et al: Quantitative immunohistochemical analysis of insulin-like growth factor binding protein-3 in human prostatic adenocarcinoma: a prognostic study. J Urol 159: 2220 –2225, 1998. 121. De Marzo AM, Coffey DS, and Nelson WG: New concepts in tissue specificity for prostate cancer and benign prostatic hyperplasia. Urology 53: 29 –39, 1999. 122. Brolin J, Skoog L, and Ekman P: Immunohistochemistry and biochemistry in detection of androgen, progesterone, and estrogen receptors in benign and malignant human prostatic tissue. Prostate 20: 281–295, 1992. 123. Bonkhoff H, Fixemer T, and Remberger K: Relation between Bcl-2, cell proliferation, and the androgen receptor status in prostate tissue and precursors of prostate cancer. Prostate 34: 251–258, 1998. 124. Hiramatsu M, Maehara I, Orikasa S, et al: Immunolocalization of oestrogen and progesterone receptors in prostatic hyperplasia and carcinoma. Histopathology 28: 163–168, 1996. 125. Bonkhoff H, Fixemer T, Hunsicker I, et al: Estrogen receptor expression in prostate cancer and premalignant prostatic lesions. Am J Pathol 155: 641– 647, 1999. 126. Brooks JD, Weinstein M, Lin X, et al: CG island methylation changes near the GSTP1 gene in prostatic intraepithelial neoplasia. Cancer Epidemiol Biomarkers Prev 7: 531–536, 1998. 127. Brawer MK, Peehl DM, Stamey TA, et al: Keratin immunoreactivity in the benign and neoplastic human prostate. Cancer Res 45: 3663–3667, 1985. 128. Shappell SB, Boeglin WE, Olson SJ, et al: 15-lipoxygenase-2 (15-LOX-2) is expressed in benign prostatic epithelium and reduced in prostate adenocarcinoma. Am J Pathol 155: 235–245, 1999. 129. Gupta S, Srivastava M, Ahmad N, et al: Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate 42: 73–78, 2000. 130. Shurbaji MS, Kuhajda FP, Pasternack GR, et al: Expression of oncogenic antigen 519 (OA-519) in prostate cancer is a potential prognostic indicator. Am J Clin Pathol 97: 686 – 691, 1992. 131. Epstein JI, Carmichael M, and Partin AW: OA-519 (fatty acid synthase) as an independent predictor of pathologic state in adenocarcinoma of the prostate. Urology 45: 81– 86, 1995. 132. Zhang SX, Bentel JM, Ricciardelli C, et al: Immunolocalization of apolipoprotein D, androgen receptor and prostate specific antigen in early stage prostate cancers. J Urol 159: 548 –554, 1998. 133. Chaib H, Rubin MA, Mucci NR, et al: Isolation and characterization of AIPC, a PDZ domain-containing protein UROLOGY 57 (Supplement 4A), April 2001

highly expressed in human primary prostate tumors. Proc Am Assoc Cancer Res 41: 318, 2000. 134. Bostwick DG, Burke HB, Wheeler TM, et al: The most promising surrogate endpoint biomarkers for screening candidate chemopreventive compounds for prostatic adenocarcinoma in short-term phase II clinical trials. J Cell Biochem Suppl 19: 283–289, 1994. 135. Sakr WA, and Grignon DJ: Prostate cancer: indicators of aggressiveness. Eur Urol 32: 15–23, 1997. 136. Bostwick DG: Practical clinical application of predictive factors in prostate cancer. A review with an emphasis on quantitative methods in tissue specimens. Anal Quant Cytol Histol 20: 323–342, 1998. 137. Hawkins V, Doll D, Bumgarner R, et al: PEDB: the prostate expression database. Nucleic Acids Res 27: 204 –208, 1999. 138. Chavez-Eng CM, Constanzer ML, and Matuszewski BK: Picogram determination of a novel dopamine D4 receptor antagonist in human plasma and urine by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 691: 77– 85, 1997. 139. McNeal JE, and Bostwick DG: Intraductal dysplasia: a premalignant lesion of the prostate. Hum Pathol 17: 64 –71, 1986. 140. Bostwick DG: Prospective origins of prostate carcinoma. Prostatic intraepithelial neoplasia and atypical adenomatous hyperplasia. Cancer 78: 330 –336, 1996. 141. Knudson AG: Stem cell regulation, tissue ontogeny, and oncogenic events. Semin Cancer Biol 3: 99 –106, 1992. 142. De Marzo AM, Nelson WG, Meeker AM, et al: Stem cell features of benign and malignant prostate epithelial cells. J Urol 160: 2381–2392, 1998. 143. Verhagen AP, Ramaekers FC, Aalders TW, et al: Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 52: 6182– 6187, 1992. 144. Ames BN: Mutagenesis and carcinogenesis: endogenous and exogenous factors. Environ Mol Mutagen 14: 66 –77, 1989. 145. Weitzman SA, and Gordon LI: Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis. Blood 76: 655– 663, 1990. 146. Bartsch H, and Frank N: Blocking the endogenous formation of N-nitroso compounds and related carcinogens. IARC Sci Publ 139: 189 –201, 1996. 147. Smith CJ, and Gardner WA Jr: Inflammation-proliferation: possible relationships in the prostate. Prog Clin Biol Res 239: 317–325, 1987. 148. Gardner WA, and Bennett BD: The prostate-overview: recent insights and speculations, in Weinstein RS, and Gardner WA (Eds): Pathology and Pathobiology of the Urinary Bladder and Prostate. Baltimore, Williams & Wilkins, 1992, pp 129 –148. 149. Platz EA: Prostatitis and prostate cancer. New Dev Prostate Cancer Treatment 3: 71–73, 1998. 150. Franks LM: Atrophy and hyperplasia in the prostate proper. J Pathol Bacteriol 68: 617– 621, 1954. 151. Liavag I: Atrophy and regeneration in the pathogenesis of prostatic carcinoma. Acta Pathol Microbiol Scand 73: 338 –350, 1968. 152. McNeal JE: Normal histology of the prostate. Am J Surg Pathol 12: 619 – 633, 1988. 153. Bennett BD, Richardson PH, and Gardner WA: Histopathology and cytology of prostatitis, in Lepor H, and Lawson RK (Eds): Prostate Diseases. Philadelphia, W.B. Saunders Company, 1993, pp 399 – 414. 113

154. McNeal JE: Prostate, in Sternberg SS (Ed): Histology for Pathologists. Philadelphia, Lippincott-Raven, 1997, pp 997–1017. 155. Sharma OP, Adlercreutz H, Strandberg JD, et al: Soy of dietary source plays a preventive role against the pathogenesis of prostatitis in rats. J Steroid Biochem Mol Biol 43: 557–564, 1992. 156. Smigel K: Vitamin E reduces prostate cancer rates in Finnish trial: U.S. considers follow-up. J Natl Cancer Inst 90: 416 – 417, 1998. 157. Putzi MJ, and De Marzo AM: Morphological transitions between proliferative inflammatory atrophy (PIA) and

114

high grade prostatic intraepithelial neoplasia (PIN). Urology 56: 828 – 832, 2000. 158. Ruska KM, Sauvageot J, and Epstein JI: Histology and cellular kinetics of prostatic atrophy. Am J Surg Pathol 22: 1073–1077, 1998. 159. Gage W, Marchi VL, Nelson WG, et al: Modes of tissue injury in proliferative inflammatory atrophy (pia) of the prostate: macrophages and inducible nitric oxide synthetase (iNOS). J Urol 163(suppl): 24, 2000. 160. Kononen J, Bubendorf L, Kallioniemi A, et al: Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4: 844 – 847, 1998.

UROLOGY 57 (Supplement 4A), April 2001