Comparative gene and protein expression in primary cultures of epithelial cells from benign prostatic hyperplasia and prostate cancer.

Primary cultures are widely used to investigate the disease-specific biology of prostate cancer and benign prostatic hyperplasia (BPH). To identify genes differentially expressed between epithelial cells cultured from adenocarcinomas versus BPH tissues, we used probe array technology. Gene expression profiles were evaluated on Affymetrix Human Cancer G110 Array Chips containing approximately 1900 cancer-related genes. After defined statistical analysis, genes that were over-expressed in cancer cultures were identified. Protein expression of four of the differentially expressed genes was measured in immunoblots, and the expression of two other genes was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). While no gene or protein was consistently over-expressed in all cancer versus BPH cell cultures, cytokeratin 16 protein was highly elevated in several of the cancer cultures, suggesting that a hyperproliferative phenotype may be characteristic of prostate cancer cells.

The prostate specific antigen era in the United States is over for prostate cancer: what happened in the last 20 years?

PURPOSE: We assessed how well preoperative serum prostate specific antigen (PSA) reflects the largest cancer in consecutive untreated radical prostatectomies during the last 20 years at Stanford University. MATERIALS AND METHODS: A total of 1,317 consecutive radical prostatectomies were divided into 4, 5-year periods between August 1983 and July 2003, and examined sequentially in 3 mm step sections by 1 pathologist. The largest cancer and 5 other histological variables in each prostate were measured. Preoperative clinical stages were tabulated for each 5-year period. Means, Pearson correlation coefficients, % change and multiple regression were used to compare selected variables. RESULTS: Most parameters decreased linearly during the 20 years, including palpable nodules on digital rectal examination from 91% to 17%, mean age from 64 to 59 years, mean serum PSA from 25 to 8 ng/ml, and index (largest) cancer volume from 5.3 to 2.4 cc. Percent Gleason grade 4/5 of the largest cancer averaged 27% to 35% and prostate weight 44 to 53 gm. Contrasting August 1983 to December 1988 with January 1999 to July 2003, 6 histological cancer parameters had statistically significant relationships to serum PSA in the first period. In the last 5 years serum PSA was related only to prostate size. CONCLUSIONS: Serum PSA was related to prostate cancer 20 years ago. In the last 5 years serum PSA has only been related to benign prostatic hyperplasia. There is an urgent need for serum markers that reflect the size and grade of this ubiquitous cancer.

Frequent HIN-1 promoter methylation and lack of expression in multiple human tumor types.

HIN-1 (high in normal-1) is a candidate tumor suppressor identified as a gene silenced by methylation in the majority of breast carcinomas. HIN-1 is highly expressed in the mammary gland, trachea, lung, prostate, pancreas, and salivary gland, and in the lung, its expression is primarily restricted to bronchial epithelial cells. In this report, we show that, correlating with the secretory nature of HIN-1, high levels of HIN-1 protein are detected in bronchial lavage, saliva, plasma, and serum. To determine if, similar to breast carcinomas, HIN-1 is also silenced in tumors originating from other organs with high HIN-1 expression, we analyzed its expression and promoter methylation status in lung, prostate, and pancreatic carcinomas. Nearly all prostate and a significant fraction of lung and pancreatic carcinomas showed HIN-1 hypermethylation, and the majority of lung and prostate tumors lacked HIN-1 expression. In lung carcinomas, the degree of HIN-1 methylation differed among tumor subtypes (P = 0.02), with the highest level of HIN-1 methylation observed in squamous cell carcinomas and the lowest in small cell lung cancer. In lung adenocarcinomas, the expression of HIN-1 correlated with cellular differentiation status. Hypermethylation of the HIN-1 promoter was also frequently observed in normal tissue adjacent to tumors but not in normal tissue from noncancer patients, implying that HIN-1 promoter methylation may be a marker of premalignant changes. Thus, silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that elimination of HIN-1 function may contribute to several forms of epithelial tumorigenesis.

Volume indexes of total, free, and complexed prostate-specific antigen enhance prediction of extraprostatic disease extension in men with nonpalpable prostate cancer.

OBJECTIVES: To analyze the ability of volume-adjusted total, complexed, and free prostate-specific antigen (PSA) to predict organ-confined cancer at radical prostatectomy in patients with nonpalpable disease. METHODS: Collected sera were assayed for total PSA (tPSA), complexed PSA (cPSA), and free PSA (fPSA) in 78 men who underwent radical prostatectomy with nonpalpable prostate cancer. The pathologic results (organ-confined versus extraprostatic extension [EPE]), tPSA, cPSA, fPSA/tPSA ratio, cPSA/tPSA ratio, fPSA/cPSA ratio, tPSA density (tPSAD), cPSA density (cPSAD), and fPSA density (fPSAD) were compared by the Mann-Whitney U test and receiver operating characteristic curves. RESULTS: Fifteen men (19.2%) had pathologic EPE. After stratifying the patients on the basis of the Beckman tPSA, the cPSAD, tPSAD, and fPSAD were significant predictors of EPE when comparing their respective medians in individuals with tPSA greater than 4.0 ng/mL. Statistically significant differences were noted between patients with and without EPE for tPSAD (P = 0.0015), cPSAD (P = 0.0018), and fPSAD (P = 0.0022), but not for the fPSA/tPSA, cPSA/tPSA, and fPSA/cPSA ratios. The area under the receiver operating characteristic curve was similar for tPSA (0.539) and cPSA (0.542), as it was for tPSAD (0.708), cPSAD (0.700), and fPSAD (0.731). The specificity and diagnostic accuracy of tPSAD, cPSAD, and fPSAD were significantly greater than those of tPSA and cPSA (specificity P <0.001; diagnostic accuracy P <0.05). CONCLUSIONS: In men with nonpalpable prostate cancer, the density parameters of tPSA, cPSA, and fPSA performed equivalently and appeared to enhance the predictability of EPE.

Genetic profiling of Gleason grade 4/5 prostate cancer: which is the best prostatic control tissue?

PURPOSE: We examined the variation in gene expression profiles of prostate cancer caused by zone specific genes. MATERIALS AND METHODS: Ten normal central zone, 10 transition zone (benign prostatic hyperplasia) and 6 normal peripheral zone tissues from radical retropubic prostatectomies were compared to each other and to 12 peripheral zone Gleason grade 4/5 cancers. Test chips and HuGeneFL6800 (Affymetrix, Inc., Santa Clara, California) chips were used to assay the transcribed genes. Data were obtained with the Microarray Suite Version 4.0.1 (Affymetrix, Inc.) and analyzed statistically. RESULTS: Substantially different gene expression profiles were found depending upon which of the 3 zonal tissues were used as a control. All 3 profiles were compared for efficiency (ability to locate genes) and for robustness (the magnitude of difference between the control and the Gleason grade 4/5 tissue). Microscopically normal appearing peripheral zone tissue at the gene level shows many characteristics of Gleason grade 4/5 cancer. CONCLUSIONS: Gene expression profiles of prostate cancer are affected by the zonal location of the control tissue and the cancer.

Prognostic factors for multifocal prostate cancer in radical prostatectomy specimens: lack of significance of secondary cancers.

PURPOSE: We evaluated secondary cancers in the prostate in relation to predictions of pathological stage and prognosis. MATERIALS AND METHODS: A total of 222 men with T1c (impalpable) prostate cancer and 6 or more systematic needle biopsies were matched with radical prostatectomy and classified into 3 groups according to tumor multifocality and secondary cancer volumes, including a single tumor in 54 (24%), an index (largest) tumor with secondary cancers less than 0.5 cc in 86 (39%) and an index tumor with secondary cancers greater than 0.5 cc in 82 (37%). Logistic analysis was used to predict adverse histological features. Cox proportional hazards analysis was used to predict prostate specific antigen (PSA) failure after radical prostatectomy. RESULTS: There were no differences among the 3 groups with respect to preoperative serum PSA, number of positive cores, percent Gleason grade 4/5 cancer in the needle biopsy or histological features in radical prostatectomy specimens. On logistic analysis neither serum PSA nor pre-radical biopsy predicted adverse histological features in radical prostatectomy specimens. The Cox regression model showed that primary predictors of PSA failure were percent Gleason grade 4/5 cancer in the biopsy (HR = 2.6, p = 0.015) and prostatectomy (HR = 2.4, p = 0.04) specimens, and the number of positive cores (HR = 2.5, p = 0.04). When comparing PSA failure rates among the 3 groups, the multifocal group with smaller secondary cancers showed a better prognosis than the single tumor group (p = 0.019). CONCLUSIONS: Secondary cancers in multifocal prostate tumors did not adversely influence the results of preoperative clinical parameters, including PSA and needle biopsy findings. Percent Gleason grade 4/5 cancer in needle biopsies and prostatectomy specimens is the most powerful predictor of biochemical failure in men with stage T1c prostate cancer after radical prostatectomy.

Hepsin and maspin are inversely expressed in laser capture microdissectioned prostate cancer.

PURPOSE: Recent studies have shown that hepsin, a serine protease, is over expressed in prostate cancers, implicating hepsin activity in tumor invasion. Using microarray technology we have previously identified 22 genes that were up-regulated in high grade prostate cancers compared with benign prostatic hyperplasia. Of them hepsin was the most differentially over expressed. In the current report we compare hepsin to maspin (BD Transduction Laboratories, San Diego, California), a serine protease inhibitor (serpin), to measure the balance between levels of serine proteases and serpins, which are considered to be a critical determinant of net proteolytic activity. MATERIALS AND METHODS: We combined the technique of laser capture microdissection with gene expression monitoring by micro-array analysis to investigate the gene expression profiles of prostate cells of different histological types. We also studied maspin immunohistochemically. RESULTS: We observed that hepsin as well as 7 of 22 previously reported up-regulated genes demonstrated a pattern of increasing expression with increasing malignant phenotype. In contrast, the expression of maspin (a serpin) decreased with increasing malignancy of prostate cancers. Using immunohistochemistry we observed that maspin protein is expressed strongly in benign prostatic tissues and slightly in grade 3 prostate cancers, and is absent in grade 4/5 cancers. CONCLUSIONS: We conclude that the increased ratio of hepsin-to-maspin may have an important role in prostate cancer progression and invasion.

Vitamin D receptor start codon polymorphism (FokI) and prostate cancer progression.

Vitamin D plays an important role in cell growth and differentiation and is proposed to protect against cancer initiation and/or progression. The vitamin D receptor (VDR) has a thymine/cytosine (T/C) polymorphism located in the first of two potential start (ATG) codons that can be detected by a RFLP using the endonuclease FokI. The C variant, which lacks the first ATG, results in a shorter VDR and is referred to as the F allele. The T variant (f allele) initiates at the first ATG. We examined the association of the VDR FokI genotype with histopathological characteristics and prognosis of prostate cancer among 191 mostly Caucasian subjects who had undergone radical prostatectomy between 1984 and 1992. The frequencies of the FF, Ff, and ff genotypes were 41%, 38%, and 21%, respectively. Subjects with the ff genotype had a lower mean percentage of Gleason grade 4/5 cancer (30.3%) than subjects with the FF or Ff genotypes (42.8% and 43.8%, respectively; P = 0.015 by t test for ff versus FF + Ff). The data suggest that the presence of an F allele increased the risk of being diagnosed with more aggressive cancer because higher percentage of Gleason grade 4/5 is associated with worse prognosis. The age-adjusted risk of prostate-specific antigen failure was lower for the ff genotype than for the FF genotype by Cox proportional hazards analysis but did not achieve statistical significance (hazard ratio = 0.76; 95% confidence interval, 0.44-1.32). This risk reduction disappeared after further adjustment for percentage of Gleason grade 4/5, cancer volume, and preoperative serum prostate-specific antigen level (hazard ratio = 1.03; 95% confidence interval, 0.58-1.85). In conclusion, the ff genotype was associated with less aggressive histopathological findings than Ff or FF genotypes. Additional studies with a larger sample size and investigation of the functional significance of the FokI polymorphism in prostate cancer cells are warranted.

Complexed prostate-specific antigen as a staging tool: results based on a multicenter prospective evaluation of complexed prostate-specific antigen in cancer diagnosis.

Within a 7-site prospective evaluation of the Bayer complexed prostate-specific antigen PSA (cPSA) assay, we analyzed the ability of cPSA to predict extracapsular extension (ECE) before radical prostatectomy. Included in this analysis were 152 men diagnosed with cancer, who subsequently underwent radical prostatectomy. Sera were tested with the Bayer total PSA (tPSA) and cPSA assays, and the Beckman free PSA (fPSA) and tPSA assays. Treating surgical pathology result as a binary variable (organ confined vs ECE), mean tPSA, cPSA, fPSA/tPSA (f/tPSA) ratios, tPSA density (tPSAD), and cPSA density (cPSAD) were compared by receiver operating characteristic (ROC) curves and univariate analysis. In all, 28 men (18.4%) had pathologically identified ECE. Between those with and without ECE, significant differences were observed for tPSA (P = 0.0127), cPSA (P = 0.0120), tPSAD (P = 0.0001), and cPSAD (P = 0.0002), but not f/tPSA (P = 0.3774) or c/tPSA (P = 0.2882). All tested parameters except f/tPSA (P = 0.376) and c/tPSA (P = 0.288) predicted ECE (P <0.05) by logistic regression. The ROC area under the curve (AUC) was identical for tPSA and cPSA (0.621) and for tPSAD (0.692) and cPSAD (0.691). Kendall-tau correlation coefficients also demonstrated the strongest correlation with ECE for cPSAD and tPSAD. Either alone or as a tPSAD calculation, cPSA carries equivalent staging ability to tPSA. The use of f/tPSA appears to be less effective in staging than either cPSA or tPSA, whereas the use of either cPSAD or tPSAD provides maximal staging accuracy. Therefore, cPSA could be applied as an accurate predictor of ECE independently or in a nomogram along with other predictive variables.

Complexed prostate-specific antigen for early detection of prostate cancer in men with serum prostate-specific antigen levels of 2 to 4 nanograms per milliliter.

Complexed PSA (cPSA) has been shown to improve specificity in the detection of prostate cancer over that of total PSA (tPSA) testing in men with tPSA values greater than the cutoff value of 4.0 ng/mL. However, recent studies have reported a 25% incidence of prostate cancer in men with tPSA values in the 2.5- to 4.0-ng/mL range. We performed a multicenter study of cPSA in a population of men who underwent prostate biopsies because of elevated PSA levels or abnormal digital rectal examination (DRE). As part of this study, we sought to assess the clinical value of cPSA in comparison to tPSA, the free/tPSA ratio (f/tPSA) and the complexed/tPSA ratio (c/tPSA) in early detection of prostate cancer in men with tPSA values in the range of 2 to 4 ng/mL. The study was performed at 7 centers. Sera were drawn from men who underwent biopsy procedures consisting of >10 prostate tissue cores. Receiver operating characteristic (ROC) analysis was performed from the results of patients with tPSA values in the range of 2 to 4 ng/mL, including men with suspicious as well as unremarkable findings on DRE. Sera were collected and tested with the Bayer tPSA and cPSA assay and the Beckman free PSA and tPSA assays. ROC analysis was performed for all samples in the 2- to 4-ng/mL PSA range. At biopsy, 158 men had no evidence of malignancy and 57 (26.5%) were diagnosed with prostate cancer. ROC analysis indicated that the area under the curve (AUC) for cPSA was 0.64, which was statistically significantly greater than that achieved for tPSA (AUC, 0.57; P <0.0001). The AUC for f/tPSA and c/tPSA were 0.60 and 0.63, respectively, which was not statistically significantly different from that of tPSA or cPSA (P >or=0.252). A cutpoint of 2.5 ng/mL for tPSA and 2.1 ng/mL for cPSA provided a specificity of 20.3% and 34.2%, respectively, and sensitivity levels of 86%. Using cutpoints of 25% for f/tPSA and 74% for c/tPSA provided a specificity of 11.0% and 21.5%, respectively, and sensitivity levels of 97%. In all, >92% of the cancers treated with radical prostatectomy were organ confined, and the histologic grading of the tumors ranged from moderately to poorly differentiated with Gleason scores from 5 to 9. These data confirm that there is a high incidence of clinically significant prostate cancer in men with tPSA levels <4.0 ng/mL. Measurement of cPSA proved useful in stratifying men with tPSA values in the 2- to 4-ng/mL range into high- and low-risk groups for prostate cancer. The use of cPSA as a single test was found to enhance detection of prostate cancer over that of testing with tPSA and PSA ratios in men with tPSA values in the range of 2 to 4 ng/mL.

Can volume measurement of the prostate enhance the performance of complexed prostate-specific antigen?

We assessed whether volume-based complexed prostate-specific antigen (cPSA) indices could enhance prostate cancer detection in men with serum total PSA (tPSA) between 2.5 and 10.0 ng/mL. Between December 1998 and April 2000, cPSA assay was measured in 480 men who underwent transrectal ultrasound-guided prostate biopsies at 7 institutions. We compared the usefulness of cPSA and its indices with the ratio of free PSA (fPSA) to tPSA (percent fPSA) for early detection of prostate cancer. Overall, 168 men (35%) had cancer. In the 341 men with tPSA between 4.01 and 10.0 ng/mL at approximately 90% sensitivity and areas under the receiver operating characteristics curve, the performances of volume-based parameters were significantly better (P <0.05) than those of tPSA and cPSA. In the 139 men with tPSA between 2.5 and 4.0 ng/mL, at 90% sensitivity, the specificity of the ratio of cPSA to tPSA (percent cPSA) was best, followed by cPSA density (cPSAD). In the 101 men with the history of a previous prostate biopsy, at approximately 90% sensitivity, the specificity of cPSAD was significantly better than those of tPSA and percent fPSA (P <0.05). In the 371 men with a total prostate volume of >or=30 cm(3) at approximately 90% sensitivity, the specificity of the cPSAD was significantly better than that of tPSA, percent fPSA, and cPSA (P <0.05). In the 109 men with a total prostate volume of <30 cm(3), at 90% sensitivity the specificity of cPSA and cPSAD was better than that of percent fPSA. In conclusion, volume-based cPSA can modestly enhance the performance of cPSA.

Polymorphisms in the androgen receptor and type II 5 alpha-reductase genes and prostate cancer prognosis.

BACKGROUND: Cytosine-adenine-guanine repeat length of the androgen receptor gene and the A49T and V89L polymorphisms of the 5 alpha-reductase (SRD5A2) gene have been associated with prostate cancer. METHODS: We investigated the relationship of the three genetic polymorphisms to tumor grade among 211 men who had undergone radical prostatectomy. Subjects had prostate cancer <3 cm(3) with a percentage of cancer represented by Gleason grade 4 or 5 (% Gleason grade 4/5) of either > or = 20% or < or = 5%. We also examined the association between those genetic markers and prostate specific antigen (PSA) failure among 112 subjects with > or = 20% Gleason grade 4/5. RESULTS: In cross-sectional analysis, none of the polymorphisms was a significant predictor of % Gleason grade 4/5. In longitudinal analysis, the LL genotype at the V89L site was associated with statistically significant four- to sixfold increase in PSA failure risk after adjustment for clinicopathologic variables. CONCLUSIONS: We observed poorer prognosis among men with the LL genotype at codon 89 of the SRD5A2 gene. Lack of consistency between studies must be resolved before clinical utility of this marker is established.

Morphologic and clinical significance of multifocal prostate cancers in radical prostatectomy specimens.

OBJECTIVES: To examine the histologic details of small, independent cancers compared with the largest (index) tumor and their impact on prostate-specific antigen (PSA) failure in 486 men treated only by radical retropubic prostatectomy (RRP). METHODS: The tumor volume and percentage of Gleason grade 4/5 carcinoma were recorded in 3-mm step sections. Univariate statistics were calculated for the largest, total (largest plus smaller cancers), and smaller tumor volumes, number of independent foci, patient age, and follow-up. Cox hazards model determined the relative importance of all variables in relation to failure. RESULTS: The mean index tumor volume was 4.16 cm3; smaller cancer volumes averaged 0.63 cm3. The index cancer volume was gaussian in distribution; smaller tumor volumes were highly skewed toward 234 carcinomas less than 0.5 cm3. Only 17% of all cases had one carcinoma. The Cox model showed similar hazard rates of PSA failure for both the index (3.43) and the total cancer (3.74) volumes. The hazard rate for the presence of any Gleason grade 4/5 carcinoma was 6.5. As the numbers of smaller tumors increased, the PSA cure rates improved. CONCLUSIONS: The PSA failure rates (hazard ratios) were similar for the index tumor and the index plus smaller cancers, confirming that predictive estimates only need to measure the largest carcinoma. The greater the number of lesser cancers, the smaller the size of the index cancer. The extraordinary multiplicity of these small independent cancers in 3-mm step sections may explain the poor correlation between six or more biopsies with the index cancer in radical prostatectomy specimens.

Preoperative serum prostate specific antigen levels between 2 and 22 ng./ml. correlate poorly with post-radical prostatectomy cancer morphology: prostate specific antigen cure rates appear constant between 2 and 9 ng./ml.

PURPOSE: Serum prostate specific antigen (PSA) is widely used as a guide to initiate prostatic biopsies and to follow men older than 50 years old with and without prostate cancer. However, benign prostatic hyperplasia (BPH) is a common cause of serum PSA values between 2 and 10 ng./ml. A better understanding of the relationships among serum PSA, prostate cancer and BPH is important. MATERIALS AND METHODS: A total of 875 men underwent radical prostatectomy at our institution between December 1984 and January 1997. Of these men 784 had a serum PSA of 2 to 22 ng./ml., including 579 with the largest cancer located in the peripheral zone of the prostate. Of the 579 men 406 had serum PSA followups for greater than 3 years after radical prostatectomy. We examined Pearson correlations (R2) between preoperative serum PSA, and the volume of Gleason grades 4/5 and 3 to 1 cancer in 784 men, separating peripheral zone from transition zone cancers. We used broken line regression with break points of 7 and 9 ng./ml. preoperative PSA to summarize the relationship of each PSA doubling to 5 different morphological variables in 579 men with peripheral zone cancer. A 9 ng./ml. break point was used for prostate weight. Trend summaries with a local regression line for the relationships between 6 morphological variables and PSA were superimposed on full scatterplots of the 579 men with PSA less than 22 ng./ml. Cox proportional hazard models were used to examine 5-year PSA failure-free probabilities based on 406 men with minimal PSA followups greater than 3 years at break points of 7 to 9 ng./ml. PSA. RESULTS: Pearson correlation between cancer volume and preoperative serum PSA in 875 men was weak (r2 = 0.27) and driven by large cancers with serum PSA greater than 22 ng./ml. For peripheral zone cancer the overall R2 x 100 for 641 men with low and high grade cancer was 10% and only 3% for low grade cancer, that is almost no PSA produced by these peripheral zone cancers enters the serum. All morphological variables changed at rates of doubtful medical significance below a PSA of 7 to 9 ng./ml. but at rates that were significantly worse above 9 ng./ml. R2 for these relationships was never greater than 15%. Large individual morphological variations at all levels of PSA emphasize the serious limitation of PSA as a predictor of prostate cancer morphology. Below 9 ng./ml. prostate weight increased by 21% for each doubling of PSA but above 9 ng./ml. the increase was only 4.8%. CONCLUSIONS: Preoperative serum PSA has a clinically useless relationship with cancer volume and grade in radical prostatectomy specimens, and a limited relationship with PSA cure rates at preoperative serum PSA levels of 2 to 9 ng./ml. Trend summaries for prostate weight on broken line regression showed that below 9 ng./ml. BPH is a strong contender for the cause of PSA elevation, constituting the primary cause of the over diagnosis of prostate cancer.