Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4-216) recognizes nucleophosmin/B23.

BACKGROUND: The nuclear protein B23, nucleophosmin, is an RNA-associated nucleolar phosphoprotein reported to be more abundant in malignant and growing cells than in normal nondividing cells. We examined the levels of B23 in fresh human prostate tissue and in five human prostate cancer cell lines with monoclonal antibodies (mAb) to nucleophosmin (alpha-B23) and to human prostate cancer nuclear matrix proteins (PRO:4-216). METHODS: mAb PRO:4-216 and mAb alpha-B23 were used for protein level detection. Nuclear matrix proteins (NMPs) were prepared from prostate tumor and five human prostate cancer cell lines: LNCaP, TSU, DU145, PC-3, and PPC-1. The NMPs were run on one-dimensional and two-dimensional (2D) electrophoresis gels for Western blot analysis with the two mAbs. Histologic sections from paraffin-embedded normal and cancerous prostate tissue were stained immunohistochemically with both mAbs. RESULTS: PRO:4-216 and B23 mAbs identified a 40-kD protein (pI approximately 5.0) by Western blot analysis in the human prostate cancer cell lines and on two-dimensional blots of human prostate cancer NMPs. Immunohistochemical staining demonstrated large punctate nuclear dots in most cancer nuclei, while staining of normal tissue was less intense or absent. Predominant reactivity was of epithelial nuclei, with some minor reactivity of stromal nuclei. Red blood cells (RBCs) and white blood cells (WBCs) were routinely negative. CONCLUSIONS: PRO:4-216, previously characterized as recognizing prostate cancer nuclear matrix proteins, recognized B23/nucleophosmin. PRO:4-216 and alpha-B23 showed intense immunohistochemical staining of B23/nucleophosmin in cancer nuclei compared to adjacent normal cells in paraffin-embedded prostate tissue. This preliminary study showed the potential of B23 as a tumor marker for human prostate cancer.

Influence of finasteride on free and total serum prostate specific antigen levels in men with benign prostatic hyperplasia.

PURPOSE: Finasteride therapy for benign prostatic hyperplasia (BPH) results in a marked lowering of serum prostate specific antigen (PSA) levels. However, little is known about the effect of finasteride on unbound or free serum levels of PSA. Such information would be important since percent free PSA may substantially improve the cancer specificity of PSA testing. Thus, we prospectively studied the effect of finasteride therapy on total and free serum PSA levels. MATERIALS AND METHODS: In a randomized, placebo controlled, double-blind trial 40 men with histologically confirmed BPH (age range 52 to 78 years) were treated with either 5 mg. finasteride daily (26 patients) for 9 months or placebo (14) for 6 months. Prostate volume was assessed by transrectal ultrasound. Serum levels of free and total PSA were measured from archived serum samples stored at -70C at baseline and for as long as 9 months of treatment. RESULTS: In the finasteride group mean total PSA levels declined from 3.0 ng./ml. at baseline to 1.5 ng./ml. after 6 months of treatment (50% decrease, p <0.01). In the placebo group, with similar baseline levels, no significant change was observed. PSA density declined significantly in finasteride treated men (p <0.01) but not in men receiving placebo. The mean percent free PSA (13 to 17% at baseline) was not altered significantly by finasteride or placebo. CONCLUSIONS: Total PSA serum levels decreased by an average of 50% during finasteride therapy but percent free PSA did not change significantly. This information is potentially useful in the interpretation of PSA data used for early detection of prostate cancer in men receiving finasteride. However, further studies are required to demonstrate the use of percent free PSA to detect the development of cancer.

Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial.

CONTEXT: The percentage of free prostate-specific antigen (PSA) in serum has been shown to enhance the specificity of PSA testing for prostate cancer detection, but earlier studies provided only preliminary cutoffs for clinical use. OBJECTIVE: To develop risk assessment guidelines and a cutoff value for defining abnormal percentage of free PSA in a population of men to whom the test would be applied. DESIGN: Prospective blinded study using the Tandem PSA and free PSA assays (Hybritech Inc, San Diego, Calif). SETTING: Seven nationwide university medical centers. PARTICIPANTS: A total of 773 men (379 with prostate cancer, 394 with benign prostatic disease) 50 to 75 years of age with a palpably benign prostate gland, PSA level of 4.0 to 10.0 ng/mL, and histologically confirmed diagnosis. MAIN OUTCOME MEASURES: A percentage of free PSA cutoff that maintained 95% sensitivity for prostate cancer detection, and probability of cancer for individual patients. RESULTS: The percentage of free PSA may be used in 2 ways: as a single cut-off (ie, perform a biopsy for all patients at or below a cutoff of 25% free PSA) or as an individual patient risk assessment (ie, base biopsy decisions on each patient's risk of cancer). The 25% free PSA cutoff detected 95% of cancers while avoiding 20% of unnecessary biopsies. The cancers associated with greater than 25% free PSA were more prevalent in older patients, and generally were less threatening in terms of tumor grade and volume. For individual patients, a lower percentage of free PSA was associated with a higher risk of cancer (range, 8%-56%). In the multivariate model used, the percentage of free PSA was an independent predictor of prostate cancer (odds ratio [OR], 3.2; 95% confidence interval [CI], 2.5-4.1; P < .001) and contributed significantly more than age (OR, 1.2; 95% CI, 0.92-1.55) or total PSA level (OR, 1.0; 95% CI, 0.92-1.11) in this cohort of subjects with total PSA values between 4.0 and 10.0 ng/mL. CONCLUSIONS: Use of the percentage of free PSA can reduce unnecessary biopsies in patients undergoing evaluation for prostate cancer, with a minimal loss in sensitivity in detecting cancer. A cutoff of 25% or less free PSA is recommended for patients with PSA values between 4.0 and 10.0 ng/mL and a palpably benign gland, regardless of patient age or prostate size. To our knowledge, this study is the largest series to date evaluating the percentage of free PSA in a population representative of patients in whom the test would be used in clinical practice.

Differential nuclear matrix protein expression in prostate cancers: correlation with pathologic stage.

PURPOSE: Nuclear Matrix Proteins (NMP) have been shown to be tissue and cell-type specific. Several unique NMPs have been investigated in various cancerous tissues, including prostate, bladder and kidney, and some are presently utilized as tumor markers. This study was aimed at characterizing the differential NMP expression in the pathologically more aggressive prostate cancers. METHODS: High resolution two-dimensional gel electrophoresis and silver staining was used to elucidate the NMP distribution of fresh prostate cancer nuclei, obtained from 39 radical prostatectomy specimens, surgically removed from men with clinically localized prostate cancer. Based on the final pathological grading, specimens were grouped according to predicted prognosis: poor--with seminal vesicle (SV) or lymph node (LN) involvement or established capsular penetration (ECP) with gleason score >7; intermediate--organ confined (OC) or focal capsular penetration (FCP) with gleason score 7 or ECP with gleason score 6; and good--with OC or FCP and gleason score <7. RESULTS: A specific charged protein (YL-1) of molecular weight 76 kD and isoelectric range 6.0-6.6 was found to be consistently present in 19 of 19 aggressive cancers. It was present only in 1 of 10 in the group with good prognosis and weakly positive in 9 of 10 in the intermediate group. CONCLUSIONS: Within this preliminary study, the expression of YL-1 appears to be related to aggressive prostate cancer, suggesting a potential marker of poor prognosis for clinically localized prostate cancer. Further characterization of the identity and function of this NMP is needed to fully ascertain its clinical potential.

Prospective evaluation of percent free-PSA and complexed-PSA for early detection of prostate cancer.

Aims of the study: Retrospective studies investigating the use of percent free-PSA for early detection of prostate cancer were limited for various reasons: by their use of long-term stored sera, poor mix of non-cancer to cancer cases and the use of only men with PSA values between 4.0 and 10.0 ng/mL. This prospective study investigates the clinical utility of percent free-PSA and complexed-PSA for early detection of prostate cancer in 219 consecutive men presenting for prostate biopsy. Methods: Of 246 consecutive men who underwent ultrasound guided sextant biopsy of the prostate for PSA elevation and/or suspicious digital rectal exam, 219 men had serum total PSA levels between 2.0 and 20.0 ng/mL and were included in this study. Serum total, free and complexed (PSA-ACT) were measured (Hybritech Inc.). Results: Pathologic examinations demonstrated that 72% and 28% of the biopsies were non-cancer and cancer respectively. The mean percent free-PSA was statistically different between the groups (cancer 14%+/-6.4 and non-cancer 18+/-9%, P<0.001) and improved cancer detection. PSA-ACT provided only modest improvement in cancer detection over that of total PSA. Among this cohort of men, the optimal total PSA reflex range for percent free-PSA was 3.0-7.0 ng/mL (38% specificity) with a percent free-PSA cut-off of 20% (95% sensitivity) yet only affected 56% of the cases. Conclusions: PSA-ACT added very little additional value to the clinical utility of total PSA for early detection. Percent free-PSA performed well for all reflex ranges. A sensitivity and specificity of 95% and 20% respectively were obtained using a single cut-off of 25% for percent free-PSA for the group of men with total PSA values between 4.0 and 10.0 and correlated well with recently reported prospective analyses.

Molecular forms of prostate-specific antigen and human kallikrein 2 (hK2) in urine are not clinically useful for early detection and staging of prostate cancer.

OBJECTIVES: Prostate-specific antigen (PSA), a member of the human kallikrein (hK) family, is the most important tumor marker for early detection, staging, and monitoring of men with prostate cancer today. However, the sensitivity of serum PSA is not sufficient to be used alone for prostate cancer screening. Recently, it was reported that the serum-to-urinary total PSA ratio improves the detection of men with prostate cancer, especially in men with a serum total PSA level between 4.0 and 10.0 ng/mL. We tested this hypothesis by evaluating the clinical usefulness of this PSA ratio as well as the use of the different molecular forms of PSA and human kallikrein 2 (hK2) in urine for detection and staging of prostate cancer. METHODS: One hundred ten fresh, midstream urine specimens (prostate cancer 62, benign prostatic hyperplasia [BPH] 38, healthy male control 5, women 5) were collected. Serum total PSA, urine total PSA, urinary free PSA, urinary alpha 1-antichymotrypsin-bound PSA, and urinary hK2 levels were determined by monoclonal antibody assays (Hybritech Inc.). The serum-to-urinary total PSA ratio was calculated. RESULTS: The serum-to-urinary total PSA ratio did not accurately distinguish between men with BPH and men with prostate cancer. There was no significant difference between the urinary levels of any of the molecular forms of PSA or hK2 between men with prostate cancer and men with BPH. Among men with prostate cancer, neither urinary hK2 nor urinary levels of any of the molecular forms of PSA correlated with age, pathologic stage, or Gleason grade. CONCLUSIONS: In our study, the serum-to-urinary total PSA ratio did not improve the detection of men with prostate cancer. Furthermore, measurement of the molecular forms of PSA and hK2 in urine did not improve the detection or staging of prostate cancer over serum PSA alone.

Preliminary immunohistochemical characterization of a monoclonal antibody (PRO:4-216) prepared from human prostate cancer nuclear matrix proteins.

OBJECTIVES: A nuclear matrix protein (PC-1) was previously identified and reported to be present only in human prostate cancer but absent in tissue from the same prostate containing either benign prostatic hyperplasia (BPH) or normal prostate tissue. The PC-1 protein was identified by high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and exhibited a molecular mass of 56 kDa and an isoelectric point of 6.58. This work investigates the immunohistochemical characterization of PRO:4-216, a monoclonal antibody to PC-1. METHODS: Areas of the 2D-PAGE gels containing the human prostate cancer nuclear matrix proteins near PC-1 were isolated, eluted, and injected into mice to develop monoclonal antibodies. Antibodies were screened by immunofluorescence for nuclear reactivity to a human prostate cancer cell line (LnCaP) and by 1D and 2D Western blots for reactivity with prostate cancer nuclear matrix proteins. Monoclonal antibodies from the selected clones were affinity purified. The monoclonal antibody PRO:4-216 was used to analyze frozen tissue from 20 cancerous, 22 BPH, and 22 normal regions from fresh human prostate specimens. Tissue sections were analyzed for their immunohistochemical (IHC) (horseradish peroxidase) staining. RESULTS: Using a reference value for positive staining at an IHC score of greater than 50, 85% (17 of 20) of the cancerous, 5% (1 of 22) of the BPH, and 9% (2 of 22) of the normal prostate tissues stained positive. The one BPH and two normal tissues that stained positive were taken from prostates in which the adjacent cancerous tissue also demonstrated high IHC scores (greater than 225). CONCLUSIONS: These data demonstrate nuclear reactivity on fresh frozen human prostate cancer tissue for the monoclonal antibody PRO:4-216. PRO:4-216 may aid in distinguishing normal prostate and BPH from cancerous tissue.

Prostate tissue composition and response to finasteride in men with symptomatic benign prostatic hyperplasia.

PURPOSE: We sought to quantify prostate tissue changes induced by finasteride and to identify a predictor of finasteride response in men with symptomatic benign prostatic hyperplasia (BPH) via a randomized, placebo controlled, double-blind clinical trial. MATERIALS AND METHODS: Men with symptomatic BPH (52 to 78 years old) were randomly assigned to 6 months of treatment with finasteride (26) or placebo (15). Outcome measures were clinical (urinary symptom score and flow rate), chemical (serum prostate specific antigen and dihydrotestosterone levels), volumetric (transrectal ultrasound, and magnetic resonance imaging for whole and zonal prostate volumes) and histological (morphometry of prostate sextant biopsies, separated into inner and outer gland segments, to measure the percent epithelium, stroma and glandular lumen). RESULTS: In the finasteride group we found a suggestion of decreasing symptom scores and increasing flow rates (not significant) with significant decreases (p < 0.01) in prostate specific antigen (48%), dihydrotestosterone (74%) and prostate volume (21%). Finasteride treatment induced a 55% decrease in inner gland epithelium (p < 0.01) with little effect on stroma or lumina. We also found a linear correlation between pretreatment inner gland epithelial content and prostate volume decrease induced by the drug (tau = 0.58, p = 0.01). CONCLUSIONS: Finasteride treatment results in a major suppression of prostate epithelium, which is most pronounced in the inner gland. Moreover, a finasteride induced prostate volume decrease was predictable by quantification of epithelial tissues of the inner gland. These data lend additional support to the emerging concept of transition zone primacy in symptomatic BPH.

Combination of prostate-specific antigen, clinical stage, and Gleason score to predict pathological stage of localized prostate cancer. A multi-institutional update.

OBJECTIVE: To combine the clinical data from 3 academic institutions that serve as centers of excellence for the surgical treatment of clinically localized prostate cancer and develop a multi-institutional model combining serum prostate-specific antigen (PSA) level, clinical stage, and Gleason score to predict pathological stage for men with clinically localized prostate cancer. DESIGN: In this update, we have combined clinical and pathological data for a group of 4133 men treated by several surgeons from 3 major academic urologic centers within the United States. Multinomial log-linear regression was performed for the simultaneous prediction of organ-confined disease, isolated capsular penetration, seminal vesicle involvement, or pelvic lymph node involvement. Bootstrap estimates of the predicted probabilities were used to develop nomograms to predict pathological stage. Additional bootstrap analyses were then obtained to validate the performance of the nomograms. PATIENTS AND SETTINGS: A total of 4133 men who had undergone radical retropubic prostatectomy for clinically localized prostate cancer at The Johns Hopkins Hospital (n=3116), Baylor College of Medicine (n=782), and the University of Michigan School of Medicine (n=235) were enrolled into this study. None of the patients had received preoperative hormonal or radiation therapy. OUTCOME MEASURES: Simultaneous prediction of organ-confined disease, isolated capsular penetration, seminal vesicle involvement, or pelvic lymph node involvement using updated nomograms. RESULTS: Prostate-specific antigen level, TNM clinical stage, and Gleason score contributed significantly to the prediction of pathological stage (P<.001). Bootstrap estimates of the median and 95% confidence interval of the predicted probabilities are presented in the nomograms. For most cells in the nomograms, there is a greater than 25% probability of qualifying for more than one of the pathological stages. In the validation analyses, 72.4% of the time the nomograms correctly predicted the probability of a pathological stage to within 10% (organ-confined disease, 67.3%; isolated capsular penetration, 59.6%; seminal vesicle involvement, 79.6%; pelvic lymph node involvement, 82.9%). CONCLUSIONS: The data represent a multi-institutional modeling and validation of the clinical utility of combining PSA level measurement, clinical stage, and Gleason score to predict pathological stage for a group of men with localized prostate cancer. Clinicians can use these nomograms when counseling individual patients regarding the probability of their tumor being a specific pathological stage; this will enable patients and physicians to make more informed treatment decisions based on the probability of a pathological stage, as well as risk tolerance and the values they place on various potential outcomes.

The role of free/total prostate-specific antigen ratio in the prediction of final pathologic stage for men with clinically localized prostate cancer.

OBJECTIVES: The combined use of total prostate-specific antigen (PSA), clinical stage, and Gleason score accurately predicts final pathologic stage for men with clinically localized prostate cancer. Recently, the free/ total PSA ratio has been proposed as an adjunct for early detection of prostate cancer. We examined the association between free/total PSA and pathologic stage. METHODS: In a prospective study, 301 consecutive men with clinically localized prostate cancer (average age 58.8 years, range 45-72) underwent a staging pelvic lymphadenectomy and radical prostatectomy. Total PSA and free PSA were measured from preoperative sera. Pathologic stage was determined as organ-confined (OC, n = 169), capsular penetration (CP+, n = 108), seminal vesicle involvement (SV+, n = 13) and lymph node involvement (LN+, n = 11). RESULTS: Overall, 292/301 (97%) of the free/total PSA values were < 25%, and thus suspicious for prostate cancer. Combination of total PSA, Gleason score, and clinical stage predicted well OC (P = 0.00001) and LN+ (P = 0.023); whereas, replacing total PSA with free/total PSA ratio did not improve the prediction of OC (P = 0.0007) nor LN+ (P = 0.03). CONCLUSIONS: The free/total PSA ratio cutoff point of 25% had high sensitivity for prostate cancer among a group of men with clinically localized disease. The free/total PSA ratio did not significantly improve the prediction of pathologic stage provided by total PSA when used alone or in combination with Gleason score and clinical stage. These preliminary data demonstrate that free/total PSA levels provide no additional information for pathologic stage prediction when combined with Gleason score and clinical stage in men with clinically localized prostate cancer.

Analysis of percent free prostate-specific antigen (PSA) for prostate cancer detection: influence of total PSA, prostate volume, and age.

OBJECTIVES: To determine whether the use of the free-to-total PSA ratio (percent free PSA) could increase the specificity of PSA testing for prostate cancer detection in men with serum PSA concentrations between 4.0 and 10.0 ng/mL, and to assess the influence of total PSA, prostate volume, and age on percent free PSA. METHODS: Sera were obtained from 217 men with histologically confirmed diagnoses (139 prostate cancer, 78 benign). Free and total PSA concentrations were determined using Hybritech Tandem assays. RESULTS: Use of percent free PSA increased PSA specificity: 29% of negative biopsies would be spared while retaining 95% sensitivity. Percent free PSA increased with increasing age and prostate volume. Percent free PSA decreased as total PSA increased. A significant relation exists between percent free PSA and the probability of a positive biopsy; in this cohort, a patient with a low percent free PSA (< or = 10%) had a higher probability of cancer (63 +/- 9%) than a patient with a high percent free PSA (> or = 26%) (probability 2 +/- 3%). CONCLUSIONS: Percent free PSA may be used as an aid in distinguishing prostate cancer from benign disease in men with a total PSA between 4.0 and 10.0 ng/mL. Large prospective multicenter trials are required to develop consistent recommendations and determine the appropriate cutpoints and risk probabilities, controlling for total PSA, prostate volume, age, and biopsy history.

Standard versus age-specific prostate specific antigen reference ranges among men with clinically localized prostate cancer: A pathological analysis.

PURPOSE: Age-specific prostate specific antigen (PSA) references ranges have been suggested to account for the age-dependent nature of the serum PSA concentration. It has been hypothesized that reference ranges of 0 to 2.5 ng./ml.serum PSA (40-49 years), 0 to 3.5 ng./ml. (50-59 years), 0 to 4.5 ng./ml. (60 to 69 years) and 0 to 6.5 ng./ml. (70 to 79 years) would detect fewer (potentially insignificant) prostate cancers in older men and more (potentially curable) cancers in younger men. MATERIALS AND METHODS: To investigate the pathological stage of tumors that would be affected by the use of age-specific PSA references ranges, we reviewed the medical records for 4,597 men with clinically localized (stage T1c, T2, or T3a) prostate cancer, with an average age of 62 +/- 7 years (range 38 to 76), who underwent radical prostatectomy between 1984 and 1994 at our institutions. Favorable pathological results were defined as organ-confined disease or capsular perforation with a Gleason score of less than 7, and unfavorable pathological results were defined as capsular perforation with a Gleason score of 7 or more, seminal vesicle invasion or lymph node involvement. RESULTS: Overall, 18% of the men had PSA levels less than the standard PSA reference range (4.0 ng./ml.) compared to 22% when using the age-specific ranges. There were 74 more cancers detected in men younger than 60 years with the use of age-specific ranges, of which 81% had favorable pathological results. Among the men 60 years or older, 191 of 252 cancers (76%) not detected by using age specific ranges less than 3% were also stage T1c and 95% of these undetected T1c cancers were of favorable pathological status. Of those cancers not detected in older men with the age-specific ranges were of favorable pathological status. Of those cancers not detected in older men with age-specific ranges less than 3% were also stage T1c and 95% of these undetected T1c cancers were of favorable pathological status. Age-specific PSA reference ranges increased the potential for detection of prostate cancer by 18% in the younger men and decreased the detection by 22% in the older men. CONCLUSIONS: Among these men with clinically localized prostate cancer, age specific PSA references ranges increased the detection of more potentially curable tumors in young men and decreased the detection of less advanced tumors in the older men compared to the standard reference range of 4.0 ng./ml. Among older men with nonpalpable (stage T1c) tumors age-specific PSA references ranges would have detected fewer tumors. However, 95% of these "missed" tumors would have had favorable pathological findings.

Clearance rate of serum-free and total PSA following radical retropubic prostatectomy.

BACKGROUND: Our objective was to determine the clearance rate of free and total serum PSA following radical retropubic prostatectomy. METHODS: Sera were obtained from 10 men with localized prostate cancer prior to and 1, 4, 8, 24, 48, and 72 hr after radical prostatectomy. No patient received any postoperative blood transfusion. Free and total PSA were measured using the Hybritech Tandem-R (total PSA) and radioimmunometric free PSA assay. Postsurgery serum-free and total PSA concentrations were modeled using a "two-compartment" pharmacokinetic model. RESULTS: The pharmacokinetic model with an initial constant "infusion" suggests that, following release from the prostate, both free and total PSA are taken up into a second compartment for metabolism. The movement of PSA between these compartments was accurately modeled. Following surgery, there is a shift of both free and total PSA best modeled as a constant infusion into the serum at a rate of 1.97 and 1.60 ng/ml, respectively, for a period of approximately 1 hr. Following this initial constant infusion, the half-life estimations for free and total PSA are initially 1.2 and 0.75 hr, respectively, which then increase to 22 and 33 hr, respectively. CONCLUSIONS: Serum free and total PSA are cleared from the circulation following a "two-compartment" model with an initial constant "infusion." The constant "infusion" is most likely a consequence of surgical manipulation. The initial half-life estimates are < 2 hr for both free and total PSA, and later increase to 22 and 33 hr, respectively.