Purification of a Human Prostate Specific Antigen.

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

Introduction to artificial neural networks for physicians: taking the lid off the black box.

BACKGROUND: Over the past 5 years, a steady stream of publications has discussed the use of artificial neural networks (ANNs) for urologic and other medical applications. The pace of this research has increased recently, and deployed products based on this technology are now appearing. Before these tools can be widely accepted by clinicians and researchers, a deeper level of understanding of ANNs is necessary. This article attempts to lay some of the groundwork needed to facilitate this familiarity. METHODS: A short discussion of neural network history is included for background. This is followed by an in-depth discussion of how and why ANNs work. This discussion includes the relationship between ANNs and statistical regression. An investigation of issues associated with neural networks follows, applicable to both general and urologic-specific applications. RESULTS: Neural networks are computer models that have been studied extensively for over 50 years, with prostate cancer applications since 1994. From a biological viewpoint, ANNs are artificial analogues of data structures that exist in nervous systems. From a numeric viewpoint, ANNs are matrices of numbers whose values comprise knowledge that is distilled from historic databases. Many types of neural networks are analogous to well-known statistical methods. CONCLUSIONS: ANNs are complex numeric constructs, but no more complex than similar statistical methods. However, several issues associated with neural network derivation demand that developers apply rigorous engineering practices in their studies.

Prostate-specific antigen levels in young white and black men 20 to 45 years old.

OBJECTIVES: To determine the prostate-specific antigen (PSA) levels and PSA change over time in young white and black men 20 to 45 years old. METHODS: The Department of Defense Serum Repository, a serum bank that stores all residual serum from the military human immunodeficiency virus screening program at -25 degrees C, was sampled to obtain a total of 588 black and 588 white subjects 20 to 45 years old. This was a retrospective study with only demographic data available on the studied subjects. The samples used for this study were collected between June 24, 1988 and June 12, 1996. Individuals with a history of prostate disease were excluded by query of a centralized Department of Defense diagnosis database. Three serum specimens evenly distributed over a mean of 6 years were selected for each individual to determine the free and total PSA levels and PSA velocity. The Hybritech Tandem-E PSA assay was used for the total PSA measurement, and the Hybritech Tandem-R assay was used for the free PSA measurement. RESULTS: The baseline serum PSA levels differed by race (P = 0.04). The median (25th, 75th percentile) baseline serum PSA levels for black men 20 to 29, 30 to 39, and 40 to 45 were 0.38 ng/mL (0.26, 0.61), 0.45 ng/mL (0.32, 0. 67), and 0.52 ng/mL (0.37, 0.73), respectively. The median baseline serum PSA levels for the same decade groups in white men were 0.38 ng/mL (0.27, 0.57), 0.45 ng/mL (0.28, 0.68), and 0.40 ng/mL (0.26, 0. 64), respectively. The PSA velocity was higher in white men than in black men (mean 2.8%/yr and 1.6%/yr, respectively, P = 0.032). CONCLUSIONS: These results suggest that although black men 20 to 45 years old have higher baseline serum PSA levels than white men of the same age, the PSA velocity is greater in young white than in young black men. Additional work is needed to determine the clinical significance of these findings.

Development of dendritic-cell based prostate cancer vaccine.

Available treatments for metastatic prostate cancer have failed to demonstrate significant curative potential. Current efforts are now directed towards developments of novel strategies for the treatment of metastatic prostate cancer. Cancer immunotherapeutic strategies utilize patient immune system components to kill cancer cells. This review discusses progress in active specific immunotherapeutic approaches as potential alternative methods in the treatment of metastatic prostate cancer. One of the newest advances in cancer immunotherapy is the use of dendritic cells as the vehicle to deliver cancer antigens for an effective in vivo T cell activation. The development of dendritic cell-based prostate cancer vaccine, as well as results of several clinical trials in prostate cancer involving the administration of peptide-pulsed autologous dendritic cell pulsed are discussed.

Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA).

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.

Dendritic cell-based immunotherapy of prostate cancer: immune monitoring of a phase II clinical trial.

We assessed both non- and peptide-specific immune responses in prostate cancer patients before and after immunotherapy with dendritic cells exogenously pulsed with the prostate-specific membrane antigen-derived peptides, PSM-P1 and PSM-P2. For all subjects, we observed that clinical responses were strongly associated with two indicators of immunocompetence: skin test responses to recall antigens and cytokine secretion by T cells after nonspecific stimulation. In a subset of responders, we observed cytokine secretion or cytotoxicity against the immunizing peptides or an immunodominant epitope from an influenza recall antigen. The clinical results support the use of monitoring for overall immunocompetence to help determine why a patient has or has not responded to therapy. Moreover, it could be useful as an inclusion criterion to select those more likely to benefit from treatment.

Higher-dose and less frequent dendritic cell infusions with PSMA peptides in hormone-refractory metastatic prostate cancer patients.

BACKGROUND: Infusion of dendritic cells (DCs) pulsed with PSMA peptides was considered possible in hormone-refractory metastatic prostate cancer patients both with or without prior treatment with a greater number of DCs and for lesser infusions than previously administered. METHODS: DCs + PSMA peptides in patients undergoing leukapheresis were administered monthly 1-4 times, at rates greater than 20 million DCs in 17 patients not previously treated, and in 11 patients previously treated. RESULTS: Three partial responders and one complete responder were noted in the 17 previously untreated persons. DCs + PSMA peptides averaged 28.5 million cells (range in millions, 21.0-42.3). All responders received 3 or 4 infusions of greater than 22 million cells (3-4 times). In the previously treated group of 11 patients, DCs infused averaged 29.3 million cells (range in millions, 20-40.5). One new responder (bone scan) was noted. Two prior responders continued. Observation times were similar. Toxicity was minimal. CONCLUSIONS: These results suggest that DCs + PSMA peptide infusions can be given with greater numbers of DCs with a lesser number of infusions (1-4 monthly) with no loss of response rates compared to those noted previously, and without increased side effects. In previously treated patients (both relapsing and nonrelapsing), adverse effects were not noted, and new responses can be anticipated to be without harmful side effects. However, the follow-up time, and number of patients in this group, were small.

Follow-up ProstaScint scans verify detection of occult soft-tissue recurrence after failure of primary prostate cancer therapy.

BACKGROUND: A reliable imaging modality is required to uncover occult soft- tissue recurrence after failure of primary prostate cancer therapy. This retrospective study was done to evaluate the ability of the (111)Indium-labeled monoclonal antibody (ProstaScint(R)) scan in detection of prostatic bed recurrence and/or metastases to regional and/or distant lymph nodes. METHODS: One hundred sequential patients were evaluated with repeated ProstaScint(R) scans because of evidence of recurrence during the course of their disease. These 100 patients were followed closely from November 1994 and April 1999, and had concurrent bone scans and serum prostate-specific antigen (PSA) evaluations. They have had hormone therapy (n = 53) and/or experienced a rising PSA after radical prostatectomy (n = 38) or after radiation therapy (n = 56). Scan images were scored 0-3, where score 0 = negative, score 1= prostate bed uptake, score 2 = regional lymph node uptake, and score 3 = distant lymph node uptake. In each patient, the uptake of the follow-up scan(s) was compared to that of the initial scan. RESULTS: The median age was 70 years (range, 45-87), and 23 patients had a positive bone scan. The average PSA was 40.5 ng/ml (standard deviation, 223.5). There was 257 scans representing 100 patients. All patients had at least 2 scans, 35 patients had 3 scans, and 11 patients had 4 scans. No individual exhibited detectable adverse clinical reactions during or after the scan. The findings of the initial and consecutive scans were anatomically consistent in 79%, whereas in 21% there were skip metastases. In 24 patients the lesions progressed by scan and PSA, 10 patients showed progression of scan but no PSA progression, 49 patients showed no change, and 17 patients showed a remission related to adjuvant therapy. CONCLUSIONS: The consistency on repeating the scan (79%) and the high percentage of patients showing persistent uptake at the prostate bed (43%) as well as the percentage of detection of regional nodes (20%) and distant nodes (32%) reflects the importance of using the ProstaScint(R) scan in finding occult recurrences after primary treatment failure of prostate cancer. These results are similar to those reported earlier in autopsy series studies in similar populations.

Evaluation of prostate cancer patients receiving multiple staging tests, including ProstaScint scintiscans.

BACKGROUND: Multiple serum tests were performed on archival samples from patients who participated in trials to assess the ProstaScint scan staging ability. Traditional statistical analysis as well as artificial neural network (ANN) analysis were employed to evaluate individual patients and the group as a whole. The results were evaluated so that each factor was tested for prognostic value. METHODS: Data obtained from serum tests, bone scans, and ProstaScint scans were evaluated by traditional statistical methods and ANN to determine the individual value in clinical staging of prostate cancer. RESULTS: Two hundred seventy-five patients (180 postprostatectomy, 95 intact prostate) with prostate cancer (14 with distant metastases) were available for analysis. Data available included: clinical state (remission or progression), most recent clinical TNM stage, bone scan, and ProstaScint scan. Serum was tested for prostate-specific membrane antigen(PSMA), prostate-specific antigen(PSA), free PSA (fPSA), and complexed PSA (cPSA). Additional calculations included percent free PSA, and percent complexed PSA. Spearman individual statistical assessment for traditional group evaluation revealed no significant factors for T-stage. The free PSA and complex PSA had a significant association with node (N)-status. The distant metastases (M) stage correlated well with the bone scan and clinical stage. ANN analysis revealed no significant T-stage factors. N-stage factors showed a 95% sensitivity and 49% specificity. These factors included the presence or absence of a prostate, PSA serum levels, bone scan, and ProstaScint scans as major associated indicators. ANN analysis of the important variables for M-stage included ProstaScint scan score, and PSA levels (total, percent complexed, percent free, and fPSA). These factors were associated with a 95% sensitivity and 15% specificity level. CONCLUSIONS: Two hundred seventy-five patients receiving treatment for prostate cancer were evaluated by ANN and traditional statistical analysis for factors related to stage of disease. ANN revealed that PSA levels, determined by a variety of ways, ProstaScint scan, and bone scan, were significant variables that had prognostic value in determining the likelihood of nodal disease, or distant disease in prostate cancer patients.

Prostate-specific membrane antigen (PSMA): current benefits and future value.

We will review the evolution, benefits, and limitations of PSMA testing in the past, as well as its current and future value. Prostate cancer has been the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. It has a wide spectrum of biological behavior between latent (indolent) and progressive (aggressive). Further identification of prostate-specific membrane antigen (PSMA) as a prognostic proliferation marker may enhance our understanding of the types of prostate cancer. A review of PSMA testing in the past as well as currently was conducted. Studies were reviewed that deal with detection of PSMA in serum and seminal fluid, reverse transcriptase-polymerase chain reaction (RT-PCR), immunoscintigraphy, and immunohistochemical assays. PSMA is expressed primarily in benign and cancerous prostatic epithelial cells. It is up-regulated in hormone resistant states, and in metastatic situations or other clinical situations where there is tumor recurrence or extension. Based on current results, PSMA detected in the serum by western blotting can assist in the identification, staging, and monitoring of metastatic prostate cancer. In addition, PSMA shows a promising role in directed imaging and therapy of recurrent or metastatic disease.

Detection of extraprostatic prostate cells utilizing reverse transcription-polymerase chain reaction.

This article reviews the utility of reverse transcription-polymerase chain reaction (RT-PCR) in prostate cancer. RT-PCR aims to detect occult micrometastases in non-prostatic sites. Due to its exquisite analytical sensitivity, RT-PCR is able to amplify and detect even low-level, prostate-specific messages present at these extraprostatic sites. In recent years, a fair amount of data on the clinical utility of the technique had been reported. The target tissues under investigation are peripheral blood, bone marrow aspirate, and lymph nodes. Favorite markers of choice are prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and human glandular kallikrein-2 (hK2). False positives among negative controls are low. For the most part, RT-PCR is inadequate in detecting tumor cells in the peripheral blood from patients who are known to have metastatic prostate cancer. All studies showed that RT-PCR could detect PSA, PSMA or hK2 mRNAs in the circulation of patients who have organ-confined or extraprostatic disease. Most studies showed that RT-PCR utilizing current markers could not be used as a prospective test to diagnose prostate cancer. However, a few studies also showed that the detection rate could be predictive and sensitive enough to differentiate patients with organ-confined disease from those with extraprostatic disease. Data from PSA- or PSMA-RT-PCR using lymph nodes as the tissue source is more encouraging. RT-PCR was able to detect PSA and/or PSMA positive samples that have not been detected by conventional pathology.

Progress in active specific immunotherapy of prostate cancer.

Treatments available for metastatic prostate cancer have failed to demonstrate significant curative potential. Current efforts are now directed towards developments of novel strategies for the treatment of metastatic prostate cancer. Cancer immunotherapeutic strategies utilize patient immune system components to kill cancer cells. This review discusses progress in active specific immunotherapeutic approaches as potential alternative methods in the treatment of metastatic prostate cancer. Various methods of augmenting the immune response against prostate cancer are discussed including systemic cytokine adjuvant therapy, cytokine gene transduced tumor vaccines, non-antigen specific immunization, DNA and peptide vaccines plus adjuvants, as well as dendritic cell-based cancer vaccines.

Use of artificial neural networks in evaluating prognostic factors determining the response to dendritic cells pulsed with PSMA peptides in prostate cancer patients.

BACKGROUND: Our purpose was to compare the importance of over 22 measurements used in evaluating the clinical responses of patients with metastatic or locally recurrent prostate cancer, treated by dendritic cell (DC) infusions with prostate-specific membrane antigen (PSMA) peptides. METHODS: Artificial neural networks (ANNs) were employed for assessment, as well as the traditional methods of logistic regression. RESULTS: Twenty-six patients with metastatic disease and 37 patients with local recurrence were available for evaluation and comparison. ANN evaluation ranked the collective effects of DC infusion, immune responses (CD3+ cells, CD16+ cells, zeta chain+ cells), and cytokines, e.g., IL-6 and PSMA levels, very highly. Logistic regression identified all of these parameters to some degree, but in a different rank order. Patients with metastases showed a sharp rate of response secondary to the level of DC infusion, in contrast to those patients with local recurrence, in which it was more gradual. CONCLUSIONS: ANN analysis emphasizes the importance of level of DC infusion, immune parameters, cytokines, and markers such as PSMA in determining the response to PSMA peptide immunotherapy. The criteria of response were judged to be correct in 86% of metastatic patients and 83% of locally recurrent patients evaluated in this study.

American Joint Committee on Cancer prognostic factors consensus conference.

BACKGROUND: The American Joint Committee on Cancer (AJCC) published the 1st edition of the Cancer Staging Manual in 1977 and began using T (tumor extent), N (regional lymph node status), and M (the presence or absence of distant metastasis) in an organized staging scheme to express the extent of disease in a number of cancer sites. The goal of this program has been to provide physicians and others with a useful methodology to plan treatment, project prognosis, and measure outcome end results. Until recent years, this system has incorporated only elements of anatomic extent of the tumors determined by clinical and pathologic methods. At the present time an increasing number of nonanatomic cancer prognostic factors are being identified and studied. Some of these factors currently are being used for outcome predictions and treatment decisions. METHODS: To begin the process of identifying and validating these prognostic factors to refine the present TNM system, the AJCC convened a Prognostic Factors Consensus Conference to evaluate the roles of biologic, genetic, molecular, and other nonanatomic factors in staging cancer. Working groups were appointed for carcinomas of the breast, colorectum, prostate, and ovary and experts in each of these areas were invited to participate. Emphasis was placed on evaluating existing data and the correlation of these data with survival. RESULTS: None of the groups believed that there were sufficient data at the present time to merit incorporation of serum markers into the TNM system for the four tumors under consideration, although this soon might become possible in prostate carcinoma after the evaluation of survival data from multiple institutions. Recommendations were made regarding the emerging sentinel lymph node technique, the need for an increased use of histopathology in the staging of breast and ovarian carcinomas, and the use of additional histologic staining techniques for the detection of "micrometastases" in lymph nodes. A number of additional recommendations were made for changes to the TNM system that did not involve serum markers and other nonanatomic cancer prognostic factors. CONCLUSIONS: These recommendations are presented for the purpose of discussion and evaluation and do not yet represent formal proposals for a change in the AJCC TNM system of staging.

Review of phase II hormone refractory prostate cancer trials.

In men with hormone refractory metastatic prostate cancer, single agents have demonstrated an objective response when using a response rate greater than standard treatment in prior National Prostate Cancer Project (NPCP) trials. Among these agents are methotrexate, cisplatin, 5-fluorouracil, cyclophosphamide, doxorubicin, estramustine, methyl N-(2-chloroethyl)-N'-cyclohexyl-Nnitrosourea (CCNU), 5-(3,3-dimethyl1-triazenyl)1H-imidazole-4-carboxamide (DTIC), and vinblastine. Other combinations of protocols have been previously evaluated. Currently under review is a 1 4-year follow-up on an adjuvant trial (NPCP 900/1000) where all patients had a bilateral pelvic lymphadenectomy. These patients were then randomized to surgery or radiation, and received either cyclophosphamide, estramustine, or no therapy for 2 years. Clinical results showed there was no difference in disease-free survival or survival rates between those patients in the group who had T3 prostate cancer, and those with negative pelvic lymph node dissection. The drugs diethylstilbestrol, megestrol, or streptozocin have been used. In clinical trials, the single agent diethylstilbestrol usually had a better effect. Combinations of estramustine with vinblastine were also effective when compared with the results of standard treatment. When comparing patients who relapsed from hormone refractory prostate cancer, there was no significant statistical response rate difference (P = <0.05) in patients who were offered either flutamide or estramustine. To determine more accurately the positive results from these trials, the pre-prostate-specific antigen (PSA) era of stable disease must be re-evaluated.