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Objectives: To visualize human prostate cancer cells in mouse bone with bioconjugated near-infrared quantum dot (QD) probes. Near-infrared fluorescent probes using QDs can visualize tumors in deep tissues in vivo., Methods: Human prostate cancer C4-2B xenografts grown in mouse tibia were detected by prostate-specific membrane antigen antibody conjugated with QDs emitting light at the near-infrared range of 800 nm (QD800). Images in culture and in vivo were acquired using the IVIS Imaging System., Results: As few as 5000 cells can be detected subcutaneously when tagged with QD800 conjugate and injected directly into mice. QD800 conjugate injected intravenously in mice harboring C4-2B tumors in tibia detected signals from a minimum of 500 000 cells. The maximal light emission was detected 30 minutes after intravenous injection of QD800 conjugate in mice with established C4-2B tumors., Conclusions: Bioconjugated near-infrared QD probes are highly sensitive molecular imaging tools for human prostate cancer micrometastases in mice.

Objectives: To evaluate the cause and significance of elevated activated partial thromboplastin time (aPTT) in a group of patients who received a first-generation adenoviral vector (Ad-OC-TK) delivering a toxic gene to prostate cancer cells as part of a Phase I clinical trial at the University of Virginia., Methods: Eleven subjects were injected intratumorally to metastatic lesions of prostate cancer in the prostatic fossa, retroperitoneal lymph nodes, or bone (iliac, ischium, or vertebrae). After the initial laboratory evaluation, patients with elevated aPTT underwent a series of additional tests, including mixing studies, coagulation factor, prekallikrein, and high-molecular-weight kininogen, and lupus anticoagulant studies (modified Russell viper venom time) with phospholipid correction, and a Staclot LA assay., Results: Of the 11 subjects who were enrolled in the trial, 6 had elevated aPTT values. Of the 6 patients, 3 had aPTT elevation of more than 10 seconds above normal. Two of the subjects with higher values demonstrated an inhibitory pattern with the factor VIII and XI assays, and the lupus anticoagulant studies were positive. No clinical sequelae to the elevated aPTT values were observed., Conclusions: This is, to our knowledge, the first formal report of a first-generation adenoviral vector causing a slight transient elevation of the aPTT through the induction of an antiphospholipid antibody. No clinical sequelae related to elevated aPTT values were observed. The adenoviral protocol was safe; similar protocols should be aware of this phenomenon.

Objectives: To evaluate the normal distribution of serum prostate-specific antigen (PSA) levels in healthy Chinese men, because, until recently, studies conducted to establish normal serum PSA values have not involved a Chinese population., Methods: Between September 1999 and December 2001, 1096 healthy Chinese men aged 23 to 85 years, who had undergone a routine health examination, were recruited to this study. All underwent detailed clinical examinations, including serum PSA determination and digital rectal examination. All men with abnormal digital rectal examination findings and/or an abnormal serum PSA level (greater than 4.0 ng/mL) underwent transrectal ultrasound-guided sextant biopsy., Results: The median serum PSA concentration was 0.50 ng/mL (95th percentile 1.20) for men 23 to 29 years old (n = 77); 0.55 ng/mL (95th percentile 1.21) for men 30 to 39 years old (n = 189); 0.54 ng/mL (95th percentile 1.23) for men 40 to 49 years old (n = 233); 0.82 ng/mL (95th percentile 2.35) for men 50 to 59 years old (n = 177); 0.93 ng/mL (95th percentile 3.20) for men 60 to 69 years old (n = 265); and 1.17 ng/mL (95th percentile 3.39) for men 70 years old or older (n = 155). The serum PSA concentration correlated with age (P <0.001), with an increase of approximately 1.1% annually. No change occurred in the median serum PSA value and 95th percentile in men younger than 50 years old; a gradual increase was observed in men older than 50 years. In those 50 years old or older, the median and 95th percentile serum PSA values for Chinese men were significantly lower than those for other races and even for other Asian men., Conclusions: These findings further confirm that the serum PSA level correlates with age. Moreover, the distribution and cutoff value of the serum PSA level differs along ethnic lines. In addition, our findings raise the question of whether lowering the PSA cutoff may enhance the detection of cancer in Chinese men who have the lowest prostate cancer rate.

Objectives: To develop a new toxic gene therapy using the tissue-specific human chorionic gonadotropin-beta (hCG-beta) promoter for testicular cancer. Although most patients presenting with disseminated testicular tumor are cured through the use of chemotherapy with or without surgery, those patients with relapse after initial therapy present a difficult clinical problem. The serum tumor marker hCG-beta is frequently elevated in patients with testicular cancer, and the pretreatment and post-treatment levels of serum hCG-beta are highly predictive of treatment outcome., Methods: Human testicular embryonal carcinoma cell line, NEC 8, a human prostate cancer cell line, PC-3, and a human bladder cancer cell line, WH, were used in this study. A transient expression experiment was used to analyze the activity of a 729-bp hCG-beta promoter in all three cell lines. A recombinant adenovirus carrying thymidine kinase (Ad-hCG-beta-TK) under control of the hCG-beta promoter was generated. The tissue-specific activity of Ad-hCG-beta-TK was tested in vitro and in vivo., Results: The hCG-beta promoter had significantly greater activity in the hCG-beta-producing cell line (NEC 8) than in the non-hCG-beta-producing cell lines (PC-3 and WH). In vitro, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 growth but not PC-3 or WH cell growth. In vivo, Ad-hCG-beta-TK with acyclovir significantly inhibited NEC 8 subcutaneous tumor growth in nude mice., Conclusions: In this study, we explored the possibility of developing a new therapeutic agent to target and induce the killing of testicular germ cell tumor selectively by using tissue-specific hCG-beta promoters.

Changes in genomic and phenotypic expression of progressing prostate tumors and their stroma occur in a dynamic fashion based on bidirectional signaling from stromal-epithelial interactions. These interactions may underlie the ability of prostate cancer cells to survive and proliferate in the prostate and bone. By investigating the phenotypic and genotypic changes of stromal cells adjacent to cancer cells and the reciprocal changes of cancer cells, novel molecular markers may be developed to diagnose cancer earlier before pathologic appearance of cancer cells at the primary site. Attacking epithelial and stromal elements together is a unique approach to both localized and metastatic prostate cancer therapy. Co-targeting both tumor cells and stroma requires identifying a reliable tumor and tissue-specific cis-DNA element, such as osteocalcin (OC) promoter. OC expression is elevated in prostate tumor cells and in prostate and bone stromal cells interdigitating with both localized and metastatic prostate epithelium. We have previously designed an adenovirus-based therapeutic gene vehicle and demonstrated that a replication-competent adenoviral vector (Ad vector) is highly efficient in blocking the growth of cancer cells in culture, including cells without androgen receptor as well as cells that do or do not make prostate-specific antigen. In vivo, intravenous administration of an Ad-OC vector was effective against preexisting human prostate cancer subcutaneous and bone xenografts. The addition of vitamin D(3) enhanced further viral replication at target sites. Co-targeting tumor cells and stroma using systemic Ad vector is a viable and promising option for treatment of both localized and metastatic prostate cancer.

Objectives: The osteoblastic response of bone to metastatic prostate cancer is both characteristic and enigmatic. The potent vasoconstrictor endothelin-1 (ET-1), produced by prostate cancer, has been identified as a potential factor in new bone formation., Methods: Using a novel method to quantitate new bone formation induced by the WISH tumor, we examined the effects of ET-1 overexpression and endothelin receptor antagonists on the osteoblastic response., Results: WISH, a human tumor cell line derived from amnion, produces ET-1 mRNA and protein and induces abundant new bone formation and splenomegaly in vivo. Stable transfection of WISH with an ET-1 overexpression cDNA construct produced clones that secreted 18-fold more bioactive ET-1 than vector-only controls. After 14 days of growth in the lower leg of nu/nu mice, ET-1 overexpressing tumors produced significantly more new bone than vector-only controls. Conversely, areas of new bone formation were significantly less in animals treated with a selective endothelin A (ET(A)) receptor antagonist A127722., Conclusions: The activity of ET-1 in this osteoblastic model provides a unique target for therapy.

Objectives: To determine the frequency of abnormal p53 expression and to characterize confirmed p53 mutations in tumors from patients with clinically localized adenocarcinoma of the prostate., Methods: p53 protein nuclear accumulation was determined immunohistochemically in the initial diagnostic tumor specimens from 37 patients with clinically localized prostate carcinoma. Two primary antibodies were used on all specimens. Structural analysis of the p53 gene was performed using the methods of polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) and DNA sequencing., Results: In 1 of the 37 (2.7%) tumor specimens, intense p53 nuclear staining was demonstrated using either antibody PAb 1801 or CM-1. The staining in this case was heterogeneous, with approximately 40% of tumor nuclei staining for p53. This tumor specimen was microdissected and DNA was extracted. Following PCR amplification, abnormally migrating bands were noted on SSCP analysis of exon 8. DNA sequencing confirmed the mutation as a C-->A transversion in codon 281 (asp-->glu). PCR/SSCP analysis of exons 5 through 8 was also performed for seven additional tumors that were negative for p53 nuclear accumulation by immunohistochemical (IHC) methods. All of these specimens demonstrated wild-type p53., Conclusions: The results of this study confirm and extend our previous findings that p53 mutations are rare in clinically localized adenocarcinoma of the prostate. In detecting clonal p53 mutations, standard immunohistochemical technique correlates reliably with structural p53 gene analysis of the evolutionary conserved domains encompassing exons 5-8. Importantly, most reports have demonstrated that p53 mutations detected by IHC are a late step in the progression of prostate cancer and are associated with advanced disease, dedifferentiation, and the acquisition of androgen independence.

Objectives: To determine the prognostic significance of microvessel density (a measure of tumor angiogenesis) in comparison with other prognostic factors for patients with clinically localized prostatic carcinoma treated with external beam radiotherapy., Methods: Microvessel density was quantified within the initial invasive carcinoma from the diagnostic transurethral resection specimen of 25 patients with a mean follow-up of 44 months. Microvessels were identified by immunohistochemical staining of endothelial cells for factor VIII-related antigen in formalin-fixed, paraffin-embedded tissue. Microvessels were counted in a x200 field (0.754 mm2) in the area of maximal angiogenesis., Results: Microvessel density correlated with several pretreatment prognostic factors, including prostate-specific antigen (PSA) (p < 0.0001), tumor grade (p = 0.006), and ploidy (p = 0.016). The degree of tumor angiogenesis also correlated with outcome following external beam radiotherapy. The mean microvessel count in the nine tumors from patients who failed radiotherapy (ie, had rising PSA and/or clinical relapse) was 97.0 +/- 33.6 (+/- SD) per x200 field compared with 46.1 +/- 17.1 for the 16 patients with no evidence of failure (p < 0.0001). Increased microvessel density was also associated with a significantly worse actuarial outcome at 4 years using either biochemical relapse (rising PSA) or a composite endpoint of rising PSA or clinical relapse (p = 0.0003)., Conclusions: The intratumoral quantification of tumor angiogenesis may prove valuable as a prognostic indicator in patients with clinically localized prostate cancer treated with radiotherapy.