The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7.

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53(R273H)) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53(R273H)-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53(R273H) binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53(R273H)-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

The phosphatidylinositol-3 kinase pathway regulates bladder cancer cell invasion.

OBJECTIVES: To investigate the role of the phosphatidylinositol (PI)-3 kinase pathway in the invasion of bladder cancer cell lines, and to assess the activation of this pathway in primary human bladder tumours. MATERIALS AND METHODS: Human bladder cancer cells were treated with pathway specific inhibitors or were transfected with PI-3 kinase pathway components. The invasion of cultured bladder cancer cells was analysed by an invasion assay. Bladder cancer cells lines and primary human bladder tumours were analysed for pathway activation by western blotting. RESULTS: A specific inhibitor of PI-3 kinase enzyme activity, Ly294002, potently suppressed the invasive properties of three highly invasive bladder tumour cell lines. Restoration of the PTEN gene to invasive UM-UC-3 bladder tumour cells or expression of a dominant-negative version of the PI-3 kinase target, Akt, also potently inhibited invasion, indicating a central role for the PI-3 kinase/Akt pathway in this process. In addition, 55% of primary tumours from patients with bladder cancer had markedly high levels of phosphorylated Akt. CONCLUSION: Pharmacological or biochemical inhibition of the PI-3 kinase pathway drastically reduced the invasive capacity of bladder cancer cell lines; over half of primary human bladder tumours had high Akt phosphorylation, suggesting that the aberrant activation of this pathway may contribute to the invasion of a significant subset of bladder cancers.

Can single dose preoperative intrathecal morphine sulfate provide cost-effective postoperative analgesia and patient satisfaction during radical prostatectomy in the current era of cost containment?

We retrospectively analyzed the analgesic efficacy and surgical outcomes of a single preoperative intrathecal long-acting morphine sulfate injection (0.25-0.5 mg) and postoperative intravenous (i.v.) ketorolac in 62 patients who underwent radical retropubic prostatectomy (RRP). Total postoperative analgesic requirement was documented along with assessment of length of hospital stay, pain control and time for resumption of normal activity. Postoperatively, 45% of patients required only nonsteroidal agents (ketorolac), whereas 55% needed a mean of 13.3 mg of supplemental i.v. morphine sulfate. Mean hospital stay was 2.3+/-0.3 days. Eighty-two per cent of patients felt the length of hospital stay adequate. Ninety-seven per cent of patients were satisfied with anesthesia selected and 95% of patients considered pain control on postoperative days 1 and 2 as effective. All patients resumed to full physical activity by 5.3+/-0.4 weeks after surgery. We conclude that a single preoperative injection of intrathecal morphine sulfate combined with i.v. ketorolac postoperatively results in effective analgesia, diminished supplemental narcotic requirement and high patient satisfaction during radical retropubic prostatectomy.

Detection of low level HER-2/neu gene amplification in prostate cancer by fluorescence in situ hybridization.

PURPOSE: Although expression of the HER-2/neu oncogene has been correlated with tumor progression in prostate cancer, the biologic significance of detecting HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) or evidence for protein overexpression by immunohistochemistry (IHC) remains unclear. In this study, we directly compared HER-2/neu FISH and IHC to determine which may be more predictive of the response to trastuzumab. PATIENTS AND METHODS: Forty patients with prostate cancer were analyzed for gene amplification by FISH performed with HER-2/neu and chromosome 17 (CEP 17) DNA probes (Vysis). Protein expression was examined by immunofluorescence and by IHC using the DAKO HercepTest antibody protocol and a monoclonal antibody to Her-2/neu on archival paraffin sections. The patients included 30 men with primary tumors that were treated with radical prostatectomy. Of these, 15 demonstrated subsequent disease progression within 3 years. Five patients with prostatic intraepithelial neoplasia were tested, as were five with metastatic disease whose samples were obtained before androgen ablation therapy. RESULTS: None of the 30 primary prostate cancer specimens showed overexpression for HER-2/neu by immunofluorescence or by IHC with the DAKO protocol. One sample showed 3+ membrane expression with the monoclonal antibody. In contrast, low copy number gene amplifications (3-8 HER-2/neu signals/nucleus) were detected in 16 of 30 samples (53%) by FISH. Most amplified cells were diploid for CEP 17, demonstrating that amplification was not due to total cell aneuploidy. FISH and IHC determined that prostatic intraepithelial neoplasia samples were normal. Four of five (80%) metastatic samples were amplified for HER-2/neu by FISH. Nearly 70% of metastatic cancer cells among all five specimens demonstrated aneuploidy. A single lymph node metastasis showed 3+ membrane staining by IHC (DAKO). CONCLUSIONS: In contrast to breast cancer, FISH detects HER-2/neu amplification in a substantial proportion of prostate cancers that do not overexpress HER-2/neuby IHC. Although the biologic significance of this finding is uncertain, it has implications for the direction of current and planned clinical trials of trastuzumab in advanced prostate cancer, including determination of patient eligibility.

Preliminary results of three-dimensional conformal radiotherapy as salvage treatment for a rising prostate-specific antigen level postprostatectomy.

The purpose of this study is to determine the effectiveness of three-dimensional conformal radiotherapy delivered to the fossa of the prostate and seminal vesicles as salvage treatment for a prostate-specific antigen (PSA) level that becomes undetectable and subsequently begins to rise postprostatectomy. Between August 1994 and December 1997, 14 patients with prostate cancer whose PSA became undetectable after a radical prostatectomy subsequently developed a rising PSA, had no evidence of metastatic disease, and were treated with three-dimensional conformal radiotherapy at the University of California, Davis Cancer Center. Gleason scores ranged from 4 to 9 (29% of the patients had a Gleason score > or =8). The seminal vesicles were involved in three (21%) cases and the surgical margins were involved in seven (50%) cases. PSA values ranged from 0.3 to 6.7 (median: 0.7) ng/ml at the start of radiotherapy. Daily 1.8-2.0-Gy fractions were administered to total doses at isocenter ranging from 60.6 to 74.2 (median: 64.9) Gy. None of the patients received hormonal therapy. Follow-up ranged from 13 to 36 (median: 22) months. For patients with a preradiotherapy Hybritech PSA < or = 1.0 ng/ml, the Kaplan-Meier estimate of the 2-year biochemical disease-free survival rate is 67%, whereas for patients with a preradiotherapy PSA more than 1.0 ng/ml, the 2-year biochemical disease-free survival rate is 20% (p = 0.17). Because of the small number of patients, the difference is not statistically significant. A positive microscopic margin had no impact on the results obtained with salvage radiotherapy. Only one of four patients with a poorly differentiated adenocarcinoma remains free of disease. Acute toxicity was mild and did not require medication (Radiation Therapy Oncology Group grade I): four (29%) patients experienced genitourinary morbidity and three (21%) patients experienced gastrointestinal morbidity. With regard to late toxicity, one (7%) patient developed a urethral stricture requiring dilatation (Radiation Therapy Oncology Group grade III). All five patients who were potent at the start of radiotherapy remain potent. Medicare's median reimbursement for salvage three-dimensional conformal radiotherapy in this study ($7,512 in 1999 U.S. dollars) is equivalent to its reimbursement for a 17-month course of goserelin hormonal therapy. Patients with prostate cancer who develop an undetectable followed by a rising PSA postprostatectomy should be referred for salvage treatment with radiotherapy when their PSA is still less than or equal to 1.0 ng/ml. Salvage three-dimensional conformal radiotherapy is well tolerated and is less expensive than more than 17 months of goserelin.

Predicting prognosis in patients with superficial bladder cancer.

Bladder cancer is the most common urologic malignancy and is expected to affect approximately 54,000 people in 1998. Superficial bladder tumors (Tis, Ta, and T1 lesions) account for approximately 70% to 80% of these malignancies. Although many advances have been made in the management of patients with superficial bladder cancer, such disease often recurs and can progress, leading to death. It is therefore imperative to find an accurate method of identifying patients at risk for disease progression. Many recent investigations have been conducted to determine whether new biological markers will help predict disease progression and, to a lesser extent, tumor response to treatment. These new markers include DNA ploidy, S-phase, certain monoclonal antibodies, the p53 (alias TP53) tumor-suppressor gene, the retinoblastoma (Rb) gene, cell adhesion molecules, and angiogenesis. It is hoped that such prognostic indicators, coupled with cytoscopic and pathologic characteristics of the tumor, will lead to selective aggressive treatment of patients at high risk for progression while sparing low-risk patients from unnecessary procedures.

Human androgen receptor expression in prostate cancer following androgen ablation.

OBJECTIVE: Metastatic prostate cancer kills patients because their tumor cells fail to respond to combined androgen blockade (CAB) or respond and then relapse. To understand the molecular basis of androgen-insensitive growth of prostate tumor cells, we evaluated changes in human androgen receptor gene (hAR) mRNA levels in patients with prostate cancer treated with CAB. METHODS: The study was carried out using quantitative reverse-transcriptase polymerase chain reaction analysis. The level of hAR mRNA were compared to serum prostate-specific antigen and the mutant status of p53 in the tumor. RESULTS: hAR was expressed in 44 of 46 tumors from untreated patients, as opposed to 30 of 45 from those who had received CAB (p = 0.001). These 30 were from 8 of 9 stage D patients and from 22 of 36 patients on downsizing CAB therapy prior to radical prostatectomy. Expression was most often seen in high stages (56% of stage B vs. 89% of stage D) and high grades (52% of Gleason 3-7 vs. 92% of Gleason 8-10, p = 0.015). No tumor with a missense p53 mutation had hAR expression following CAB. Twenty-two patients following CAB were found to have undetectable serum prostate-specific antigen levels, while their tumor expressed hAR. CONCLUSIONS: hAR expression after CAB is seen preferentially in high-grade, high-stage tumors, the type of prostate carcinomas that fail to have a durable remission. Undetectable serum prostate-specific antigen from tumors that remain hAR positive may predict relapse after hormonal ablative therapy.

CWR22: the first human prostate cancer xenograft with strongly androgen-dependent and relapsed strains both in vivo and in soft agar.

Most patients' prostate cancers respond to androgen deprivation but relapse after periods of several months to years. Only two prostate cancer xenografts, LNCaP and PC-346, have been reported to be responsive to androgen deprivation and to relapse subsequently. Both of these tumors shrink slightly, if at all, and relapse less than 5 weeks after androgen withdrawal. After androgen withdrawal, the human primary prostate cancer xenograft CWR22 regresses markedly, and prostate-specific antigen (PSA) falls up to 3000-fold in the blood of mice. PSA usually returns to normal. In some animals, the tumor relapses and is then designated CWR22R. In these animals, PSA starts to rise approximately 2-7 months, and tumor begins to grow 3-10 months after castration. Animals with CWR22 need to be euthanized because of large tumors 6-12 weeks after the transplantation of CWR22. Androgen withdrawal prolongs life approximately 3-4-fold.

Correlation of genetic and immunodetection of TP53 mutations in malignant and benign prostate tissues.

The prognostic value of the p53 gene (TP53), the most commonly mutated gene in human cancers, has been well established for several cancer types. However, because varying frequencies of TP53 mutations have been identified in prostatic adenocarcinoma (CaP) by genetic and immunohistochemical (IHC) studies, the role of TP53 in CaP tumorigenesis is currently unresolved. These experimental discrepancies could be caused by tissue heterogeneity within prostatic neoplasms, variations in experimental protocols, or other factors. Thus, the goal of this study was to develop a reliable IHC approach for the detection of p53 in archival prostate tissue. The authors evaluated four p53 antibodies, CM-1, 1801, DO-1, and DO-7, for their ability to reveal p53. They chose two reference CaP cell lines, 26 patient specimens (including eight benign prostatic hyperplasias (BPHs), 16 CaPs, and two lymph node metastases), one prostate and nine kidney cell lines for p53 analysis. The TP53 status of these samples was characterized using single-strand conformational polymorphism (SSCP) analysis of RNA/PCR products and sequencing. IHC detection of p53 was markedly enhanced by using the combination of microwave heat-induced antigen unmasking and a cocktail of the DO-1 and DO-7 antibodies. This approach identified 14 of 15 (93%) cell lines and patient samples having TP53 missense mutations in the exons 5 to 8 region. Of the 21 patient samples and cell lines that were either normal by SSCP or expressed p53 mutations that are not expected to stain, 18 (86%) were immunonegative. Because of this good correlation between molecular and IHC analysis, this approach may help to resolve the uncertainty about TP53 in CaP tumorigenesis.

False DNA aneuploidy in canine and human neoplasms.

In most cases, the appearance of aneuploid peaks in DNA histograms may be an artefact of tissue preparation or it may reflect non-stoichiometric dye binding of a cellular subpopulation rather than true DNA aneuploidy. This report reviews how false DNA aneuploidy can be recognized and eliminated from sample submitted for DNA flow cytometric analysis.

Comparison of flow cytometry to routine testicular biopsy in male infertility.

We compared DNA flow cytometry to morphologic evaluation of routine testicular biopsies as methods of monitoring spermatogenesis. The study group consisted of 14 azoospermic men and 5 others who underwent testicular surgery unassociated with fertility problems. The findings for both studies were divided into three groups: normal, moderately abnormal, and markedly abnormal. Correlations between the findings from routine biopsy and flow cytometry were good. Of 9 patients having normal testicular morphology, 7 had normal ploidy classes by DNA flow cytometry while 2 had moderately abnormal histograms. Of 5 cases with moderately abnormal morphology, 1 had normal, 1 had moderately abnormal, and 3 had markedly abnormal ploidy distributions. In 5 cases described as Sertoli cell only, all DNA histograms were markedly abnormal, consisting almost exclusively of diploid cells. DNA flow cytometry of testicular biopsies and aspirates has been demonstrated to be a rapid, reproducible, and objective approach in evaluating the infertile male and is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testis biopsy.

Measurement variability in DNA flow cytometry of replicate samples.

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.

Deoxyribonucleic acid flow cytometry of benign prostatic disease.

Deoxyribonucleic acid flow cytometry was performed on aspirated prostatic cells from 198 patients who had benign cytological or histological findings. Unsatisfactory acellular histograms were obtained from 10.6 per cent of the cases. Three-fourths of the satisfactory samples (more than 5,000 cells after subtracting debris) showed the expected single peak deoxyribonucleic acid diploid to near diploid histograms. Unexpectedly, the remaining samples were deoxyribonucleic acid aneuploid, most having 2 peridiploid peaks (deoxyribonucleic acid index 0.82 to 1.31). Usually, proliferation was low with less than 20 per cent hyperdiploid cells and with 2.5 +/- 1.5 per cent G2 cells. In 10 per cent of the single peak histograms there was evidence of inflammation, identified as an increase in hyperdiploid cells without an increased percentage of G2 cells but with a tail of high channel values. The aforementioned histogram features were considered to be benign findings. Seven per cent of the samples had deoxyribonucleic acid histograms suggestive of prostate cancer. Of these samples 7 had diploid or peridiploid aneuploid histograms with high proliferation (more than 20 per cent hyperdiploid cells with 8.5 +/- 3.8 per cent G2 cells), while 5 had histograms with deoxyribonucleic acid aneuploidy other than peridiploidy.

RAS enzyme-linked immunoblot assay discriminates p21 species: a technique to dissect gene family expression.

The members of the RAS gene family of protooncogenes are of implied biological significance in oncogenesis. The precise role of these genes is unclear. One difficulty has been the inability to discriminate the individual p21 protein products of various ras genes in cell lines, de novo human tumors, and related normal tissues. In this report, specific proteins of the human c-Ha-ras-1, c-Ki-ras-2, and c-N-ras genes have been detected and discriminated by the differential use of various antisera recognizing these p21s. This enzyme-linked immunoblot assay utilizes a double antibody system in which monoclonal antibodies are initially used to immunoprecipitate the p21ras proteins. Immunoprecipitates are then subjected to one-dimensional Western blot analysis utilizing other antibodies raised against p21s, coupled with nonradiolabeled enzyme-linked colorimetric detection. By direct detection, the specific products of the three human ras genes can be discriminated. In addition, we describe the generation and characterization of a new anti-p21c-N-ras-specific antibody. The simultaneous expression into protein of multiple ras genes is unequivocally demonstrated in both homogeneous cell lines and heterogeneous human tissues. This new technique is also applicable for discrimination of the protein products of other gene families.

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience.

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.

Interinstitutional variability in DNA flow cytometric analysis of tumors. The National Cancer Institute's Flow Cytometry Network Experience.

Flow cytometric DNA analysis of human urinary bladder specimens may be clinically useful for prognosis in transitional cell (urothelial) carcinoma and for detecting recurrence after treatment. However, many important methodological differences exist among institutions which have described this technique, and it has not previously been shown that data from different institutions are comparable. The National Cancer Institute has created a Flow Cytometry Network to address the need for technology assessment of flow cytometry. This report describes the independent flow cytometric analysis and interpretation of "unknown" paraffin-embedded bladder tumor specimens by the five Network institutions. Although important differences in method existed among the institutions, substantial agreement was achieved in actual data generated and their interpretation. This suggests that a consensus regarding acceptable laboratory performance of this technique could be reached, which should facilitate its more widespread clinical implementation.

Chemosensitivity of murine renal carcinoma.

A non-endocrine dependent, spontaneous carcinoma of the kidney in a Wistar-Lewis rat has been studied for sensitivity to chemotherapeutic agents. Two tumour models have been employed. Subcutaneously transplanted flank nodules were used to screen single agents for antitumour activity.. A model of intraperitoneal metastatic disease was employed to test further agents which had demonstrated some effectiveness in the nodule model. Single agents that proved ineffective were streptozotocin, neocarzinostatin, chlorozotocin and carminomycin. 5-FU, bleomycin and hydroxyurea were also ineffective at the doses tested. Agents that were effective included cyclophosphamide, adriamycin, vinblastine, vindesin and maytansine. The most effective combination therapy appeared to be cyclophosphamide with vindesin and cisplatin.

Clomiphene citrate: pharmacologic treatment of hypofertile male.

Thirty-two males were treated with clomiphene citrate for a minimum of six months, a maximum of twelve months, or until pregnancy occurred. Among these patients, thirteen pregnancies occurred. Climiphene citrate appears a reasonable pharmaceutical method for management of idiopathic oligospermia.