Howard L. Adler

ELEVATED LEVELS OF CIRCULATING INTERLEUKIN-6 AND TRANSFORMING GROWTH FACTOR-beta 1 IN PATIENTS WITH METASTATIC PROSTATIC CARCINOMA

PURPOSE Specific cytokines have been found to be secreted by and influence the growth of prostate cancers in cell culture. Interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 (TGF-beta1) have all been closely associated with prostate cancer. We analyzed the levels of these cytokines in the systemic circulation of patients with varying stages of prostate cancer compared to controls. MATERIALS AND METHODS Serum IL-6, TNFalpha and GM-CSF were measured using commercially available enzyme linked immunosorbent assays in 5 groups of patients, including controls-19 men presenting to prostate cancer screening with normal digital rectal examination and serum prostate specific antigen (PSA) no greater than 2.0 ng./ml., stage pT2-19 with cancer confined to the prostate in the radical prostatectomy specimen, stage pT3-10 with extraprostatic extension and/or seminal vesicle involvement, stage N1-12 with lymph node metastases at pelvic lymph node dissection, and stage M1-9 with bone metastases. Platelet poor plasma TGF-beta1 was measured using a commercially available enzyme linked immunosorbent assay in controls and patients with stage M1 disease only because it was not available for patients with stages pT2, pT3 and N1 disease. No patient had a history of any other malignancy. All blood specimens were collected before surgery and/or androgen ablation. Statistical analysis was done with the Kruskal-Wallis analysis of variance. RESULTS Serum IL-6 and platelet poor plasma TGF-beta1 were significantly elevated in patients with clinically evident metastases (p = 0.0008 and 0.0412, respectively) while serum GM-CSF and TNFalpha were not. IL-6 and TGF-beta1 correlated with increasing serum PSA (p = 0.0335 and 0.0386, respectively). GM-CSF did not correlate with PSA or age. In multivariate analysis TNFalpha correlated with age but not PSA. CONCLUSIONS IL-6 and TGF-beta1 correlate with tumor burden as assessed by serum PSA or clinically evident metastases. Further research is needed to determine the response to androgen ablation as well as the source(s) and actions of these cytokines.

In Situ Gene Therapy for Adenocarcinoma of the Prostate: A Phase I Clinical Trial

For patients with local recurrence of prostate cancer after definitive irradiation therapy there is no treatment widely considered safe and effective. After extensive preclinical testing of prodrug gene therapy in vitro and in vivo, we conducted a phase I dose escalation clinical trial of intraprostatic injection of a replication-deficient adenovirus (ADV) containing the herpes simplex virus thymidine kinase gene (HSV-tk) injected directly into the prostate, followed by intravenous administration of the prodrug ganciclovir (GCV). Our goal was to determine safe dose levels of the vector for future trials of efficacy. Patients with a rising serum prostate-specific antigen (PSA) level and biopsy confirmation of local recurrence of prostate cancer without evidence of metastases one or more years after definitive irradiation therapy were eligible for the trial. After giving informed consent, patients received injections of increasing concentrations of ADV/HSA-tk in 1 ml into the prostate under ultrasound guidance. Ganciclovir was then given intravenously for 14 days (5 mg/kg every 12 hr). Patients were monitored closely for evidence of toxicity and for response to therapy. Eighteen patients were treated at 4 escalating doses: group 1 (n = 4) received 1 x 10(8) infectious units (IU); group 2 (n = 5) received 1 x 10(9) IU; group 3 (n = 4) received 1 x 10(10) IU; group 4 (n = 5) received 1 x 10(11) IU. Vector was detected by PCR of urine samples after treatment, increasing in frequency and duration (up to 32 days) as the dose increased. All cultures of blood and urine specimens were negative for growth of adenovirus. Minimal toxicity (grade 1-2) was encountered in four patients. One patient at the highest dose level developed spontaneously reversible grade 4 thrombocytopenia and grade 3 hepatotoxicity. Three patients achieved an objective response, one each at the three highest dose levels, documented by a fall in serum PSA levels by 50% or more, sustained for 6 weeks to 1 year. This study is the first to demonstrate the safety of ADV/HSV-tk plus GCV gene therapy in human prostate cancer and the first to demonstrate anticancer activity of gene therapy in patients with prostate cancer. Further trials are underway to identify the optimal distribution of vector within the prostate and to explore the safety of repeat courses of gene therapy.

Prostate-Specific Antigen Response and Systemic T Cell Activation After In Situ Gene Therapy in Prostate Cancer Patients Failing Radiotherapy

In an extended phase I/II study we evaluated 36 prostate cancer patients with local recurrence after radiotherapy who received single or repeated cycles of replication-deficient adenoviral vector (ADV)-mediated herpes simplex virus-thymidine kinase (HSV-tk) plus ganciclovir (GCV) in situ gene therapy with respect to serum PSA levels, alterations in immune cells, and numbers of apoptotic cells in needle biopsies. An initial cycle of HSV-tk plus GCV gene therapy caused a significant prolongation of the mean serum PSA-doubling time (PSADT) from 15.9 to 42.5 months (p = 0.0271) and in 28 of the injected patients (77.8%) there was a mean PSA reduction (PSAR) of 28%. It took a mean of 8.5 months for the PSA to return to the initial PSA (TR-PSA) value. A repeated cycle of gene therapy failed to significantly extend PSADT but did result in significant increases in PSAR (29.4%) and TR-PSA (10.5 months). Moderately increased serum adenovirus antibody titers were generally observed 2 weeks after initial vector injection. Also at this time there was a statistically significant increase in the mean percent of CD8(+) T cells positive for the HLA-DR marker of activation in peripheral blood (p = 0.0088). Studies using prostate biopsies obtained at the same time point demonstrated that vector DNA was detectable by PCR in most samples yet all patients remained positive for prostate cancer in at least one biopsy core. Further analysis demonstrated a correlation between the level of CD8(+) cells and the number of apoptotic cells in biopsies containing cancer cells (p = 0.042). We conclude that repeated cycles of in situ HSV-tk plus GCV gene therapy can be administered to prostate cancer patients who failed radiotherapy and have a localized recurrence. Biological responses to this experimental therapy including increases in PSADT, PSAR, and TR-PSA, and activated CD8(+) T cells present in the peripheral blood, were demonstrated. Interestingly, the density of CD8(+) cells in posttreatment biopsies correlated with the number of apoptotic cells.

Membrane type 1-matrix metalloproteinase promotes human prostate cancer invasion and metastasis

Development of metastases requires cancer cells to breach underlying basement membrane, migrate through interstitial stroma and gain access to blood or lymphatic vessels. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Expression of MT1-MMP in human prostate cancer correlates with the stage of this disseminated disease. The mechanism underlying this observation, however, still remains to be understood. To study the role of MT1-MMP in prostate cancer dissemination, endogenous and recombinant MT1-MMP expressed in human prostate cancer cell lines (DU-145 and LNCaP) were examined. Using FITC-labeled Matrigel, a soluble basement membrane extract coated coverslips, LNCaP cells stably expressing a chimera of MT1-MMP and Green Fluorescent Protein (MT1-GFP) degraded Matrigel and readily migrated over degraded substrates. The degradation of Matrigel by LNCaP cells expressing MT1-GFP was sensitive to MMP inhibitors, CT-1746 and TIMP-2, but not TIMP-1. Cell migration was dramatically enhanced by expression of MT1-MMP. By employing surgical orthotopic implantation of LNCaP cells stably expressing MT1-GFP into the prostate gland of immunodeficient mice, we demonstrated that MT1-MMP promotes lymph node and lung metastasis of prostate cancer cells. Together, these results emphasize the pivotal role of MT1-MMP in prostate cancer dissemination and confirm that MT1-MMP is a suitable target to prevent cancer metastasis.

Noninvasive Detection of Prostate Cancer by Quantitative Analysis of Telomerase Activity

Purpose: Prostate cancer is the most common male malignancy and the second leading cause of male cancer death; therefore, there is urgent necessity for noninvasive assays for early detection of prostate cancer. Obtaining prostate tumor samples surgically is problematic because the malignancy is heterogeneous and multifocal and early-stage tumors are nonpalpable. In contrast, exfoliated cells represent the cancer status of the entire gland better due to the general tendency of cancer cells to exfoliate into biological fluids. The purpose of this study was to clarify whether quantitative analysis of telomerase activity in exfoliated cells in urine could serve as a reliable molecular marker of prostate malignancy. Experimental Design: We analyzed prospectively post-prostatic examination–exfoliated cells from the urine of 56 patients undergoing routine prostate screening. Epithelial cells were isolated and enriched by immunomagnetic separation. Telomerase activity was analyzed by quantitative real-time PCR telomeric-repeat amplification protocol assay using Opticon MJ research instrument. Results: We report now that all prostate cancer patients revealed high levels of telomerase activity thereby showing 100% of the assay sensitivity. In contrast, the majority of patients with clinically confirmed benign prostatic hyperplasia (BPH) did not express any telomerase activity (70% of all BPH patients), most likely presenting cancer-free cases, or expressed low levels of activity (18%). However, about 12% of BPH patients revealed high levels of telomerase activity that potentially can reflect hidden prostate cancer. Conclusions: We suggest that the quantitative analysis of telomerase activity can be useful for the selection of prostate cancer and cancer-free cases.

CORRELATION OF PREOPERATIVE PLASMA IGF-I LEVELS WITH PATHOLOGIC PARAMETERS AND PROGRESSION IN PATIENTS UNDERGOING RADICAL PROSTATECTOMY

OBJECTIVES To test whether preoperative insulin-like growth factor (IGF)-I levels could predict pathologic stage and prognosis of prostate cancer in patients undergoing radical prostatectomy. METHODS The study group consisted of 120 consecutive patients who underwent radical prostatectomy for clinically localized prostate cancer. Preoperative plasma IGF-I levels were measured using the DSL-IGF-I Elisa assay. Surgically removed prostate specimens were analyzed pathologically, using a whole-mount step-section technique. Preoperative plasma IGF-I levels were compared with final pathologic parameters and with prostate-specific antigen (PSA) progression-free survival. Preoperative IGF-I levels in this cohort were also compared with IGF-I levels measured in 20 healthy men without any cancer and in 10 men with untreated, metastatic prostate cancer. RESULTS Plasma IGF-I levels predicted neither organ-confined disease (P = 0.5611) nor the risk of PSA progression (P = 0.8125) at a median follow-up of 48.6 months after prostatectomy. Furthermore, IGF-I levels did not correlate with preoperative PSA level (P = 0. 2811) or final Gleason score (P = 0.4906). IGF-I levels in radical prostatectomy patients were not significantly higher than those in healthy subjects or in patients with metastatic disease (mean 156.7 +/- 66 ng/mL, 148.6 +/- 49 ng/mL, and 148.6 +/- 93 ng/mL, respectively; P = 0.8442). CONCLUSIONS Circulating IGF-I levels may predict the future risk of developing prostate cancer, but our study found no association with other established markers of biologically aggressive disease or with disease progression in patients with clinically localized prostate cancer.

Prostate Cancer Cell Adhesion to Bone Marrow Endothelium The Role of Prostate-Specific Antigen

Bone metastasis is the most frequent complication of prostate cancer (PC). Elucidation of the biological basis of this specificity is required for the development of approaches for metastatic inhibition. We investigated the possibility that the preferential attachment of PC cells to bone marrow endothelium (as opposed to endothelium from other organs) affects this specificity. We selected, from peptide phage-displayed libraries, peptide ligands to surfaces of PC cells (C4-2B) attenuated (30–40%) binding of C4-2B cells to bone marrow endothelial cells (BMECs). We then determined the molecules on the surface of C4-2B cells interacted with the selected peptides using column affinity chromatography and a cDNA expression phage-displayed library generated from C4-2B cells in T7 phage. We identified a phage from the cDNA library that specifically bound to one of the selected peptides-L11. This phage displayed the amino acid sequence homologous for the COOH-terminal portion of prostate-specific antigen (PSA). To examine the possible direct involvement of PSA in the interactions between PC and BMECs, we performed a cell–cell adhesion assay. Antibodies to PSA attenuated PC cells adhesion to BMECs. In addition, exogenous proteolytically active PSA modulated this adhesion. Finally, inactivation of mRNA coding PSA by a small interfering RNA (siRNA) diminished C4-2B cell adhesion to BMECs. These results indicate that PSA expressed as secreted and surface-associated molecules in C4-2B cells is involved in cell–cell interactions and/or digests components of bone marrow endothelium for preferential adhesion and penetration of PC cells. The suggested experimental approach is a promising strategy for identification of cell surface molecules involved in intercellular interactions.

EXTRAPERITONEAL ENDOSCOPIC PELVIC LYMPH NODE DISSECTION A REVIEW OF 125 PATIENTS

PURPOSE We evaluated the efficacy of a totally extraperitoneal approach to endoscopic pelvic lymph node dissection. MATERIALS AND METHODS Extraperitoneal endoscopic pelvic lymphadenectomy was performed in 125 patients with clinically localized prostate cancer. All patients were candidates for brachytherapy, cryotherapy or radical perineal prostatectomy. The first 65 patients underwent lymphadenectomy regardless of local clinical stage, prostate specific antigen (PSA) or tumor grade. The last 60 patients met 2 of 3 selection criteria, consisting of clinical local stage T2b or greater, prostate specific antigen greater than 20 and Gleason score 7 or higher. Patients were evaluated for morbidity and mortality, nodal yield, operative time, conversion rate to transperitoneal laparoscopic or open lymphadenectomy and hospital stay. RESULTS Mean operative time was 104 minutes, mean length of stay was 2.1 days and mean nodal yield was 10.2. Of the patients 19.2% had positive nodes, and positive nodal yield increased to 32.9% when selection criteria were used. Of the cases 4% were converted to a transabdominal laparoscopic approach and 2.4% to open lymphadenectomy. Symptomatic lymphoceles required percutaneous drainage in 2.4% of the patients. One patient died of massive pulmonary embolism. CONCLUSIONS This study demonstrates that the extraperitoneal endoscopic pelvic lymph node dissection is an effective and relatively safe method of surgically staging prostate cancer. It compares favorably to other methods of surgical staging.

Deficits in urological knowledge among medical students and primary care providers: potential for impact on urological care.

PURPOSE Recent data indicate a decline in the urological education of third and fourth year medical students. To determine if this decline has an impact on the treatment of patients we performed a survey to evaluate the general level of knowledge, attitudes and practices with regard to common urological issues seen in a general medical practice among medical students and faculty involved in primary care at an academic institution. MATERIALS AND METHODS A confidential questionnaire was distributed to attendings, residents and fellows, and the clinical medical students at our academic institution to ascertain how they evaluate and treat patients with common urological complaints. All responses were entered into SPSS statistical software. RESULTS A total of 300 surveys were distributed, 150 of which were returned with complete information for data analysis. Knowledge with regard to various conditions including hematuria, recognition of an age specific abnormality in serum prostate specific antigen and overactive bladder was low for all groups. Furthermore, respondents demonstrated a low likelihood of requesting formal urological evaluation for these conditions. Exposure to a urology elective in medical school had a positive impact on some areas of urological evaluation. CONCLUSIONS General urological knowledge with regard to the primary care setting is insufficient. The potential for impact on patient care is enormous. These data highlight the need for a definitive urological curriculum in medical school as well as continued education at the resident and faculty level with regard to evaluation, management and recognition of when to request formal urological evaluation in the primary care setting.

A new partial volume segmentation approach to extract bladder wall for computer-aided detection in virtual cystoscopy

We propose a new partial volume (PV) segmentation scheme to extract bladder wall for computer aided detection (CAD) of bladder lesions using multispectral MR images. Compared with CT images, MR images provide not only a better tissue contrast between bladder wall and bladder lumen, but also the multispectral information. As multispectral images are spatially registered over three-dimensional space, information extracted from them is more valuable than that extracted from each image individually. Furthermore, the intrinsic T1 and T2 contrast of the urine against the bladder wall eliminates the invasive air insufflation procedure. Because the earliest stages of bladder lesion growth tend to develop gradually and migrate slowly from the mucosa into the bladder wall, our proposed PV algorithm quantifies images as percentages of tissues inside each voxel. It preserves both morphology and texture information and provides tissue growth tendency in addition to the anatomical structure. Our CAD system utilizes a multi-scan protocol on dual (full and empty of urine) states of the bladder to extract both geometrical and texture information. Moreover, multi-scan of transverse and coronal MR images eliminates motion artifacts. Experimental results indicate that the presented scheme is feasible towards mass screening and lesion detection for virtual cystoscopy (VC).