Leonard G. Gomella

Effect of dutasteride on the risk of prostate cancer

BACKGROUND We conducted a study to determine whether dutasteride reduces the risk of incident prostate cancer, as detected on biopsy, among men who are at increased risk for the disease. METHODS In this 4-year, multicenter, randomized, double-blind, placebo-controlled, parallel-group study, we compared dutasteride, at a dose of 0.5 mg daily, with placebo. Men were eligible for inclusion in the study if they were 50 to 75 years of age, had a prostate-specific antigen (PSA) level of 2.5 to 10.0 ng per milliliter, and had had one negative prostate biopsy (6 to 12 cores) within 6 months before enrollment. Subjects underwent a 10-core transrectal ultrasound-guided biopsy at 2 and 4 years. RESULTS Among 6729 men who underwent a biopsy or prostate surgery, cancer was detected in 659 of the 3305 men in the dutasteride group, as compared with 858 of the 3424 men in the placebo group, representing a relative risk reduction with dutasteride of 22.8% (95% confidence interval, 15.2 to 29.8) over the 4-year study period (P<0.001). Overall, in years 1 through 4, among the 6706 men who underwent a needle biopsy, there were 220 tumors with a Gleason score of 7 to 10 among 3299 men in the dutasteride group and 233 among 3407 men in the placebo group (P=0.81). During years 3 and 4, there were 12 tumors with a Gleason score of 8 to 10 in the dutasteride group, as compared with only 1 in the placebo group (P=0.003). Dutasteride therapy, as compared with placebo, resulted in a reduction in the rate of acute urinary retention (1.6% vs. 6.7%, a 77.3% relative reduction). The incidence of adverse events was similar to that in studies of dutasteride therapy for benign prostatic hyperplasia, except that in our study, as compared with previous studies, the relative incidence of the composite category of cardiac failure was higher in the dutasteride group than in the placebo group (0.7% [30 men] vs. 0.4% [16 men], P=0.03). CONCLUSIONS Over the course of the 4-year study period, dutasteride reduced the risk of incident prostate cancer detected on biopsy and improved the outcomes related to benign prostatic hyperplasia. (ClinicalTrials.gov number, NCT00056407.)

MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets

Small non‐coding microRNAs (miRNAs) contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers. However, the specific role of miRNAs in the metastatic process is still unknown. To seek a specific miRNA expression signature characterizing the metastatic phenotype of solid tumours, we performed a miRNA microarray analysis on 43 paired primary tumours (ten colon, ten bladder, 13 breast, and ten lung cancers) and one of their related metastatic lymph nodes. We identified a metastatic cancer miRNA signature comprising 15 overexpressed and 17 underexpressed miRNAs. Our results were confirmed by qRT‐PCR analysis. Among the miRNAs identified, some have a well‐characterized association with cancer progression, eg miR‐10b, miR‐21, miR‐30a, miR‐30e, miR‐125b, miR‐141, miR‐200b, miR‐200c, and miR‐205. To further support our data, we performed an immunohistochemical analysis for three well‐defined miRNA gene targets (PDCD4, DHFR, and HOXD10 genes) on a small series of paired colon, breast, and bladder cancers, and one of their metastatic lymph nodes. We found that the immunohistochemical expression of these targets significantly follows the corresponding miRNA deregulation. Our results suggest that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets. Copyright

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- And tissue-specific microRNAs

Significance MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology. Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.

Dual roles of PARP-1 promote cancer growth and progression

UNLABELLED PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)-positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. SIGNIFICANCE These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting.

Retroperitoneal and pelvic extraperitoneal laparoscopy : An international perspective

OBJECTIVES To assess technical preferences and current practice trends of retroperitoneal and pelvic extraperitoneal laparoscopy. METHODS A questionnaire survey of 36 selected urologic laparoscopic centers worldwide was performed. RESULTS Twenty-four centers (67%) responded. Overall, 3988 laparoscopic procedures were reported: transperitoneal approach (n = 2945) and retroperitoneal/extraperitoneal approach (n = 1043). Retroperitoneoscopic/extraperitoneoscopic procedures included adrenalectomy (n = 74), nephrectomy (n = 299), ureteral procedures (n = 166), pelvic lymph node dissection (n = 197), bladder neck suspension (n = 210), varix ligation (n = 91), and lumbar sympathectomy (n = 6). Mean number of total laparoscopic procedures performed in 1995 per center was 41 (range 5 to 86). Major complications occurred in 49 (4.7%) patients and included visceral complications in 26 (2.5%) patients and vascular complications in 23 (2.2%). Open conversion was performed in 69 (6.6%) patients, electively in 41 and emergently in 28 (visceral injuries, n = 16; vascular injuries, n = 1 2). Retroperitoneoscopy/extraperitoneoscopy is gaining in acceptance worldwide: in 1993, the mean estimated ratio of transperitoneal laparoscopic cases versus retroperitoneoscopic/ extraperitoneoscopic cases per center was 74:26; however, in 1996 the ratio was 49:51. CONCLUSIONS Retroperitoneoscopy and pelvic extraperitoneoscopy are important adjuncts to the laparoscopic armamentarium in urologic surgery. The overall major complication rate associated with retroperitoneoscopy/extraperitoneoscopy was 4.7%.

Changes in circulating carcinoma cells in patients with metastatic prostate cancer correlate with disease status.

OBJECTIVES To investigate the diurnal variations in circulating tumor cells (CTCs) in metastatic carcinoma of the prostate (CAP) and to determine whether the change in CTCs correlated with disease progression. METHODS Samples were prepared by immunomagnetic selection of cells from 7 mL of blood targeting the epithelial cell adhesion molecule and differential fluorescent labeling of the collected cells using a nucleic acid dye, antibodies directed against the common leukocyte (CD45), and cytokeratin antigens. Events that stained with the nucleic acid dye and expressed cytokeratin but lacked CD45 were defined as CTCs by multiparameter flow cytometry. RESULTS Male controls (n = 22) exhibited 0.8 +/- 1.2 events per 7 mL blood compared with 5.9 +/- 4.7 in 10 samples from patients with localized CAP and 46.6 +/- 65.6 events in 10 samples from patients with metastatic CAP. Diurnal testing of 8 cases demonstrated stable levels of CTCs. Ten patients were serially analyzed during a 6-month period for serum prostate-specific antigen and CTCs. The correlation between the prostate-specific antigen level and CTC number was fair. Slow disease progression was found in 4 patients with low CTC numbers (3.0 +/- 3) but it was significantly higher than the control group (P <0.002). Rapid disease progression occurred in 6 patients who demonstrated high CTC numbers (68.5 +/- 71.9). Two patients received chemotherapy that caused substantial fluctuations in the CTCs with less pronounced changes in the prostate-specific antigen level. CONCLUSIONS We conclude that the level of CTCs can be quantified in the circulation of patients with metastatic CAP and that the change in CTCs correlates with disease progression with no diurnal variations.

Pelvic lymphadenectomy can be omitted in selected patients with carcinoma of the prostate: development of a system of patient selection☆

OBJECTIVES The prevalence of pelvic lymph node metastases in men with clinically localized prostate cancer has decreased dramatically over the past decade, possibly due to efforts at early detection. With a significantly lower incidence of pelvic node involvement, it may be possible to identify a segment of patients for whom pelvic lymph node dissection (PLND) may be omitted. This study was conducted to develop a method to select patients for whom PLND could be omitted. METHODS We analyzed serum prostate-specific antigen (PSA), clinical stage, biopsy Gleason score, and final pathologic stage in 481 men with clinically localized prostate cancer. These variables were compared to the risk of positive pelvic lymph nodes. RESULTS Logistic regression analysis determined that combining all three variables provided the best determination of final pathologic stage. A series of probability curves have been created to estimate the risk of positive lymph nodes in a given patient. Based on the distribution of patients in this study and using these probability functions, PLND could be avoided in up to 50% of patients with localized prostate cancer diagnosed by contemporary methods. CONCLUSIONS In properly selected patients, pelvic lymphadenectomy can be omitted in the staging and treatment of localized prostate cancer.

Proepithelin Promotes Migration and Invasion of 5637 Bladder Cancer Cells through the Activation of ERK1/2 and the Formation of a Paxillin/FAK/ERK Complex

The growth factor proepithelin (also known as progranulin, acrogranin, PC-derived growth factor, or granulin-epithelin precursor) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. Proepithelin is overexpressed in a great variety of cancer cell lines and clinical specimens of breast, ovarian, and renal cancer as well as glioblastomas. In this study, we have investigated the effects of proepithelin on bladder cancer cells using human recombinant proepithelin purified to homogeneity from 293-EBNA cells. Although proepithelin did not appreciably affect cell growth, it did promote migration of 5637 bladder cancer cells and stimulate in vitro wound closure and invasion. These effects required the activation of the mitogen-activated protein kinase pathway and paxillin, which upon proepithelin stimulation formed a complex with focal adhesion kinase and active extracellular signal-regulated kinase. Our results provide the first evidence for a role of proepithelin in stimulating migration and invasion of bladder cancer cells, and support the hypothesis that this growth factor may play a critical role in the establishment of the invasive phenotype.

PHASE I STUDY OF INTRAVESICAL VACCINIA VIRUS AS A VECTOR FOR GENE THERAPY OF BLADDER CANCER

PURPOSE Vaccinia virus is a DNA poxvirus previously used as a vaccine to eradicate smallpox. The virus has a high efficiency of infection, replicates in the cytoplasm without chromosomal integration and can transport a large amount of recombinant DNA without losing infectivity. Therefore, it is an excellent choice as a vector for gene delivery in vivo. Large quantities of vaccinia have been injected into dermal, subcutaneous and peripheral lymph node melanoma metastases without significant side effects, and with efficient infection of the tumor cells and recombinant gene transfection. To determine if vaccinia, when given intravesically, can effectively infect bladder mucosa and tumor with acceptable toxicity, we performed a phase I trial of intravesical vaccinia in patients with muscle invasive transitional cell carcinoma before radical cystectomy. MATERIALS AND METHODS After documenting immune competence and demonstration of a major reaction after revaccination, patients received 3 increasing doses of intravesical Dryvax vaccinia virus (Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania) that was provided by the Centers for Disease Control. Approximately 24 hours after the third dose, cystectomy was performed and the tissue was examined microscopically. RESULTS There were 4 patients who were treated. The 3 patients who received the highest doses (100 x 106 plaque forming units) had significant mucosal and submucosal inflammatory infiltration by lymphocytes, eosinophils, and plasma cells into tumor and normal tissue. Dendritic cells were recruited to the site after exposure to the vaccinia. Significant mucosal edema and vascular ectasia were seen. Tumor and normal urothelial cells showed evidence of viral infection, including enlarged vacuolated cells with cytoplasmic inclusions. There were no clinical or laboratory manifestations of vaccinia related toxicity except mild dysuria. Of the 4 patients 3 survived and were free of disease at 4-year followup. CONCLUSIONS Our study demonstrates that vaccinia virus can be administered safely into the bladder with recruitment of lymphocytes and induction of a brisk local inflammatory response. To our knowledge, this is the first report of direct delivery of live virus into the human bladder. The role of wild type vaccinia as immunotherapy for bladder cancer warrants further study. Furthermore, these data support the exploration of recombinant vaccinia as a putative gene therapy vector for intravesical infection and transfection of bladder tumor cells with cytokine or other genes, an approach that our group pioneered and most recently studied in patients with superficial melanoma.