National Trends and Impact of Regionalization of Radical Cystectomy on Survival Outcomes in Patients with Muscle Invasive Bladder Cancer.

OBJECTIVE: To evaluate national trends and the effect of surgical volume on perioperative mortality and overall survival (OS)in patients undergoing radical cystectomy (RC) for muscle invasive bladder cancer (MIBC). METHODS: We investigated the National Cancer Database to identify patients with localized MIBC (cT2a-T4, M0) who underwent RC from 2004 to 2014. Demographics, 30- and 90-day mortality rates, as well as OS were analyzed. Hospitals were stratified into low-, medium-, and high-volume centers according to median number of RCs performed per year. Multivariate logistic regression models were fitted to identify independent predictors of perioperative mortality. Kaplan-Meier survival curves were generated to evaluate OS. Cox proportional hazard modeling was performed to identify independent predictors of OS. RESULTS: A total of 24,763 patients with localized MIBC who underwent RC from 2004 to 2014 were included in the study. Overall, most (70.85%) RCs occurred at low-volume hospitals, whereas only 15.83% were performed at high-volume hospitals. Thirty-day mortality rates were 2.87%, 2.19%, and 1.83% (P < .01); and 90-day mortality rates were 8.25%, 6.9%, and 5.9% (P < .01) at low-, medium-, and high-volume hospitals, respectively. Multivariate analyses identified RC volume as an independent predictor of 30- and 90-day mortality. RC in high-volume hospitals was associated with a 35% risk reduction in 30-day mortality (odds ratio 0.65, 95% confidence interval [CI] 0.49-0.85; P < .01), and a 26% risk reduction in 90-day mortality (0.74, 95% CI, 0.63-0.87; P < .01). CONCLUSIONS: Treatment at high-volume centers offers improved outcomes and OS benefit. However, in the United States, only 16% of RCs are performed in high-volume hospitals.

Pathological downstaging following radical cystectomy for muscle-invasive bladder cancer: Survival outcomes in the setting of neoadjuvant chemotherapy versus transurethral resection only.

INTRODUCTION: Neoadjuvant chemotherapy (NAC) improves survival for patients undergoing radical cystectomy for muscle-invasive bladder cancer (MIBC). The overall survival (OS) advantage with NAC is primarily seen in patients who achieve pathological downstaging. However, a substantial number of patients achieve pathological downstaging following transurethral resection (TUR) without NAC. OBJECTIVES: To analyze the OS outcomes in patients who achieve pathological downstaging in the setting of NAC vs. TUR only. MATERIALS AND METHODS: We reviewed the National Cancer Database (NCDB) for patients diagnosed with MIBC who underwent radical cystectomy between 2004 and 2014. Patients who achieved complete downstaging (CD) (pT0N0) or noninvasive downstaging (NID) (pT0/Tis/TaN0) were further analyzed. OS was evaluated by comparing those who underwent NAC to those who underwent TUR only. RESULTS: A total of 24,763 patients with MIBC were identified. 1,781 (7.2%) patients had NID and 1,015 (4.1%) had CD. Of all patients, 3,838 (15.5%) underwent NAC. In patients with NID, 757 (42.5%) underwent NAC and 1024 (57.5%) had cystectomy after TUR only. In patients with CD, 465 (45.8%) had NAC, while 550 (54.2%) had TUR only. In both NID and CD, cT2 patients were more likely to have TUR only (P = 0.019, P < 0.001), cT3 patients were more likely to receive NAC (P = 0.008, P < 0.001). Compared to the TUR only group, NAC was associated with improved 5-year OS in those with NID, 77% compared to 68% (HR 0.68, 95% CI [0.52-0.90]), as well as those with CD, 80% vs. 70% (HR 0.59, 95% CI [0.39-0.89]). CONCLUSIONS: NAC was associated with significant overall survival benefit in the subset of patients who achieved CD and NID at radical cystectomy. Overall, NAC was underutilized in patients with MIBC.

Increased expression of TRIP13 drives the tumorigenesis of bladder cancer in association with the EGFR signaling pathway.

Thyroid hormone receptor interactor 13 (TRIP13) is a crucial regulator of the spindle apparatus checkpoint and double-stranded break repair. The abnormal expression of TRIP13 was recently found in several human cancers, whereas the role of TRIP13 in the development of bladder cancer (BCa) has not been fully elucidated. Here, we reported that TRIP13 expression was elevated in BCa tissues compared with normal bladder tissues. Notably, the increased expression of TRIP13 was correlated with advanced tumor stage, lymph node metastasis, distant metastasis and reduced survival in BCa patients. Knockdown of TRIP13 in bladder cancer cells suppressed proliferation, induced cell cycle arrest, promoted apoptosis, and impaired cell motility, ultimately inhibiting tumor xenograft growth. Mechanistic investigations revealed that TRIP13 directly bound to epidermal growth factor receptor (EGFR), modulating the EGFR signaling pathway. Furthermore, TRIP13 expression was positively correlated with EGFR expression in BCa specimens, and the high expression of both TRIP13 and EGFR predicted poor survival. Overall, our results underscore the crucial role of TRIP13 in the tumorigenesis of BCa and provide a novel biomarker and therapeutic target for BCa treatment.

Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production.

We elucidate the role of p21/Waf-1, a cyclin-dependent kinase inhibitor, on the oncolytic infection and replication cycle of adenovirus by studying both mRNA and adenoviral proteins expression. We found that infection in the absence of p21 causes a significant increase in adenoviral genomes and late gene expression. Similarly, the oncolytic adenoviral infected p21−/− cells have earlier formation of replication foci and robust replication kinetics that were not observed in the wild type p21/Waf-1 intact cells. These findings suggest a culmination that the presence of intact p21 in host cells causes defects in the oncolytic viral life cycle which results in the production of immature and noninfectious particles.

Contemporary surgical outcomes of venous tumour thrombectomy managed with intraoperative Doppler ultrasound for kidney cancer.

INTRODUCTION: Radical nephrectomy (RN) with venous tumour thrombectomy (VTT) carries a significant morbidity and mortality risk. Examination of a contemporary single-institution series permits the development of a management algorithm and an audit its results. We report outcomes following the use of intraoperative colour Doppler ultrasound and our surgical pathway. METHODS: We retrospectively reviewed the records of all patients who underwent RN with VTT for kidney cancer between January 1, 2013 and October 1, 2016. Surgical complications, postoperative complications (Clavien-Dindo classification ≥3), 90-day readmission rates, and outcomes are reported. Multivariate linear regression, logistic regression, and Cox proportional hazard modelling were used to identify associations. RESULTS: Fifty-eight patients underwent RN with VTT. Of these, 26 (45%) patients had Mayo Clinic level III or IV thrombus and nineteen required venovenous/cardiopulmonary bypass. Three patients required patch grafting. The median length of hospital stay was eight days and there were 20 major complications. The 30-day readmission rate was 21% and the 90-day mortality rate was 8.9%. In multivariate analysis, low serum albumin and age-adjusted Charlson comorbidity score predicted length of stay. Increased intraoperative blood loss was significantly associated with increasing body mass index, serum creatinine, tumour thrombus level, and a history of significant weight loss >9.1kg. Low serum hematocrit predicted 90-day mortality. CONCLUSIONS: Intraoperative colour Doppler ultrasound is a useful tool and can facilitate caval preservation. Caval grafting can be avoided in most cases. Venovenous bypass can be avoided in many level III cases. Early therapeutic anticoagulation should be instituted with caution.

XBSeq2: a fast and accurate quantification of differential expression and differential polyadenylation.

BACKGROUND: RNA sequencing (RNA-seq) is a high throughput technology that profiles gene expression in a genome-wide manner. RNA-seq has been mainly used for testing differential expression (DE) of transcripts between two conditions and has recently been used for testing differential alternative polyadenylation (APA). In the past, many algorithms have been developed for detecting differentially expressed genes (DEGs) from RNA-seq experiments, including the one we developed, XBSeq, which paid special attention to the context-specific background noise that is ignored in conventional gene expression quantification and DE analysis of RNA-seq data. RESULTS: We present several major updates in XBSeq2, including alternative statistical testing and parameter estimation method for detecting DEGs, capacity to directly process alignment files and methods for testing differential APA usage. We evaluated the performance of XBSeq2 against several other methods by using simulated datasets in terms of area under the receiver operating characteristic (ROC) curve (AUC), number of false discoveries and statistical power. We also benchmarked different methods concerning execution time and computational memory consumed. Finally, we demonstrated the functionality of XBSeq2 by using a set of in-house generated clear cell renal carcinoma (ccRCC) samples. CONCLUSIONS: We present several major updates to XBSeq. By using simulated datasets, we demonstrated that, overall, XBSeq2 performs equally well as XBSeq in terms of several statistical metrics and both perform better than DESeq2 and edgeR. In addition, XBSeq2 is faster in speed and consumes much less computational memory compared to XBSeq, allowing users to evaluate differential expression and APA events in parallel. XBSeq2 is available from Bioconductor: http://bioconductor.org/packages/XBSeq/.

Identification of miR-30b-3p and miR-30d-5p as direct regulators of androgen receptor signaling in prostate cancer by complementary functional microRNA library screening.

The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3'-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3'-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d-5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa.

Characterization of a novel metastatic prostate cancer cell line of LNCaP origin.

BACKGROUND: The LNCaP cell line was originally isolated from the lymph node of a patient with metastatic prostate cancer. Many cell lines have been derived from LNCaP by selective pressures to study different aspects of prostate cancer progression. When injected subcutaneously into male athymic nude mice, LNCaP and its derivatives rarely metastasize. METHODS: Here, we describe the characteristics of a new LNCaP derivative, JHU-LNCaP-SM, which was generated by long term passage in normal cell culture conditions. RESULTS: Short tandem repeat (STR) analysis and genomic sequencing verified JHU-LNCaP-SM derivation from parental LNCaP cells. JHU-LNCaP-SM cells express the same mutated androgen receptor (AR) but unlike LNCaP, are no longer androgen dependent for growth. The cells demonstrate an attenuated androgen responsiveness in transcriptional assays and retain androgen sensitive expression of PSA, AR, and PSMA. Unlike parental LNCaP, JHU-LNCaP-SM cells quickly form subcutaneous tumors in male athymic nude mice, reliably metastasize to the lymph nodes and display a striking intra-tumoral and spreading hemorrhagic phenotype as tumor xenografts. CONCLUSIONS: The JHU-LNCaP-SM cell line is a new isolate of LNCaP, which facilitates practical, preclinical studies of spontaneous metastasis of prostate cancer through lymphatic tissues.

A functional screen identifies miRNAs that inhibit DNA repair and sensitize prostate cancer cells to ionizing radiation.

MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents or through predicted miRNA regulation of DNA repair genes. We hypothesized that additional DNA damage regulating miRNAs could be identified by screening a library of 810 miRNA mimetics for the ability to alter cellular sensitivity to ionizing radiation (IR). A prostate cancer Metridia luciferase cell model was applied to examine the effects of individual miRNAs on IR sensitivity. A large percentage of miRNA mimetics were found to increase cellular sensitivity to IR, while a smaller percentage were protective. Two of the most potent IR sensitizing miRNAs, miR-890 and miR-744-3p, significantly delayed IR induced DNA damage repair. Both miRNAs inhibited the expression of multiple components of DNA damage response and DNA repair. miR-890 directly targeted MAD2L2, as well as WEE1 and XPC, where miR-744-3p directly targeted RAD23B. Knock-down of individual miR-890 targets by siRNA was not sufficient to ablate miR-890 radiosensitization, signifying that miR-890 functions by regulating multiple DNA repair genes. Intratumoral delivery of miR-890 mimetics prior to IR therapy significantly enhanced IR therapeutic efficacy. These results reveal novel miRNA regulation of DNA repair and identify miR-890 as a potent IR sensitizing agent.

Real-time, near-infrared fluorescence imaging with an optimized dye/light source/camera combination for surgical guidance of prostate cancer.

PURPOSE: The prostate-specific membrane antigen (PSMA) is a surface glycoprotein overexpressed on malignant prostate cells, as well as in the neovasculature of many tumors. Recent efforts to target PSMA for imaging prostate cancer rely on suitably functionalized low-molecular-weight agents. YC-27 is a low-molecular-weight, urea-based agent that enables near-infrared (NIR) imaging of PSMA in vivo. EXPERIMENTAL DESIGN: We have developed and validated a laparoscopic imaging system (including an optimized light source, LumiNIR) that is capable of imaging small tumor burdens with minimal background fluorescence in real-time laparoscopic extirpative surgery of small prostate tumor xenografts in murine and porcine models. RESULTS: In a mouse model, we demonstrate the feasibility of using real-time NIR laparoscopic imaging to detect and surgically remove PSMA-positive xenografts. We then validate the use of our laparoscopic real-time NIR imaging system in a large animal model. Our novel light source, which is optimized for YC-27, is capable of detecting as little as 12.4 pg/mL of the compound (2.48-pg YC-27 in 200-µL agarose). Finally, in a mouse xenograft model, we demonstrate that the use of real-time NIR imaging can reduce positive surgical margins (PSM). CONCLUSIONS: These data indicate that a NIR-emitting fluorophore targeted to PSMA may allow improved surgical treatment of human prostate cancer, reduce the rate of PSMs, and alleviate the need for adjuvant radiotherapy postoperatively.

A novel approach for detecting viable and tissue-specific circulating tumor cells through an adenovirus-based reporter vector.

BACKGROUND: Circulating tumor cells (CTCs) hold great promise as biomarkers and are a direct source of tumor cells through a simple blood draw. However, CTCs are rare and their detection requires sensitive and specific methods to overcome the overwhelming hematocyte population. Therefore, CTC detection remains technically challenging. METHODS: An assay was developed for detecting viable and tissue-specific CTCs using a tropism-enhanced and conditionally replicating reporter adenovirus (CTC-RV). Adenoviral replication was made prostate-specific by placing the E1A gene under the control of the probasin promoter and prostate-specific antigen enhancer (PSE-PBN). Viral tropism was expanded through capsid-displayed integrin targeting peptides. A secreted reporter, humanized Metridia Luciferase (hMLuc), was engineered for expression during the major late phase of viral replication. The assay involves red blood cell lysis, cell collection, viral infection, and subsequent quantification of reporter activity from cellular media. Assay and reporter stability, cell specificity and sensitivity were evaluated in cell dilution models in human blood. RESULTS: A conditionally replicating prostate-selective adenovirus reporter and CTC assay system were generated. The secreted reporter, MLuc, was found to be stable for at least 3 days under assay conditions. CTC detection, modeled by cell dilution in blood, was selective for androgen receptor positive prostate cancer (PCa) cells. Serial dilution demonstrated assay linearity and sensitivity to as few as three cells. Prostate cancer cell viability declined after several hours in anticoagulated blood at ambient temperatures. CONCLUSIONS: Conditionally replicative adenoviral vectors and secreted reporters offer a functional method to detect viable CTCs with cell specificity and high sensitivity.

Evaluation of prostate-specific membrane antigen as an imaging reporter.

UNLABELLED: Genetic reporters provide a noninvasive method to monitor and evaluate a population of cells. The ideal properties of a gene reporter-probe system include biocompatibility, lack of immunogenicity, low background expression or signal, and high sensitivity of detection. The prostate-specific membrane antigen (PSMA) is an attractive candidate for a genetic reporter as it is a human transmembrane protein with a selective expression pattern, and there are several PSMA imaging agents available for clinical and preclinical applications. We evaluated the use of PSMA as a genetic imaging reporter by comparison to 2 clinically established reporters, the mutant herpes simplex virus type I thymidine kinase and the human sodium-iodide symporter. METHODS: Adenoviruses expressing each reporter were constructed and validated in vitro for expression and function. To compare PSMA with existing imaging reporters, a bilateral Matrigel suspension model was established with nude mice bearing cells equally infected with each reporter or control adenovirus. Dynamic PET was performed, and time-activity curves were generated for each reporter-probe pair. RESULTS: A comparison of peak target-to-background ratios revealed that PSMA offered the highest ratio relative to the control Matrigel suspension as well as muscle. Further, as proof of concept, PSMA was applied as an imaging reporter to monitor adenoviral liver transduction with both nuclear and optical imaging probes. CONCLUSION: These preliminary studies support further development of PSMA as a noninvasive genetic reporter.

Prevalence of the HOXB13 G84E prostate cancer risk allele in men treated with radical prostatectomy.

OBJECTIVE: To determine the prevalence and clinical correlates of the G84E mutation in the homeobox transcription factor, or HOXB13, gene using DNA samples from 9559 men with prostate cancer undergoing radical prostatectomy. PATIENTS AND METHODS: DNA samples from men treated with radical prostatectomy at the University of Michigan and John Hopkins University were genotyped for G84E and this was confirmed by Sanger sequencing. The frequency and distribution of this allele was determined according to specific patient characteristics (family history, age at diagnosis, pathological Gleason grade and stage). RESULTS: Of 9559 patients, 128 (1.3%) were heterozygous carriers of G84E. Patients who possessed the variant were more likely to have a family history of prostate cancer than those who did not (46.0 vs 35.4%; P = 0.006). G84E carriers were also more likely to be diagnosed at a younger age than non-carriers (55.2 years vs 58.1 years; P < 0.001). No difference in the proportion of patients diagnosed with high grade or advanced stage tumours according to carrier status was observed. CONCLUSIONS: In the present study, carriers of the rare G84E variant in HOXB13 were both younger at the time of diagnosis and more likely to have a family history of prostate cancer compared with homozygotes for the wild-type allele. No significant differences in allele frequency were detected according to selected clinical characteristics of prostate cancer. Further investigation is required to evaluate the role of HOXB13 in prostate carcinogenesis.

Mechanism of growth inhibition of prostate cancer xenografts by valproic acid.

UNLABELLED: Valproic Acid (VPA), a histone deacetylase inhibitor, has been demonstrated to cause a marked decrease in proliferation of prostate cancer (PCa) cells in vitro and a significant reduction in tumor volume in vivo. The goal of this study is to better understand the VPA-induced growth inhibition in vivo, by studying expression of various markers in PCa xenografts. METHODS: For in vitro experiments, PCa cells were treated with 0, 0.6, and 1.2 mM VPA for 14 days. For in vivo models, experimental animals received 0.4% VPA in drinking water for 35 days. Tissue microarray was generated using cell pellets and excised xenografts. RESULTS: VPA treatment causes cell cycle arrest in PCa cells in vivo, as determined by increase in p21 and p27 and decrease in cyclin D1 expression. Increased expression of cytokeratin18 was also seen in xenografts. LNCaP xenografts in treated animals had reduced androgen receptor (AR) expression. While decreased proliferation was found in vitro, increase in apoptosis was found to be the reason for decreased tumor growth in vivo. Also, an anti-angiogenic effect was observed after VPA treatment. CONCLUSION: VPA inhibits tumor growth by multiple mechanisms including cell cycle arrest, induction of differentiation, and inhibition of growth of tumor vasculature.

A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the ß-actin promoter and enhancer.

Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human ß-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.

Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts.

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen-positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

Does valproic acid induce neuroendocrine differentiation in prostate cancer?

Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatment in vivo. In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting.

Adenovirus targeting to prostate-specific membrane antigen through virus-displayed, semirandom peptide library screening.

The convergence of phage-displayed peptide libraries and recombinant viral vectors launched a promising new direction in targeted viral gene therapeutics, but the translation of targeting peptides to functional cancer therapeutic agents has been challenging. Here, we report progress in developing a successful strategy to optimize targeted viral infection through adenovirus-displayed, semirandom peptide libraries. A phage-derived peptide targeting the prostate-specific membrane antigen (PSMA) was genetically incorporated into the adenoviral capsid Fiber protein and flanked by random peptide cassettes. The resulting adenovirus library was biopanned against PSMA-expressing cells and tumors to identify a PSMA-retargeted adenovirus. While the initial peptide alone could not target viral infection, the selected virus preferentially infects PSMA-expressing cells through the targeting peptide and infects LNCaP tumors after intravenous injection. Our results indicate that virus-displayed, semirandom peptide libraries can be used to optimize targeting infection. This approach represents a novel principle for developing targeted agents in a variety of disease models.

Cryoimmunotherapy in urologic oncology.

Cryoablation is gaining acceptance as a primary treatment of localized as well as a salvage therapy of metastatic urologic malignancies. Anecdotal clinical reports suggest cryoablation can induce a systemic anti-tumor immune response; this phenomenon has been confirmed in animal models. To capitalize on this stimulatory effect of cryotherapy for control of advanced malignancies, it must be further intensified. This article reviews the existing evidence regarding cryoimmunology and discusses the mechanisms for generation of an anti-tumor immune response. Several immunotherapy approaches that can be combined with cryoablation to devise a cryoimmunotherapeutic strategy with potential to affect the progression of metastatic disease are described.

Prostate stem cell antigen enhancer and uroplakin II promoter based bladder cancer targeted tissue-specific vector.

PURPOSE: To construct a dual specific vector which contains prostate stem cell antigen enhancer (PSCAE) and uroplakin II (UPII) promoter targeted bladder cancer. METHODS: UPII promoter and PSCAE were amplified by polymerase chain reaction (PCR). Luciferase gene (LUC) was obtained from plasmid pBK-CMV-LUC. PSCAE, UPII promoter and LUC were inserted into shuttle plasmid to create Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. Rp-UPII-LUC and Rp-PSCAE-UPII-LUC were cotransfected with pCMV-beta-gal into various cell lines at the presence or absence of androgen receptor agonist R1881 and androgen receptor antagonist flutamide. Luminescence was detected with luciferase assay kit and counted on liquid scintillation counter. RESULTS: Bladder cancer cells showed higher LUC activity than non-bladder cancer cells after transfected with plasmids Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. PSCAE could improve the LUC activity in both AR positive and AR negative bladder cancer cells but not in non-bladder cancer cells and normal human urothelial (NHU) cells. R1881 could increase the LUC activity in AR positive bladder cancer cells but not in AR negative bladder cancer cells and non-bladder cancer cells. Flutamide could not inactivate PSCAE in bladder cancer cells. CONCLUSIONS: PSCAE can improve target gene expression in bladder cancer cells but not in non-bladder cancer cells and NHU cells. PSCAE maintains a certain level of androgen independent activity in bladder cancer cells. PSCAE is active in both AR positive and AR negative bladder cancer cells. The results suggest that combination of PSCAE with UPII promoter is feasible in constructing bladder cancer-specific vectors.