Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance.

PURPOSE: Latent grade group ≥2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group ≥2 cancers. MATERIALS AND METHODS: Low- and intermediate-risk prostate cancer patients were assigned to training (n=333) and validation (n=202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with cross-validation, we evaluated methods for training classifiers of pathological grade group ≥2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort. RESULTS: PRONTO-e comprises 353 mRNA and copy number alteration features. PRONTO-m includes 94 clinical, mRNAs, copy number alterations, and methylation features at 14 and 12 loci, respectively. In independent validation, PRONTO-e and PRONTO-m predicted grade group ≥2 with respective true-positive rates of 0.81 and 0.76, and false-positive rates of 0.43 and 0.26. Both classifiers were resistant to sampling error and identified more upgrading cases than a well-validated presurgical risk calculator, CAPRA (Cancer of the Prostate Risk Assessment; P < .001). CONCLUSIONS: Two grade group classifiers with superior accuracy were developed by incorporating RNA and DNA features and validated in an independent cohort. Upon further validation in biopsy samples, classifiers with these performance characteristics could refine selection of men for active surveillance, extending their treatment-free survival and intervals between surveillance.

Bladder-sparing Radiotherapy for Muscle-invasive Bladder Cancer: A Qualitative Study to Identify Barriers and Enablers.

AIMS: Bladder-sparing radiotherapy for muscle-invasive bladder cancer (MIBC) may be underutilised in North America. To understand factors driving practice we used the Theoretical Domains Framework (TDF) to identify barriers and enablers of bladder-sparing radiotherapy utilisation. MATERIALS AND METHODS: A convenience sample of Canadian urologists, medical oncologists and radiation oncologists participated in individual semi-structured 1 h interviews. An interview guide was developed using the TDF to assess barriers and enablers of bladder-sparing radiotherapy use. Interviews were recorded and transcribed. Two investigators independently identified barriers and enablers and assigned them to specific themes. Participant recruitment continued until saturation. RESULTS: In total, 71 physicians were invited to participate and 34 (48%) agreed to be interviewed; 13 urologists, 11 radiation oncologists and 10 medical oncologists. We identified the following barriers to the use of bladder-sparing radiotherapy (relevant TDF domains in parentheses): (1) beliefs that radiotherapy has inferior survival compared with cystectomy (beliefs about consequences); (2) lack of referral from urology to radiation oncology (behavioural regulation; memory, attention and decision-making); (3) lack of 'champions' who advocate for radiotherapy (social and professional role); and (4) inadequate multidisciplinary collaboration (environmental context and resources). Predominant enablers to the use of bladder-sparing radiotherapy included: (1) 'champions' who believe in the value of radiotherapy (social and professional role); (2) beliefs by urologists that radiation oncologists should present radiotherapy options to all patients (social and professional role); (3) institutional policy that all MIBC patients should be seen by multiple specialists (environmental context and resources); (4) system facilitators of radiation oncology referral (i.e. nurse navigator) (environmental context and resources); and (5) patient-driven consultations seeking alternatives to cystectomy (social influences). CONCLUSIONS: These findings identify important barriers and enablers to the use of bladder-sparing radiotherapy in MIBC. Physician beliefs, access to multidisciplinary care and institutional context should be considered in efforts to increase the use of bladder-sparing radiotherapy.

Patterns of Referral to Radiation Oncology among Patients with Bladder Cancer: a Population-based Study.

AIMS: Radical radiotherapy is a reasonable alternative to cystectomy for some patients with invasive bladder cancer, and postoperative radiotherapy may be indicated in patients at high risk of local recurrence. Here we describe pre- and postoperative radiation oncology consultation among patients with bladder cancer in Ontario. MATERIALS AND METHODS: Records of radiotherapy and surgery were linked to the Ontario Cancer Registry (OCR) to identify all patients who received treatment with curative intent for bladder cancer between 1994 and 2008. Billing records were linked to the OCR to determine which patients were seen by radiation oncology before radical therapy or after cystectomy. Factors associated with radiation oncology consultation were explored by logistic regression. RESULTS: In total, 5259 patients with bladder cancer underwent treatment with curative intent in Ontario between 1994 and 2008. Of these, 3879 had primary cystectomy and 1380 had primary radiotherapy. Thirty-two per cent (1698/5259) of all patients were seen by radiation oncology. Independent factors associated with radiation oncology consultation included advanced age (P < 0.001), greater comorbidity (P < 0.001) and earlier year of diagnosis (P < 0.001). Rates also varied widely across geographical regions (range 20-57%); this variation was highly significant on multivariate analysis (P < 0.001). Only 10% (370/3759) of patients with cystectomy had a preoperative radiation oncology consultation. Ten per cent of patients treated by cystectomy (386/3879) were seen by radiation oncology in the postoperative setting; rates varied widely across regions (range 6-44%). These geographical variations were highly significant in the multivariate analysis (P < 0.001), which also showed that younger patients, those with higher stage (pT or pN), and those with positive margins, were more likely to have a postoperative radiation oncology consultation (all P < 0.001). Only 19% (80/420) of cases with positive margins had a postoperative radiation oncology consultation. CONCLUSIONS: One third of all patients with muscle-invasive bladder cancer in routine practice were seen in consultation by radiation oncology. Few patients who undergo cystectomy have the benefit of either a preoperative or a postoperative opinion about the potential role of radiotherapy in their management. Closer collaboration between radiation oncologists and urologists is warranted.

Curative therapy for bladder cancer in routine clinical practice: a population-based outcomes study.

AIMS: Definitive therapy of bladder cancer involves cystectomy or radiotherapy; controversy exists regarding optimal management. Here we describe the management and outcomes of patients treated in routine practice. MATERIALS AND METHODS: Treatment records were linked to the Ontario Cancer Registry to identify all cases of bladder cancer in Ontario treated with cystectomy or radiotherapy in 1994-2008. Practice patterns are described in three study periods: 1994-1998, 1999-2003, 2004-2008. Logistic regression, Cox model and propensity score analyses were used to evaluate factors associated with treatment choice and survival. RESULTS: In total, 3879 cases (74%) underwent cystectomy and 1380 (26%) were treated with primary radiotherapy. Cystectomy use increased over time (66, 75, 78%), whereas radiotherapy decreased (34, 25, 22%), P < 0.001. There was substantial regional variation in the proportion of cases undergoing radiotherapy (range 16-51%). Five year cancer-specific survival (CSS) and overall survival were 40 and 36% for surgical cases and 35 and 26% for radiotherapy cases (P < 0.001). In multivariate Cox model and propensity score analyses, there was no significant difference in CSS between surgery and radiotherapy (hazard ratio 0.99, 95% confidence interval 0.91-1.08); radiotherapy was associated with slightly inferior overall survival (hazard ratio 1.08, 95% confidence interval 1.00-1.16). CONCLUSION: Utilisation of cystectomy for bladder cancer in routine practice has increased over time with no evidence of a significant difference in CSS between radiotherapy and cystectomy.

Delivery of perioperative chemotherapy for bladder cancer in routine clinical practice.

BACKGROUND: Few articles have documented regimens and timing of perioperative chemotherapy for bladder cancer in routine practice. Here, we describe practice patterns in the general population of Ontario, Canada. METHODS: In this retrospective cohort study, treatment and physician billing records were linked to the Ontario Cancer Registry to describe use of neoadjuvant (NACT) and adjuvant (ACT) chemotherapy among all patients with muscle-invasive bladder cancer treated with cystectomy in Ontario 1994-2008. Time to initiation of ACT (TTAC) was measured from cystectomy. Multivariate Cox regression was used to identify factors associated with overall (OS) and cancer-specific survival (CSS). RESULTS: Of 2944 patients undergoing cystectomy, 4% (129/2944) and 19% (571/2944) were treated with NACT and ACT, respectively. Five-year OS was 25% [95% confidence interval (CI) 17% to 34%] for NACT, 29% (95% CI 25% to 33%) for ACT cases. Among patients with identifiable drug regimens, cisplatin was used in 82% (253/308) and carboplatin in 14% (43/308). The most common regimens were gemcitabine-cisplatin (54%, 166/308) and methotrexate, vinblastine, doxorubicin, cisplatin (MVAC) (21%, 66/308). Mean TTAC was 10 weeks; 23% of patients had TTAC >12 weeks. TTAC >12 weeks was associated with inferior OS [hazard ratio (HR) 1.28, 95% CI 1.00-1.62] and CSS (HR 1.30, 95% CI 1.00-1.69). In adjusted analyses, OS and CSS were lower among patients treated with carboplatin compared with those treated with cisplatin; OS HR 2.14 (95% CI 1.40-3.29) and CSS HR 2.06 (95% CI 1.26-3.37). CONCLUSIONS: Most patients in the general population receive cisplatin, and this may be associated with superior outcomes to carboplatin. Initiation of ACT beyond 12 weeks is associated with inferior survival. Patients should start ACT as soon as they are medically fit to do so.

In vitro modulation of natural killer activity of human peripheral blood mononuclear cells against prostate tumor cell line.

OBJECTIVE: Natural killer (NK) cells have long been known to be involved in the recognition and lysis of tumor cells but the mechanisms contributing to deficient NK activity in patients with cancer remains unclear. Manipulation of them is likely essential to the success of cancer immunotherapy protocols although optimal stimulation and maintenance of NK activity remains elusive. Here we studied the stimulatory effects of PHA and K562, on NK activity of human peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: The NK activity of PBMCs against DU-145 was determined with 51Cr-release assay. The PBMCs were stimulated with PHA, either on one occasion or repetitively on three occasions (1-PHA-PBMC or 3-PHA-PBMC, respectively), and were incubated with irradiated K562 (iK562). The expression of CD56, NKG2D and MICA/B were detected on PBMCs and cell lines with flow cytometry. RESULTS: PHA stimulation increased the proportion of CD56+ cells and upregulated NKG2D expression on 1-PHA-PBMC and 3-PHA-PBMC, but co-incubation with iK562 decreased NKG2D expression on 1-PHA-PBMC without change of NKG2D expression on the 3-PHA-PBMC. NK activity of 1-PHA-PBMC appeared to decrease with co-incubation with iK562 compared to a significant increase in activity of 3-PHA-PBMC. A similar increase in interferon-γ (IFN-γ) secretion from 3-PHA-PBMC was demonstrated compared to 1-PHA-PBMC. DISCUSSION AND CONCLUSION: Our results demonstrated that varying the mitogen exposure to PBMCs affect the influence of iK562 on NK activity. This effect appeared to be unrelated to the subsequent expression of NKG2D or IFN-γ secretion. These results may be beneficial in the development of future cancer immunotherapy protocols.

Discrete Fourier analysis of ultrasound RF time series for detection of prostate cancer.

In this paper, we demonstrate that a set of six features extracted from the discrete Fourier transform of ultrasound Radio-Frequency (RF) time series can be used to detect prostate cancer with high sensitivity and specificity. Ultrasound RF time series refer to a series of echoes received from one spatial location of tissue while the imaging probe and the tissue are fixed in position. Our previous investigations have shown that at least one feature, fractal dimension, of these signals demonstrates strong correlation with the tissue microstructure. In the current paper, six new features that represent the frequency spectrum of the RF time series have been used, in conjunction with a neural network classification approach, to detect prostate cancer in regions of tissue as small as 0.03 cm2. Based on pathology results used as gold standard, we have acquired mean accuracy of 91%, mean sensitivity of 92% and mean specificity of 90% on seven human prostates.

Intra-operative inferior vena cava syndrome in a patient with autosomal dominant polycystic kidney disease.

The renal and hepatic cysts characteristic of autosomal dominant polycystic kidney disease can exert a mass effect on surrounding structures. If this involves the inferior vena cava (IVC), patients usually present with signs and symptoms characteristic of congestive heart failure. However, the absence of these signs or symptoms does not exclude a potentially hemodynamically significant IVC syndrome. This case report describes a patient with no pre-operative evidence of congestive heart failure or IVC compression, who subsequently experienced intra-operative hypotension and hypoxemia due to an IVC syndrome.

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors.

Prostate-specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5-PSA) that is safe and effectively activates a PSA-specific T-cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5-PSA induced PSA-specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock-transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding beta-galactosidase (Ad5-lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8(+) T lymphocytes. Although Ad5-PSA immunization prior to RM11psa challenge was protective, Ad5-PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm(3) were efficiently eliminated if Ad5-PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2, and tumor necrosis factor-alpha. In this case, antitumor immunity was still dominated by CD8(+) T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5-PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods.

Vaccines in urologic malignancies.

The prospect of activating the immune system to combat neoplastic disease has stimulated the interest of clinicians and scientists for over 100 years. Despite a few notable exceptions (especially with urologic malignancies), immunotherapy has not fully reached its considerable therapeutic potential for the treatment of cancer. Tumors undoubtedly express antigens that may act as targets for antitumor immunity, and advances in molecular biology and tumor immunology have recently revived the possibility of a cancer vaccine. This improved understanding has resulted in numerous successes with active immunotherapy in animal models and has facilitated the clinical testing of cancer vaccines. Ongoing advances in the identification of unique, tumor-specific antigens and their presentation to stimulate T cells will be necessary for the emergence of these novel vaccine therapies for cancer patients. Herein we review the current concepts of tumor immunology, including observations on cell types probably involved with the immune surveillance of tumors, the presentation and recognition of "foreign" antigens, and possible mechanisms of tumor escape from the immune response, all of which are critical to the understanding of new initiatives for cancer vaccine therapy. Finally, we review some of the successes and limitations of vaccine therapy for urologic malignancies.

Evaluation of gene transfer efficiency by viral vectors to murine bladder epithelium.

PURPOSE: In pre-clinical gene therapy studies of bladder cancer there is tremendous variation in the ability of viral vectors to deliver genetic material to bladder epithelium. Possible explanations for this variability may involve the physical parameters of delivering vectors in these experimental models. We examined the effects of intravesical volume and pressure during instillation as well as chemical modification of the bladder epithelium on subsequent gene expression in the bladder in mice. MATERIALS AND METHODS: Female C57B1/6 mice underwent intravesical instillation of the replication restricted canarypox virus (ALVAC) recombinant for the reporter genes luciferase or beta-galactosidase. Similar viral titers were instilled at different volumes and a pressure transducer measured intravesical pressure when the vector was instilled. Also, various agents, including 0.6 N hydrochloric acid, 0.4% oxychlorosene, poly-L-lysine and 0.25 M. ammonium chloride, were used to modify the bladder surface before vector instillation and then assayed for transgene expression. RESULTS: As expected, maximum intravesical pressure measured during instillation was significantly greater in mice instilled with a higher volume (33.1 versus 9.8 mm. Hg). Significantly more gene expression was detected in bladders instilled with a higher volume of viral vectors (p <0.05). Likewise, higher instillation pressures resulted in higher transgene expression in distant organs. Modification of the bladder epithelium with agents such as oxychlorosene and poly-L-lysine resulted in elevated gene expression with only minimal increases in systemic activity. CONCLUSIONS: Significant differences in gene expression are achieved by varying physical parameters during intravesical instillation. Increased gene expression associated with larger volume instillation may be responsible for some reported variability of gene transfer to the bladder. Alternate manipulations, such as modifying the bladder surface, may be done to enhance gene transfer to the urothelium without increasing systemic distribution.

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix.

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.

Biomarker distribution after injection into the canine prostate: implications for gene therapy.

OBJECTIVE: To evaluate the distribution of biomarkers after transrectal injection into the canine prostate and to report a method for enhancing the distribution of gene expression. MATERIALS AND METHODS: Carbon black was first used to evaluate the histopathological distribution in canine prostate of single or multiple injections via the transurethral, transperineal and transrectal routes. The distribution of canarypox virus (ALVAC) vector-delivered gene expression was then compared using both fluid-phase injection techniques and delivery in a solid carrier composed of a gelatine sponge matrix. RESULTS: After transurethral administration, carbon black was detected as scattered particles in ducts and acini, mostly in the periphery of the gland. Direct transrectal injection of carbon black resulted in a localized collection at the site of injection, with only a minimal peri-acinar distribution. Transrectal injection of the fluid-phase (virus suspended in diluent) ALVAC vector encoding the beta-galactosidase gene resulted in a similar distribution, with limited gene expression at the site of injection and in the needle track. Delivery of the same number of virus particles in the gelatine sponge matrix resulted in qualitatively greater gene expression. CONCLUSIONS: Direct injection of the canine prostate with biomarkers, including viral vectors, in the fluid-phase results in very localized gene expression, while the distribution was more widespread after delivery in a gelatine sponge matrix.

Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model.

BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P =.037 for the RM-1 model and P =.03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-alpha was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols.

Transurethral radiofrequency hot balloon thermal therapy in chronic nonbacterial prostatitis.

There is no universally successful therapy for chronic nonbacterial prostatitis. Successful outcomes of treatment have been independently reported with transurethral heat therapy and balloon dilation. The THERP transurethral "hot balloon" uses balloon dilation of the prostatic urethra and radiofrequency heating of the prostate. Patients with refractory chronic nonbacterial prostatitis were assessed with validated prostatitis-specific symptom indices prior to, 3, and 6 months after 900 seconds of THERP treatment. Although early results appeared promising the long-term results in four patients led to early termination of the study. Although one patient did have improvement at 6 months, no patient reported improvement at 9 months, and the adverse events (urinary retention, retrograde ejaculation, hematuria, urethral stricture, worsening of symptoms) of this treatment in prostatitis patients was significant. This study demonstrated no sustainable efficacy and substantial adverse effects for the use of combination balloon dilation and heat therapy for patients with chronic nonbacterial prostatitis.

Antenatal hydronephrosis: thresholds of renal pelvic diameter to predict insignificant postnatal pelviectasis.

We tested the parameters of gestational age and renal pelvic anteroposterior (AP) diameter of antenatally detected pelvicaliectasis for their ability to determine insignificant postnatal renal pelvic dilatation. A retrospective analysis of 10,365 antenatal sonograms revealed 121 kidneys with pelvicaliectasis, from which 99 sonograms with sufficient postnatal follow-up were reviewed. Gestational ages were classified as <20, 20-30, or >30 weeks. Thresholds of renal pelvic AP diameter in each gestational period that were predictive of postnatal insignificance were determined to be <6, <8, and <10 mm, respectively. Insignificance postnatally was defined as no or minimal renal pelvic splitting (Society for Fetal Urology grade