Characterization of Schu S4 aro mutants as live attenuated tularemia vaccine candidates.

There is a need for development of an effective vaccine against Francisella tularensis, as this potential bioweapon has a high mortality rate and low infectious dose when delivered via the aerosol route. Moreover, this Tier 1 agent has a history of weaponization. We engineered targeted mutations in the Type A strain F. tularensis subspecies tularensis Schu S4 in aro genes encoding critical enzymes in aromatic amino acid biosynthesis. F. tularensis Schu S4ΔaroC, Schu S4ΔaroD, and Schu S4ΔaroCΔaroD mutant strains were attenuated for intracellular growth in vitro and for virulence in vivo and, conferred protection against pulmonary wild-type (WT) F. tularensis Schu S4 challenge in the C57BL/6 mouse model. F. tularensis Schu S4ΔaroD was identified as the most promising vaccine candidate, demonstrating protection against high-dose intranasal challenge; it protected against 1,000 CFU Schu S4, the highest level of protection tested to date. It also provided complete protection against challenge with 92 CFU of a F. tularensis subspecies holarctica strain (Type B). Mice responded to vaccination with Schu S4ΔaroD with systemic IgM and IgG2c, as well as the production of a functional T cell response as measured in the splenocyte-macrophage co-culture assay. This vaccine was further characterized for dissemination, histopathology, and cytokine/chemokine gene induction at defined time points following intranasal vaccination which confirmed its attenuation compared to WT Schu S4. Cytokine, chemokine, and antibody induction patterns compared to wild-type Schu S4 distinguish protective vs. pathogenic responses to F. tularensis and elucidate correlates of protection associated with vaccination against this agent.

Cytogenetic analysis of 130 renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations.

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO.

Diabetes Is a Risk Factor for Inflatable Penile Prosthesis Infection: Analysis of a Large Statewide Database.

INTRODUCTION: Although diabetes mellitus (DM) is often discussed as a risk factor for inflatable penile prosthesis (IPP) infection, the link between DM diagnosis and IPP infection remains controversial. High-quality population-based data linking DM to an increased risk of IPP infection have not been published. AIM: To evaluate the association of DM with IPP infection in a large public New York state database. METHODS: The New York Statewide Planning and Research Cooperative System (SPARCS) database was queried for men who underwent initial IPP insertion from 1995-2014. Diabetic patients were identified using ICD-9-CM codes. Patients presenting for first operation with diagnosis or Current Procedural Terminology codes suggestive of prior IPP surgery were excluded. Chi-squared analyses were performed to compare infection rates in diabetics and non-diabetics within the pre- and postantibiotic impregnated eras. Multivariate Cox proportional hazards models were constructed to evaluate whether or not DM was independently associated with IPP infection in the time periods before (1995-2003) and after (2004-2014) the widespread availability of antibiotic impregnated penile prostheses. MAIN OUTCOME MEASURE: Time to prosthesis infection was measured. RESULTS: 14,969 patients underwent initial IPP insertion during the study period. The overall infection rate was 343/14,969 (2.3%). Infections occurred at a median 3.9 months after implant (interquartile ratio: 1.0-25.0 months). Infectious complications were experienced by 3% (133/4,478) of diabetic patients and 2% (210/10,491) of non-diabetic patients (P < .001). Diabetes was associated with a significantly increased IPP infection risk on multivariable analysis controlling for age, race, comorbidities, insurance status, annual surgeon volume, and era of implantation (Hazard Ratio: 1.32, 95% CI: 1.05-1.66, P = .016). CONCLUSION: Our analysis supports the notion that DM is a risk factor for IPP infection. This has important implications for patient selection and counseling, and raises the question of whether this increased risk can be mitigated by optimization of glycemic control before surgery. Lipsky MJ, Onyeji I, Golan R, et al. Diabetes Is a Risk Factor for Inflatable Penile Prosthesis Infection: Analysis of a Large Statewide Database. Sex Med 2019;7:35-40.

The Prevalence of Bladder Cancer During Cystoscopy for Asymptomatic Microscopic Hematuria.

OBJECTIVE: To investigate the rate of bladder cancer in patients undergoing cystoscopic evaluation for asymptomatic microscopic hematuria (AMH) in order to identify groups at sufficiently low-risk for bladder cancer in whom invasive testing may be avoided. METHODS: We performed a retrospective review of patients who underwent cystoscopic evaluation for AMH between 2010 and 2018. Age, gender, smoking status, history of pelvic radiation, and number of red blood cells per high-power field on urine microscopy were recorded. We used logistic regression to explore the association between specific risk factors and a diagnosis of bladder cancer on cystoscopy. RESULTS: Among the 2118 patients who underwent cystoscopy for AMH, 25 patients (1.2%) were diagnosed with a bladder cancer, all of which were nonmuscle invasive urothelial carcinoma. There were no bladder cancers detected in patients under the age of 50. Older age and positive smoking history were significantly associated with bladder cancer. CONCLUSION: Bladder cancer was an uncommon finding on cystoscopy among patients being evaluated for AMH, especially in younger patients. We confirmed several known risk factors for bladder cancer, including older age and smoking history. Further studies are required to evaluate the utility of cystoscopy for identifying latent bladder cancers in low-risk patients.

Improved Tolerability of a Salmonella enterica Serovar Typhimurium Live-Attenuated Vaccine Strain Achieved by Balancing Inflammatory Potential with Immunogenicity.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA We determined that S Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (109 CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD50 (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

The National Resident Matching Program Code of Conduct: What Is the Perceived Degree of Compliance During the Urology Match Process?

OBJECTIVE: To assess the incidence of violations to the National Resident Matching Program Code of Conduct during the Urology Match. MATERIALS AND METHODS: We sent a survey to all 285 applicants to a single urologic program during 2017 Match cycle; the questions pertained to illegal interview questions, postinterview communication, second-look qualifications, and the applicant's perceived impact of these factors. RESULTS: At total of 166 responses were obtained (response rate 58%). Ninety-six (58%) applicants received follow-up communication from at least 1 program, the majority from multiple programs. Of those who received postinterview communication, 13% reported verbal communication, and 19% felt misled by communication to believe they had a higher chance of matching at a program. Fifty (30%) respondents did a second-look visit, and 44% reported feeling obligated to do so in order to match. Finally, 141 of 166 (85%) applicants reported being asked illegal questions regarding personal life, rank list, and/or other interviews. Female applicants reported being asked illegal questions significantly more frequently than male applicants (P < .01). CONCLUSION: During the 2017 Urology Match, a high proportion of applicants experienced violations of the National Resident Matching Program Code of Conduct. Violations included illegal questions, postinterview written and verbal communication, and pressure to do second-look visits. These findings corroborate numerous anecdotal reports, and may provide the groundwork to improve the fairness of the residency application process for the future.

Assessment of Discomfort and Pain in Patients Undergoing Fusion Magnetic Resonance Imaging-guided vs TRUS-guided Prostate Biopsy.

OBJECTIVE: To investigate patient pain perception from receiving magnetic resonance imaging fusion-guided prostate biopsy (FBx) in addition to transrectal ultrasound-guided template biopsy (TBx) vs pain from standard TBx alone. MATERIALS AND METHODS: Patients undergoing FBx + TBx or TBx alone from April 2016 to February 2017 completed a validated pain survey after biopsy. Responses were graded from 0 to 10 (0: no pain or willing to return for repeat procedure; 10: excruciating pain or not willing to return for repeat procedure if necessary). Procedures were performed by a single urologist with a 1% lidocaine periprostatic nerve block. Pain scores between groups were compared via Mann-Whitney U test. RESULTS: A total of 170 patients were included, with 96 FBx + TBx and 74 TBx. For FBX + TBx and TBx, mean age was 68.6 (±9.7) and 66.1 (±8.3) (P = .08), and median number of cores was 14.5 (8-22) and 12 (6-14) (P < .001), respectively. Both groups had mild pain from the procedure overall (median pain score 3 [range 0-9]), the probe insertion (2 [0-8]), and the biopsies themselves (3 [1-10]). If necessary, both groups were very willing to come back for the same procedure again (1 [0-10]). CONCLUSION: Patients reported no difference in pain or discomfort with FBx + TBx relative to TBx alone. Both procedures were mildly painful with patients very willing to return for repeat biopsy if necessary. Patients' pain experience should not influence whether additional FBx is performed.

Trends in Penile Prosthetics: Influence of Patient Demographics, Surgeon Volume, and Hospital Volume on Type of Penile Prosthesis Inserted in New York State.

INTRODUCTION: Penile prostheses (PPs) are a discrete, well-tolerated treatment option for men with medical refractory erectile dysfunction. Despite the increasing prevalence of erectile dysfunction, multiple series evaluating inpatient data have found a decrease in the frequency of PP surgery during the past decade. AIMS: To investigate trends in PP surgery and factors affecting the choice of different PPs in New York State. METHODS: This study used the New York State Department of Health Statewide Planning and Research Cooperative (SPARCS) data cohort that includes longitudinal information on hospital discharges, ambulatory surgery, emergency department visits, and outpatient services. Patients older than 18 years who underwent inflatable or non-inflatable PP insertion from 2000 to 2014 were included in the study. OUTCOMES: Influence of patient demographics, surgeon volume, and hospital volume on type of PP inserted. RESULTS: Since 2000, 14,114 patients received PP surgery in New York State; 12,352 PPs (88%) were inflatable and 1,762 (12%) were non-inflatable, with facility-level variation from 0% to 100%. There was an increasing trend in the number of annual procedures performed, with rates of non-inflatable PP insertion decreasing annually (P < .01). More procedures were performed in the ambulatory setting over time (P < .01). Important predictors of device choice were insurance type, year of insertion, hospital and surgeon volume, and the presence of comorbidities. CLINICAL IMPLICATIONS: Major influences in choice of PP inserted include racial and socioeconomic factors and surgeon and hospital surgical volume. STRENGTHS AND LIMITATIONS: Use of the SPARCS database, which captures inpatient and outpatient services, allows for more accurate insight into trends in contrast to inpatient sampling alone. However, SPARCS is limited to patients within New York State and the results might not be generalizable to men in other states. Also, patient preference was not accounted for in these analyses, which can play a role in PP selection. CONCLUSIONS: During the past 14 years, there has been an increasing trend in inflatable PP surgery for the management of erectile dysfunction. Most procedures are performed in the ambulatory setting and not previously captured by prior studies using inpatient data. Kashanian JA, Golan R, Sun T, et al. Trends in Penile Prosthetics: Influence of Patient Demographics, Surgeon Volume, and Hospital Volume on Type of Penile Prosthesis Inserted in New York State. J Sex Med 2018;15:245-250.

Outcomes Following Clinical Complete Response to Neoadjuvant Chemotherapy for Muscle-invasive Urothelial Carcinoma of the Bladder in Patients Refusing Radical Cystectomy.

OBJECTIVE: To investigate survival outcomes of patients with muscle-invasive bladder cancer (MIBC) that demonstrate complete clinical response (cT0) to neoadjuvant chemotherapy (NAC) and then reject subsequent radical cystectomy (RC). METHODS: A retrospective chart review identified patients with MIBC who were cT0 after platinum-based NAC. cT0 was defined as negative cytology, cystoscopy with transurethral resection of bladder tumor, and imaging. cT0 patients refusing for RC were followed up with cytology, cystoscopy with biopsy, and cross-sectional imaging. RESULTS: Forty-eight patients were identified with MIBC that were cT0 after NAC. Seven patients underwent immediate RC, whereas 41 elected bladder preservation with close surveillance. Of those remaining 41 patients, mean age was 68 ± 11 years with median follow-up of 35 months. NAC regimens were 46% methotrexate/vinblastine/doxorubicin/cisplatin, 39% gemcitabine/cisplatin, and 15% other platinum-based therapies. Five-year cancer-specific survival was 87%, disease-free survival was 58%, and cystectomy-free survival was 79%. A total of 19 patients (46%) relapsed with 5.4-month median recurrence time. CONCLUSION: Bladder preservation may be a reasonable option in a highly select subset of patients with MIBC who are complete clinical responders after NAC. For those patients that were cT0 after NAC and refused or were ineligible for RC, 5-year disease-free survival was nearly 60% and cancer-specific survival was nearly 90%. Future studies should focus on identifying clinical and molecular factors associated with a durable pathologic complete response after NAC.

Timing and Predictors of Early Urologic and Infectious Complications After Renal Transplant: An Analysis of a New York Statewide Database.

OBJECTIVES: The most common complications after renal transplant are urologic and are a cause of significant morbidity in a vulnerable population. We sought to characterize the timing and predictors of urologic complications after renal transplant using a statewide database. MATERIALS AND METHODS: We queried the New York Statewide Planning and Research Cooperative System database to identify patients who underwent renal transplant from 2005 to 2013. Postoperative complications included hydronephrosis, ureteral stricture, vesicoureteral reflux, nephrolithiasis, and urinary tract infections. Cox proportional hazards model was used to assess independent predictors of urologic complications. RESULTS: In total, 9038 patients were included in the analyses. Urologic complications occurred in 11.3% of patients and included hydronephrosis (12.0%), nephrolithiasis (2.8%), ureteral stricture (2.4%), and vesicoureteral reflux (1.5%). We found that 23% experienced at least one urinary tract infection. On multivariate analysis, predictors of urologic complications included medicare insurance, hypertension, and prior urinary tract infection. Graft recipients from living donors were less likely to experience urologic complications than deceased-donor kidney recipients (P < .001). CONCLUSIONS: Urologic complications occur in a significant proportion of renal transplants. Further study is needed to identify risk factors for complications after renal transplantation to decrease morbidity in this vulnerable population.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

Preoperative Antibiotics Before Bladder Biopsy: Are They Necessary?

OBJECTIVE: To compare postoperative infectious outcomes of bladder biopsies performed in the office without antibiotic prophylaxis vs those done with preoperative antibiotic prophylaxis in the operating room (OR). MATERIALS AND METHODS: Our institutional review board-approved database was retrospectively reviewed for patients who underwent bladder biopsy in the office or in the OR between July 2014 and August 2015. All patients with bladder biopsies performed in the OR and none in the office received preoperative antibiotic prophylaxis. Patient characteristics and post-procedural outcomes including bacteriuria, urinary tract infection (UTI), and febrile UTI were recorded. The rates of these outcomes were compared between the 2 groups using the chi-square test. Patients were excluded from analysis if they experienced a UTI or were prescribed antibiotics within 30 days before their procedure. RESULTS: In all, 216 biopsies were identified (106 in the office and 110 in the OR). No difference was noted in the rate of UTI (0.94% vs 0.91%, P = .98), or febrile UTI (0% vs 0.91%, P = .33) between those undergoing bladder biopsy in the office and those in the OR. There was no difference in the incidence of new urinary symptoms (2.8% vs 5.5%, P = .33) or post-procedural bacteriuria (3.8% vs 3.6%, P = .96). CONCLUSION: Since the introduction of the mandated use of antibiotics for routine procedures such as bladder biopsy, antibiotic use has markedly increased. Our data suggest that the preoperative antibiotic prophylaxis that is recommended may not confer benefit to select patients. At a time when antibiotic stewardship is of utmost importance, guidelines regarding its use should be reconsidered.

Understanding Simple Cystectomy for Benign Disease: A Unique Patient Cohort With Significant Risks.

OBJECTIVE: To explore patient characteristics and complications of simple cystectomy for benign disease. A secondary objective was to compare these parameters to those in radical cystectomy. METHODS: Current Procedural Terminology codes were used to identify patients within the National Surgical Quality Improvement Program who underwent cystectomy (2005-2014). International Classification of Disease, Ninth Revision, codes were used to classify patients with benign or malignant diagnoses. Perioperative complications (30 days) were identified and logistic regression analysis was used to identify factors associated with morbidity. RESULTS: We identified 389 patients who had a cystectomy for benign diagnosis. A total of 235 patients (60.4%) had complications. The most frequently reported complication was bleeding (requiring a transfusion within 72 hours) in 150 patients (38.6%). Other complications were wound infection (63, 16.2%), respiratory complication (29, 7.5%), wound dehiscence (8, 2.1%), renal complication (9, 2.3%), cardiovascular complication (6, 1.5%), and postoperative deep vein thrombosis (8; 2.1). On multivariate analysis, diabetes (odds ratio 1.9, P = .04) and smoking (odds ratio 1.8, P = .03) were associated with increased odds of any complication. Compared with those with cystectomy for malignancy, this cohort was younger, with higher American Society of Anesthesiologists scores, and chronic kidney disease stages, and the complication risk was similar (60.4% vs 57.7%, P = .3). CONCLUSION: Our data suggest that the benign and radical cystectomy patients are different patient populations, with benign patients being younger with a higher American Society of Anesthesiologists class. Even in benign disease, cystectomy is not without risk, and patients should be counseled accordingly.

Virulence of invasive Salmonella Typhimurium ST313 in animal models of infection.

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea.

Predictors of biochemical recurrence after primary focal cryosurgery (hemiablation) for localized prostate cancer: A multi-institutional analytic comparison of Phoenix and Stuttgart criteria.

BACKGROUND: The Phoenix definition (PD) and Stuttgart definition (SD) designed to determine biochemical recurrence (BCR) in patients with postradiotherapy and high-intensity focused ultrasound organ-confined prostate cancer are being applied to follow patients after cryosurgery. We sought to identify predictors of BCR using the PD and SD criteria in patients who underwent primary focal cryosurgery (PFC). MATERIALS AND METHODS: We performed a retrospective review of patients who underwent PFC (hemiablation) at 2 referral centers from 2000 to 2014. Patients were followed up with serial prostate-specific antigen (PSA). PSA levels, pre- and post-PFC biopsy, Gleason scores, number of positive cores, and BCR (PD = [PSA nadir+2ng/ml]; SD = [PSA nadir+1.2ng/ml]) were recorded. Patients who experienced BCR were biopsied, monitored carefully or treated at the discretion of the treating urologist. Cox regression and survival analyses were performed to assess time to BCR using PD and SD. RESULTS: A total of 163 patients were included with a median follow-up of 36.6 (interquartile range: 18.9-56.4) months. In all, 64 (39.5%) and 98 (60.5%) experienced BCR based on PD and SD, respectively. On multivariable Cox regression, the number of positive pre-PFC biopsy cores was an independent predictor of both PD (hazard ratio [HR] = 1.4, P = 0.001) and SD (HR = 1.3, P = 0.006) BCRs. Post-PFC PSA nadir was an independent predictor of BCR using the PD (HR = 2.2, P = 0.024) but not SD (HR = 1.4, P = 0.181). Survival analysis demonstrated a 3-year BCR-free survival rate of 56% and 36% for PD and SD, respectively. Of those biopsied after BCR, 14/26 (53.8%) using the PD and 18/35 (51.4%) using the SD were found to have residual/recurrent cancer. Of those with prostate cancer on post-PFC biopsy, 57.1% of those with BCR by the PD and 66.7% of those with BCR by the SD were found to have a Gleason score ≥7. CONCLUSION: Both the PD and the SD may be used to determine BCR in post-PFC patients. However, the ideal definition of BCR after PFC remains to be elucidated.

Urine Exosomes for Non-Invasive Assessment of Gene Expression and Mutations of Prostate Cancer.

PURPOSE: The analysis of exosome/microvesicle (extracellular vesicles (EVs)) and the RNA packaged within them (exoRNA) has the potential to provide a non-invasive platform to detect and monitor disease related gene expression potentially in lieu of more invasive procedures such as biopsy. However, few studies have tested the diagnostic potential of EV analysis in humans. EXPERIMENTAL DESIGN: The ability of EV analysis to accurately reflect prostate tissue mRNA expression was examined by comparing urinary EV TMPRSS2:ERG exoRNA from pre-radical prostatectomy (RP) patients versus corresponding RP tissue in 21 patients. To examine the differential expression of TMPRSS2:ERG across patient groups a random urine sample was taken without prostate massage from a cohort of 207 men including prostate biopsy negative (Bx Neg, n = 39), prostate biopsy positive (Bx Pos, n = 47), post-radical prostatectomy (post-RP, n = 37), un-biopsied healthy age-matched men (No Bx, n = 44), and young male controls (Cont, n = 40). The use of EVs was also examined as a potential platform to non-invasively differentiate Bx Pos versus Bx Neg patients via the detection of known prostate cancer genes TMPRSS2:ERG, BIRC5, ERG, PCA3 and TMPRSS2. RESULTS: In this technical pilot study urinary EVs had a sensitivity: 81% (13/16), specificity: 80% (4/5) and an overall accuracy: 81% (17/21) for non-invasive detection of TMPRSS2:ERG versus RP tissue. The rate of TMPRSS2:ERG exoRNA detection was found to increase with age and the expression level correlated with Bx Pos status. Receiver operator characteristic analyses demonstrated that various cancer-related genes could differentiate Bx Pos from Bx Neg patients using exoRNA isolated from urinary EVs: BIRC5 (AUC 0.674 (CI:0.560-0.788), ERG (AUC 0.785 (CI:0.680-0.890), PCA3 (AUC 0.681 (CI:0.567-0.795), TMPRSS2:ERG (AUC 0.744 (CI:0.600-0.888), and TMPRSS2 (AUC 0.637 (CI:0.519-0.754). CONCLUSION: This pilot study suggests that urinary EVs have the potential to be used as a platform to non-invasively differentiate patients with prostate cancer with very good accuracy. Larger studies are needed to confirm the potential for clinical utility.

The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence.

A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common.

Lessons Learned from Routine Intraoperative Ureteral Margin Frozen Sections during Radical Cystectomy.

INTRODUCTION: We examined the practice patterns of intraoperative ureteral frozen section during radical cystectomy and the impact of ureteral margin positivity on operative characteristics and oncologic outcomes. METHODS: The records of patients who underwent radical cystectomy at our institution from 2004 to 2011 were identified. Intraoperative ureteral frozen section characteristics were examined, including number, laterality, positivity, conversion to negative and final permanent section status. Logistic regression analysis was performed for predictors of operative time, change in urinary diversion, and biopsy confirmed upper tract recurrence and metastasis. RESULTS: A total of 590 intraoperative ureteral frozen sections were sent for analysis from 241 patients (mean age 69 years). The sections were positive in 12.9% of cases and conversion to negative was accomplished in 82%. Multiple sections were associated with longer operating time (561 vs 511 minutes, p=0.011). Sensitivity for the sections was 100% and specificity was 93.6%. Taking multiple ureteral resections did not alter the planned urinary diversion in any patient or increase perioperative complication rates. At a mean followup of 22±19.8 months, 7 patients (3%) experienced upper tract recurrence. Intraoperative ureteral frozen section conversion to negative was associated with improved overall survival but not with upper tract recurrence. CONCLUSIONS: Our practice of taking intraoperative ureteral frozen sections provided excellent sensitivity and specificity, and the prolonged operative time did not translate into increased perioperative complications. Conversion of positive to negative was associated with improved overall survival, independent of patient comorbidities and post-operative complications. No association was seen with upper tract recurrence but this was likely due to our high conversion rate to negative margins (82%), negative permanent section ureteral margin status in 97% of cases and the long followup time needed to demonstrate an association.

A rabbit model of non-typhoidal Salmonella bacteremia.

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.