Assessment of conservative dietary management as a method for normalization of 24-h urine pH in stone formers.

Low urine pH is a metabolic risk factor for stone formation. While medical therapy is typically prescribed (as urinary alkalinization), patients typically prefer dietary modifications. We aimed to assess capacity to alter urine pH with dietary management alone. We analyzed a retrospective cohort of stone formers seen between 2000 and 2015 with multiple 24-h urine collections (24hUC). Patients ≥ 18 years old with low urine pH (< 6.0) were included; those prescribed alkalinizing agents or thiazides were excluded. Demographic data, 24hUC parameters, and medications were abstracted. 24hUC was utilized to calculate gastrointestinal alkali absorption (GIAA). The primary outcome was urine pH ≥ 6.0 on second 24hUC. Predictors were selected utilizing multivariable logistic regression. The database consisted of 2197 stone formers; 224 of these met inclusion criteria. On second 24hUC, 124 (55.4%) achieved a favorable pH ≥ 6.0. On univariable analysis, a second pH ≥ 6.0 was associated with high initial pH, low initial sulfate, younger age, increase in citrate/GIAA/urine volume, and decrease in ammonium (P < 0.02). On multivariable analysis, high initial pH (OR = 23.64, P < 0.001), high initial GIAA (OR = 1.03, P = 0.001), lower initial sulfate (OR = 0.95, P < 0.001), increase in urine volume (OR = 2.19, P = 0.001), increase in GIAA (OR = 8.6, P < 0.001), increase in citrate (OR = 2.7, P = 0.014), decrease in ammonium (OR = 0.18, P < 0.001), and younger age (OR = 0.97, P = 0.025) were associated with a second pH ≥ 6.0. The analysis demonstrated a corrected AUC of 0.853. These data suggest that certain dietary recommendations (increases in urine volume, citrate, GIAA, and decreased acid load) may normalize urine pH in a select group of patients. This may allow urologists to counsel patients with low urine pH on possibility of success with dietary modification alone.

An in vitro evaluation of laser settings and location in the efficiency of the popcorn effect.

To examine different locations and laser settings' effects on the efficiency of the "popcorn" method of laser lithotripsy, which consists of placing the laser in a group of small stones and firing continuously to break them into smaller particles. Pre-fragmented BegoStones were created between 2 and 4 mm to mimic typical popcorning conditions. A 0.5 g collection of fragments was placed into 3D-printed models (a spherical calyx and ellipsoid pelvis model) and a 200-µm laser fiber was positioned above the stones. The laser was fired for 2 min with irrigation, with 5 trials at each setting: 0.2 J/50 Hz, 0.5 J/20 Hz, 0.5 J/40 Hz, 1 J/20 Hz, 0.2 J/80 Hz, 0.5 J/80 Hz. After drying, fragmentation efficiency was determined by calculating the mass of stones reduced to sub-2 mm particles. Statistical analysis was performed with ANOVA and Student's t test. The trials within the calyx model were significantly more efficient compared to the pelvis (0.19 vs 0.15 g, p = 0.01). When comparing laser settings, there was a difference between groups by one-way ANOVA [F(5,54) = 8.503, p = 5.47 × 10-6]. Post hoc tests showed a power setting of 0.5 J/80 Hz was significantly more efficient than low-power settings 0.2 J/50 Hz and 0.5 J/20 Hz (p < 0.05). Additionally, 0.2 J/50 Hz was significantly less efficient than 0.5 J/40 Hz, 1 J/20 Hz, and 0.2 J/80 Hz. Popcorning is most efficient in smaller spaces; we recommend displacement of stones into a calyx before popcorning. No difference was seen between high-power settings, although 0.5 J/40 Hz and 0.5 J/80 Hz performed best, suggesting that moderate energy popcorning methods with at least 0.5 J per pulse are most efficient.

Effect of Laser Settings and Irrigation Rates on Ureteral Temperature During Holmium Laser Lithotripsy, an In Vitro Model.

PURPOSE: Ureteroscopic laser lithotripsy requires irrigation for adequate visualization and temperature control during treatment of ureteral stones. However, there are little data on how different irrigation and laser settings affect the ureteral wall and surrounding tissues. This effect has become an important consideration with the advent of high-powered lasers. We therefore evaluated the effect of laser settings and irrigation flow on ureteral temperature in an in vitro setting. MATERIALS AND METHODS: To mimic ureteroscopic laser lithotripsy, we simulated clinically relevant irrigation flow rates and fired a Holmium:Yttrium-aluminum-garnet (Ho:YAG) laser while monitoring "intraureteral" temperature. The probe tip of a thermometer was placed 1 mm from the tip of a 200 µm laser fiber, which was fired for 60 seconds at 0.2 J/50 Hz, 0.6 J/6 Hz, 0.8 J/8 Hz, 1 J/10 Hz, and 1 J/20 Hz within a tubing system that allowed for specified room temperature flow rates (100, 50, and 0 mL/minute). We recorded temperatures every 5 seconds. The maximum temperature was noted, and each laser/flow trial was duplicated. Averaged maximum temperatures were compared using analysis of variance across irrigation settings. RESULTS: At 100 cc/minute, only the 1 J/20 Hz laser setting produced a significantly higher maximum temperature (p < 0.01), although this finding was not clinically significant at a maximum of 30.7°C. At a lower irrigation rate of 50 cc/minute, the 1 J/20 Hz setting was again the only significantly higher maximum temperature (p < 0.05), although this temperature crossed the toxic threshold at a maximum of 43.4°C. With no flow, all maximum temperatures reached over 43°C, with 0.8 J/8 Hz, 1 J/10 Hz, and 1 J/20 Hz each statistically higher than the lower-energy settings (p < 0.05). The maximum temperature at 1 J/20 Hz with no irrigation was over 100°C. CONCLUSIONS: Despite increasing laser settings, adequate irrigation can maintain relatively stable temperatures within an in vitro ureteral system. As irrigation rates decrease, even lower power laser settings produce a clinically significant increase in maximum temperature, potentially causing ureteral tissue injury.

Next-Generation Single-Use Ureteroscopes: An In Vitro Comparison.

INTRODUCTION: Single-use ureteroscopes have been gaining popularity in recent years. We compare the optics, deflection, and irrigation flow of two novel single-use flexible ureteroscopes-the YC-FR-A and the NeoFlex-with contemporary reusable and single-use flexible ureteroscopes. METHODS: Five flexible ureteroscopes, YC-FR-A (YouCare Tech, China), NeoFlex (Neoscope, Inc., USA), LithoVue (Boston Scientific, USA), Flex-Xc (Karl Storz, Germany), and Cobra (Richard Wolf, Germany), were assessed in vitro for image resolution, distortion, field of view, depth of field, color representation, and grayscale imaging. Ureteroscope deflection and irrigation were also compared. RESULTS: The YC-FR-A showed a resolution of 5.04 lines/mm and 4.3% image distortion. NeoFlex showed a resolution of 17.9 lines/mm and 14.0% image distortion. No substantial difference was demonstrated regarding the other optic characteristics between the two. Across all tested ureteroscopes, single-use or reusable, the digital scopes performed best with regard to optics. The YC-FR-A had the greatest deflection at baseline, but lacks two-way deflection. The NeoFlex had comparable deflection at baseline to reusable devices. Both ureteroscopes had substantial loss of deflection with instruments in the working channel. The YC-FR-A had the greatest irrigation rate. The NeoFlex has comparable irrigation to contemporary ureteroscopes. CONCLUSIONS: The YouCare single-use fiberoptic flexible ureteroscope and NeoFlex single-use digital flexible ureteroscope perform comparably to current reusable ureteroscopes, possibly making each a viable alternative in the future. Newer YouCare single-use flexible ureteroscopes with a digital platform and two-way deflection may be more competitive, while the NeoFlex devices are undergoing rapid improvement as well. Further testing is necessary to validate the clinical performance and utility of these ureteroscopes, given the wide variety of single-use devices under development.

Acquired resistance to the second-generation androgen receptor antagonist enzalutamide in castration-resistant prostate cancer.

Enzalutamide (MDV3100) is a second generation Androgen Receptor (AR) antagonist with proven efficacy in the treatment of castration resistant prostate cancer (CRPC). The majority of treated patients, however, develop resistance and disease progression and there is a critical need to identify novel targetable pathways mediating resistance. The purpose of this study was to develop and extensively characterize a series of enzalutamide-resistant prostate cancer cell lines. Four genetically distinct AR-positive and AR-pathway dependent prostate cancer cell lines (CWR-R1, LAPC-4, LNCaP, VCaP) were made resistant to enzalutamide by long-term culture (> 6 months) in enzalutamide. Extensive characterization of these lines documented divergent in vitro growth characteristics and AR pathway modulation. Enzalutamide-resistant LNCaP and CWR-R1 cells, but not LAPC-4 and VCAP cells, demonstrated increased castration-resistant and metastatic growth in vivo. Global gene expression analyses between short-term enzalutamide treated vs. enzalutamide-resistant cells identified both AR pathway and non-AR pathway associated changes that were restored upon acquisition of enzalutamide resistance. Further analyses revealed very few common gene expression changes between the four resistant cell lines. Thus, while AR-mediated pathways contribute in part to enzalutamide resistance, an unbiased approach across several cell lines demonstrates a greater contribution toward resistance via pleiotropic, non-AR mediated mechanisms.

Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue.

BACKGROUND: Personalization of cancer therapy requires molecular evaluation of tumor tissue. Traditional tissue preservation involves formalin fixation, which degrades the quality of nucleic acids. Strategies to bank frozen prostate tissue can interfere with diagnostic studies. PAXgene is an alternative fixative that preserves protein and nucleic acid quality. METHODS: Portions of prostates obtained from autopsy specimens were fixed in either 10% buffered formalin or PAXgene, and processed and embedded in paraffin. Additional sections were immediately embedded in OCT and frozen. DNA and RNA were extracted from the formalin-fixed, PAXgene-fixed, or frozen tissue. Quantitative PCR was used to compare the quality of DNA and RNA obtained from all three tissue types. In addition, 5 µm sections were cut from specimens devoid of cancer and from prostate cancer specimens obtained at prostatectomy and fixed in PAXgene. They were either stained with hematoxylin and eosin or interrogated with antibodies for p63, PSA and p504. RESULTS: Comparable tissue morphology was observed in both the formalin and PAXgene-fixed specimens. Similarly, immunohistochemical expression of the P63, PSA and P504 proteins was comparable between formalin and PAXgene fixation techniques. DNA from the PAXgene-fixed tissue was of similar quality to that from frozen tissue. RNA was also amplified with up to 8-fold greater efficiency in the PAXgene fixed tissue compared to the formalin-fixed tissue. CONCLUSIONS: Prostate specimens fixed with PAXgene have preserved histologic morphology, stain appropriately, and have preserved quality of nucleic acids. PAXgene fixation facilitates the use of prostatectomy tissue for molecular biology techniques such as next-generation sequencing.

Low-grade prostate cancer diverges early from high grade and metastatic disease.

Understanding the developmental relationship between indolent and aggressive tumors is central to understanding disease progression and making treatment decisions. For example, most men diagnosed with prostate cancer have clinically indolent disease and die from other causes. Overtreatment of prostate cancer remains a concern. Here we use laser microdissection followed by exome sequencing of low- and high-grade prostate cancer foci from four subjects, and metastatic disease from two of those subjects, to evaluate the molecular relationship of coincident cancer foci. Seventy of 79 (87%) high-confidence somatic mutations in low-grade disease were private to low-grade foci. In contrast, high-grade foci and metastases harbored many of the same mutations. In cases in which there was a metastatic focus, 15 of 80 (19%) high-confidence somatic mutations in high-grade foci were private. Seven of the 80 (9%) were shared with low-grade foci and 65 (82%) were shared with metastatic foci. Notably, mutations in cancer-associated genes and the p53 signaling pathway were found exclusively in high-grade foci and metastases. The pattern of mutations is consistent with early divergence between low- and high-grade foci and late divergence between high-grade foci and metastases. These data provide insights into the development of high-grade and metastatic prostate cancer.

Sox2 is an androgen receptor-repressed gene that promotes castration-resistant prostate cancer.

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways.