Assessment and validation of a screening questionnaire for the diagnosis of pediatric bladder and bowel dysfunction.

INTRODUCTION: Pediatric bladder and bowel dysfunction (BBD) is a common problem in children. However, the current ability to diagnosis and quantify pediatric BBD is limited as only a few validated instruments exist. In addition, the current questionnaires are limited by their lack of psychometric processing and methods of validation. To address these issues, the authors developed a new questionnaire to objectively diagnose pediatric BBD symptoms. This study aimed to evaluate the performance of this newly devised objective instrument in diagnosing and quantifying the symptomatology of BBD in children. MATERIALS AND METHODS: An 18-item, 5-point questionnaire was developed using both a literature review and expert opinions. The total questionnaire score could range from 0 to 72. Questions were subgrouped into six symptom categories: (1) nocturnal enuresis, (2) lower urinary tract symptoms, (3) urinary holding, (4) infrequent urination, (5) bowel symptoms, and (6) daytime urinary incontinence. The questionnaire also assessed the degree of bother associated with the symptoms. Patients were divided into cases and controls, and these two groups were compared. DISCUSSION/RESULTS: A total of 1265 new patients (758 cases and 507 controls) completed the new BBD questionnaire. The mean age of the whole study cohort was 9.5 years (range, 3-19 years). The total mean questionnaire score was significantly higher at 23 (3-58) in the cases, compared with 8 (0-35) in the controls (p < 0.001) (Summary Figure). Reliability analysis of the 18-item instrument showed a Cronbach's alpha reliability coefficient of 0.80 for the scale. CONCLUSIONS: This new instrument provides a valid and reliable method for diagnosis of pediatric BBD and classification of patients into subcategories of BBD based on their specific symptoms.

Predictors of becoming overweight among pediatric patients at risk for urinary tract infections.

BACKGROUND: The association between obesity and urinary dysfunction in childhood has been described, albeit through retrospective analysis, making temporal relationships difficult to establish. OBJECTIVE: The objective of this study was to determine risk factors for significant weight gain in children at risk for recurrent urinary tract infections. STUDY DESIGN: A secondary analysis of the Randomized Intervention for Children with Vesicoureteral Reflux and Careful Urinary Tract Infection Evaluation trials was conducted. The outcome of interest in these children was significant increase in body mass index (BMI) percentile (>85th BMI percentile for sex and age) in previously normal-weight children. Multivariable logistic regression was used to determine the independent effects of predetermined risk factors. RESULTS: In total, 446 patients were included in the study. Most patients aged less than 1 year at study entry (229, 51%), and 399 (89%) of patients were female. Eighty-four patients (17%) became clinically overweight. Patients assigned to prophylactic antibiotics were not more likely to gain significant BMI percentiles (adjusted odds ratio [aOR] = 1.1, 95% confidence interval [CI]=0.6-1.8). Significant BMI percentiles were gained in Hispanic/Latino patients compared with whites (aOR = 3.3, 95% CI=1.7-6.4), in children who were infants at study enrollment compared with non-infants (aOR = 2.1, 95% CI=1.2-3.8), and in those with persistent reflux during the study period (aOR = 2.1, 95% CI=1.0-4.3). Neither patients assigned to prophylactic antibiotics (aOR = 1.1, 95% CI=0.6-1.8) nor patients with bladder and bowel dysfunction (BBD) (aOR = 1.2, 95% CI=0.6-2.3) were more likely to gain significant BMI percentiles. DISCUSSION: Significant BMI percentile gain is common in patients at risk for UTIs. Hispanic/Latino ethnicity, persistent reflux, and younger age, specifically infants than non-infants, were identified as independent risk factors for becoming overweight in this population. Exposure to prophylactic antibiotics and BBD were not associated with becoming overweight. CONCLUSION: Risk for becoming overweight should be discussed when managing patients at risk for UTIs, especially in the subpopulations identified.

Impact of continuous low-dose antibiotic prophylaxis on growth in children with vesicoureteral reflux.

BACKGROUND: Continuous antibiotic prophylaxis (CAP) is a mainstay of treatment for children with vesicoureteral reflux (VUR). There has been an increasing focus on the effect of antibiotics on gut microbiota and subsequent repercussions on growth. Continuous antibiotic prophylaxis is generally considered safe; however, its impact on growth in children with VUR remains unknown. OBJECTIVE: This objective of this study was to determine whether CAP altered height, weight, or body mass index (BMI) in children with VUR. STUDY DESIGN: Children diagnosed with primary VUR were identified. Demographics including weight and height percentiles, BMI and BMI percentiles, age, gender, antibiotic class, prior antibiotic use, urinary tract infection history, and breakthrough infections were tested in univariate and multivariate analyses. Primary outcome was change in BMI as well as weight, height, and BMI percentiles. RESULTS: One hundred eighty patients (146 girls, 34 boys) were prescribed CAP at mean age of 29.2 ± 26.2 months. Mean follow-up on CAP was 4.1 ± 3.3 years, with median follow-up of 3.08 years. Mean increase in weight percentile was 1.49 (2.02 males, 1.37 females; P = 0.46). Mean decrease in height percentile was -4.44 (-2.18 males, -4.95 females; P = 0.51). Age at diagnosis (P = 0.004) and history of prior treatment courses of antibiotics (P = 0.007) were associated with a significant BMI increase (Fig. 1). Body mass index percentile increased from 58.4 to 66.5; however, this increase was only significant in children aged above 1 year (P < 0.0001). Of note, children above 1 year of age were significantly more likely to have a history of prior treatment courses of antibiotics (58% vs 32%; P < 0.0001), and when controlling for prior antibiotic use, the increase in BMI percentile in those over 1 year of age did not reach significance. DISCUSSION: The use of antibiotics has been associated with alterations in pediatric growth parameters in both animal models and clinical studies. However, little information exists on the impact of prophylactic-dosed antibiotics on growth. While this study is limited by the retrospective analysis and small sample size, it was found that the use of CAP did impact growth parameters, with a stronger effect seen in children who had received prior treatment courses of antibiotics before the infection leading to the initiation of CAP. CONCLUSION: Continuous antibiotic prophylaxis was correlated with significant increase in BMI in children with prior antibiotic usage and a significant increase in BMI percentile in children aged above 1 year. Continuous antibiotic prophylaxis was also associated with decreased height percentiles, particularly in patients aged less than 1 year, though it did not reach statistical significance. Further analysis is needed to investigate whether these effects on weight, height, and BMI are persistent and clinically significant.

The Iowa Voiding Improvement Partnership experience: Early observations with a collaborative pediatric uro-psychologic clinic.

INTRODUCTION: Bladder and bowel dysfunction (BBD) are common problems in children presenting for pediatric urology referral. Psychiatric issues may be present in these children, making their treatment difficult. In 2013, the University of Iowa Voiding Improvement Partnership (VIP) Clinic was established for the treatment of these patients. STUDY OBJECTIVE: This study sought to evaluate early experience with this specialized clinic, to determine the pre-existing urologic and psychologic conditions seen in these clinic patients, and to evaluate the clinical outcomes after VIP treatment. STUDY DESIGN: A retrospective, Institutional Review Board-approved review of all patients seen in the VIP Clinic was performed. The following were evaluated: patient demographics, underlying urologic and psychologic diagnosis, and treatment decisions. All patients were asked to complete the University of Iowa Pediatric Bladder and Bowel Dysfunction questionnaire at each visit. Questionnaire scores from the patients' first and most recent clinic visits were compared. RESULTS: To date, 66 patients have been evaluated at the VIP Clinic, accounting for 112 clinic visits. The mean age of the VIP patients was 8.5 years (range, 4-16) and 59% of the patients were female. Pre-existing urological conditions and psychological conditions are shown in the Summary Table. A large number (62%) of patients required further psychological evaluation, secondary to concern for an undiagnosed psychiatric issue. In addition, the clinic had improved patients' BBD symptoms over time. When first evaluated in clinic, patients had an average Iowa BBD Questionnaire score of 31 (range, 47-13), which improved to an average score of 25 (range, 47-7) (P = 0.03). In addition, 23% of the patients improved to where they could be discharged from uro-psychologic care. CONCLUSIONS: It was feasible to establish the present multidisciplinary uro-psychology clinic. Such a clinic may unearth undiagnosed psychological issues, and improve bowel and bladder dysfunction in these difficult-to-treat patients.

Time to resolution: A prospective evaluation from the Society for Fetal Urology hydronephrosis registry.

INTRODUCTION: The resolution rate of prenatal urinary tract dilation (UTD) has been documented in several retrospective studies. The present study analyzed prospective observational registry data, with the aim of determining time to resolution among patients prenatally identified with mild postnatal UTD. MATERIALS AND METHODS: A total of 248 subjects, from four centers, were prospectively enrolled from 2008 to 2015. Exclusion criteria included other anomalies (n = 69), fewer than two ultrasounds, and/or <3 months follow-up (n = 26). Resolution was defined as Outcome A (SFU 0) and Outcome B (SFU 0/1). Fisher's exact test, Mann-Whitney U or Kruskal-Wallis test and Kaplan-Meier were used for analysis. RESULTS/DISCUSSION: The median follow-up for 179 (n = 137 males) subjects was 15 months (IQR 7-24), range 0-56 months. VCUG was performed in 100 (57%) and VUR identified in 15 (15%). There was no association with reflux and resolution (P = 0.72). For resolution assessment (n = 153), lower grades were likely to resolve and demonstrated a higher rate in the Outcome B classification. Time to resolution also favored lower grades, with the majority resolving within 2 years (Figure). Surgical intervention was performed in 14 (8%). It is interesting to note that regardless of grade of UTD, there was no difference in frequency of US or the time that RUS was first performed. Practitioners performed the first RUS of life within a narrow window ranging from 0.27 RUS/month for Grade 1 UTD to 0.30 RUS/month for Grade 4 UTD. It was speculated that this practice pattern occurrence likely reflected the deficiency in knowledge by primary care providers, and identified a potential opportunity for education. The SFU registry substantiates that the vast majority of patients will demonstrate transient UTD and most cases that do not resolve will improve within 2 years of life. This data could be used to further an evidenced-based approach towards the evaluation and management of prenatal UTD, as outlined in the multidisciplinary consensus statement for prenatal urinary tract dilation. CONCLUSIONS: This prospective registry confirms that the majority of prenatal UTD is transient, resolution occurs within the first 3 years of life, and most patients will not need intervention. Redefining SFU 1 as normal increased the resolution rate. A large proportion of patients were not evaluated with a VCUG, therefore impact of VUR could not be determined.

Inter-rater reliability of distal ureteral diameter ratio compared to grade of VUR.

INTRODUCTION AND OBJECTIVE: Management of vesicoureteral reflux (VUR) remains controversial, and reflux grade constitutes an important prognostic factor. Recent work has suggested that distal ureteral diameter ratio (UDR) is a predictive factor relative to clinical outcome independent of grade. Previous studies have noted significant inter-rater variability with grading of VUR. The present study compared inter-rater reliability of reflux grade and UDR in children with primary VUR. STUDY DESIGN: Four pediatric urologists independently reviewed, in a blinded fashion, voiding cystourethrograms. For each renal unit, grade was assigned according to the standardized international scale. The UDR was calculated by dividing the largest ureteral diameter within the false pelvis by the distance between L1-L3 vertebral bodies. Correlation within each rater was determined using Pearson's correlation coefficient. Reliability of VUR grade and UDR were calculated using two-way ANOVA model inter-rater agreement. RESULTS: Four independent raters reliably measured VUR grade (ICC = 0.87, 95% CI = 0.78-0.93) and UDR (ICC = 0.95, 95% CI = 0.92-0.97). While UDR and grade were equally reliable measures, UDR had a tighter confidence interval. For each rater, grade and UDR were well correlated (r = 0.73-0.84; P < 0.0001). For higher-grade reflux, grade was more variable than UDR (Summary Figure). Using empirical thresholds, the increased variability of grade compared with UDR may lead to significant differences in clinical decision-making among physicians (P = 0.022). DISCUSSION: Known discordance with grading reflux emphasizes the need for a more objective VUR measurement, as clinicians and parents often opt for clinical intervention based on both clinical course and the likelihood of spontaneous resolution. While ICC for UDR and grade were not significantly different, the confidence intervals for grade were wider due to greater variability among grade measurements. This suggests that using UDR measurements may lead to more accurate characterization of VUR and ultimately more consistent clinical decision-making across providers. CONCLUSIONS: Ureteral diameter ratio has good inter-rater reliability among pediatric urologists, with less clinically relevant variability than VUR grade. Ureteral diameter ratio is a more objective and reliable measure than grade, and may be more useful in clinical decision-making.

Bone mineral content and areal density, but not bone area, predict an incident fracture risk: a comparative study in a UK prospective cohort.

We studied a prospective UK cohort of women aged 20 to 80 years, assessed by dual-energy X-ray absorptiometry (DXA) at baseline. Bone mineral content (BMC) and areal bone mineral density (aBMD), but not bone area (BA), at femoral neck, lumbar spine and the whole body sites were similarly predictive of incident fractures. BACKGROUND: Low aBMD, measured by DXA, is a well-established risk factor for future fracture, but little is known about the performance characteristics of other DXA measures such as BA and BMC in fracture prediction. We therefore investigated the predictive value of BA, BMC and aBMD for incident fracture in a prospective cohort of UK women. METHODS: In this study, 674 women aged 20-80 years, recruited from four GP practices in Southampton, underwent DXA assessment (proximal femur, lumbar spine, total body) between 1991 and 1993. All women were contacted in 1998-1999 with a validated postal questionnaire to collect information on incident fractures and potential confounding factors including medication use. Four hundred forty-three women responded, and all fractures were confirmed by the assessment of images and radiology reports by a research nurse. Cox proportional hazard models were used to explore the risk of incident fracture, and the results are expressed as hazard ratio (HR) per 1 SD decrease in the predictor and 95% CI. Associations were adjusted for age, BMI, alcohol consumption, smoking, HRT, medications and history of fracture. RESULTS: Fifty-five women (12%) reported a fracture. In fully adjusted models, femoral neck BMC and aBMD were similarly predictive of incident fracture. Femoral neck BMC: HR/SD = 1.64 (95%CI: 1.19, 2.26; p = 0.002); femoral neck aBMD: HR/SD = 1.76 (95%CI: 1.19, 2.60; p = 0.005). In contrast, femoral neck BA was not associated with incident fracture, HR/SD = 1.15 (95%CI: 0.88, 1.50; p = 0.32). Similar results were found with bone indices at the lumbar spine and the whole body. CONCLUSIONS: In conclusion, BMC and aBMD appear to predict incident fracture with similar HR/SD, even after adjustment for body size. In contrast, BA only weakly predicted the future fracture. These findings support the use of DXA aBMD in fracture risk assessment, but also suggest that factors which specifically influence BMC will have a relevance to the risk of the incident fracture.

Management of functional nonretentive fecal incontinence in children: Recommendations from the International Children's Continence Society.

BACKGROUND: Fecal incontinence (FI) in children is frequently encountered in pediatric practice, and often occurs in combination with urinary incontinence. In most cases, FI is constipation-associated, but in 20% of children presenting with FI, no constipation or other underlying cause can be found - these children suffer from functional nonretentive fecal incontinence (FNRFI). OBJECTIVE: To summarize the evidence-based recommendations of the International Children's Continence Society for the evaluation and management of children with FNRFI. RECOMMENDATIONS: Functional nonretentive fecal incontinence is a clinical diagnosis based on medical history and physical examination. Except for determining colonic transit time, additional investigations are seldom indicated in the workup of FNRFI. Treatment should consist of education, a nonaccusatory approach, and a toileting program encompassing a daily bowel diary and a reward system. Special attention should be paid to psychosocial or behavioral problems, since these frequently occur in affected children. Functional nonretentive fecal incontinence is often difficult to treat, requiring prolonged therapies with incremental improvement on treatment and frequent relapses.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study.

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome. METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone. FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions. INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

Utility of the distal ureteral diameter on VCUG for grading VUR.

INTRODUCTION: For children with VUR the grade of vesicoureteral reflux (VUR) remains one of the most predictive factors relative to outcome. However, the subjective nature of the currently accepted international reflux grading system (IRGS) leads to inter-observer variation. The potential of a direct measurement of the distal ureter on the voiding cystourethrogram (VCUG) normalized to the L1-L3 vertebral body distance (ureteral diameter ratio - UDR) to augment the ability of IRGS to predict the ultimate clinical outcome has previously been reported in a group of 79 children. OBJECTIVE: The goal of this current review was to expand the previous review and analysis in order to assess the predictive ability of the UDR with respect to earlier clinical outcome and to compare this ability to the grade of VUR while controlling for other variables. STUDY DESIGN: This retrospective review of the VCUG of 157 children with primary VUR included 124 girls and 33 boys with a mean age of 2.7 years (7 days-13.5 years). In addition to the UDR, other variables that were analyzed included: age, gender, VUR grade, laterality, history of febrile urinary tract infection (UTI) or multiple UTIs prior to diagnosis, and bladder-bowel dysfunction (BBD). Cox regression analysis was utilized and a generalized logit model for 2-year outcome was also fitted to compare the effect of UDR and VUR grade using Wald Chi-squared analysis. RESULTS: The 2-year outcome after the VCUG was defined as: persistent VUR (47%), spontaneous VUR resolution (15%), or operative intervention (38%). Reasons for operative intervention included: breakthrough UTI (1/3), decreased relative renal function associated with renal scarring (1/3), and failure to resolve, along with parental preference (1/3). Increasing UDR was significantly associated with increased grade and a decreased chance of spontaneous resolution. It was unlikely for a child with Grade 4, 3, or 2 VUR to have spontaneous resolution if their UDR was above 0.25, 0.3, or 0.35, respectively. In addition, higher grades of VUR, older age, and bilateral VUR were significantly associated with failure to spontaneously resolve VUR. As seen in Figure, children with grades 2 and 3 VUR less than 2 years of age had a better chance of spontaneous resolution with a larger UDR than children 2 years of age or older. When adjusting for age, grade, laterality and multiple UTIs as covariates, each unit increase of UDR of 0.1 was significantly associated with either persistent VUR (OR = 1.73, 95% CI = 1.02-2.95, P = 0.043) or the need for surgical intervention (OR = 2.40, 95% CI = 1.39-4.17, P = 0.002) compared to spontaneous resolution. When testing the effect of UDR and grade of reflux in the same model, UDR was noted to have a larger effect on predicting failure to spontaneously resolve VUR than grade (Wald Chi-Squared 13.6; P = 0.001 vs 3.62; P = 0.46, respectively). DISCUSSION: The UDR is a readily available objective measurement on the VCUG that has demonstrated ability to enhance the International Reflux Grading System. Limitations of the current review include operative intervention in 12% of the children for failure to improve or resolve VUR. This surgical intervention inhibits determination of spontaneous resolution rates. The findings in this study reflect those in a series of children from a single institution and, therefore, may be impacted by clinical practice bias and geographic variations. Subsequent multi-institutional studies could further define the potential of UDR as either an independent or additive predictive factor for grading VUR that will further permit individualized patient management. CONCLUSION: In this single institution series, UDR was highly correlated with VUR grade; however, UDR proved more predictive of spontaneous resolution, persistence, or operative intervention than grade.

Oxidative methane activation over yttrium stabilised zirconia.

The methane C-H bond is extremely stable, requiring significant energy input in reforming processes. We present a novel mechanism for energetically favourable methane C-H bond breaking over yttrium stabilised zirconia in the presence of oxygen, based on results of Density Functional Theory (DFT) and HSE06 hybrid functional calculations. We argue that this mechanism will be relevant to C-H activation over many metal oxide catalyst materials.

Focal amplification of the androgen receptor gene in hormone-naive human prostate cancer.

BACKGROUND: Androgen receptor (AR)-gene amplification, found in 20-30% of castration-resistant prostate cancer (CRPCa) is proposed to develop as a consequence of hormone-deprivation therapy and be a prime cause of treatment failure. Here we investigate AR-gene amplification in cancers before hormone deprivation therapy. METHODS: A tissue microarray (TMA) series of 596 hormone-naive prostate cancers (HNPCas) was screened for chromosome X and AR-gene locus-specific copy number alterations using four-colour fluorescence in situ hybridisation. RESULTS: Both high level gain in chromosome X (≥4 fold; n=4, 0.7%) and locus-specific amplification of the AR-gene (n=6, 1%) were detected at low frequencies in HNPCa TMAs. Fluorescence in situ hybridisation mapping whole sections taken from the original HNPCa specimen blocks demonstrated that AR-gene amplifications exist in small foci of cells (≤ 600 nm, ≤1% of tumour volume). Patients with AR gene-locus-specific copy number gains had poorer prostate cancer-specific survival. CONCLUSION: Small clonal foci of cancer containing high level gain of the androgen receptor (AR)-gene develop before hormone deprivation therapy. Their small size makes detection by TMA inefficient and suggests a higher prevalence than that reported herein. It is hypothesised that a large proportion of AR-amplified CRPCa could pre-date hormone deprivation therapy and that these patients would potentially benefit from early total androgen ablation.

Prognostic value of PTEN loss in men with conservatively managed localised prostate cancer.

BACKGROUND: The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate. METHODS: The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer. RESULTS: The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60-4.73; P=3.1 × 10(-14)). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2-24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses. CONCLUSION: In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.

Neuroendocrine differentiation does not have independent prognostic value in conservatively treated prostate cancer.

In vitro studies have implicated neuroendocrine differentiation in the development of hormone resistant prostate cancer following administration of androgen blockers. Studies on clinical material are equivocal. We wished to understand the significance of neuroendocrine differentiation in our large and well-characterised cohort of clinically localised prostate cancer, treated conservatively. Immunohistochemical expression of chromogranin-A was assessed semi-quantitatively on tissue samples of 806 patients in a tissue microarray approach. The correlation of expression with 10-year prostate cancer survival was examined. Multivariate analysis including contemporary Gleason score was performed and sub-group analysis of early hormone treated patients was also undertaken. Chromogranin-A expression correlated with high Gleason score (χ(2) = 28.35, p < 0.001) and early prostate cancer death (HR = 1.61, 95 %CI = 1.15-2.27, p < 0.001). In univariate analysis, NE differentiation correlated significantly with outcome (HR = 1.61, 95 % CI 1.15-2.27, p < 0.001) However in multivariate analysis including Gleason score, chromogranin-A expression was not an independent predictor of survival (HR = 0.97, 95 %CI = 0.89-1.37, p = 0.87). Although chromogranin-A expression was higher in patients with early hormone therapy (χ(2) = 7.25, p = 0.007), there was no association with prostate cancer survival in this sub-group (p = 0.083). Determination of neuroendocrine differentiation does not appear to have any bearing on the outcome of prostatic carcinoma and does not add to the established prognostic model.

Splice variant PRKC-ζ(-PrC) is a novel biomarker of human prostate cancer.

BACKGROUND: Previously, using gene-knockdown techniques together with genome expression array analysis, we showed the gene protein Kinase C (PKC)-zeta (PRKCZ) to mediate the malignant phenotype of human prostate cancer. However, according to NCBI, the gene has undergone several major iterations. Therefore, to understand the relationship between its structure and biological activities, we have analysed its expressed sequence in prostate cancer cell lines and tissues. METHODS: Transcriptome-walking and targeted PCR were used to sequence the mRNA transcribed from PRKCZ. Hydropathy analysis was employed to analyse the hypothetical protein sequence subsequently translated and to identify an appropriate epitope to generate a specific monoclonal antibody. RESULTS: A novel sequence was identified within the 3'-terminal domain of human PRKCZ that, in prostate cancer cell lines and tissues, is expressed during transcription and thereafter translated into protein (designated PKC-ζ(-PrC)) independent of conventional PKC-ζ(-a). The monoclonal antibody detected expression of this 96 kD protein only within malignant prostatic epithelium. INTERPRETATION: Transcription and translation of this gene sequence, including previous intronic sequences, generates a novel specific biomarker of human prostate cancer. The presence of catalytic domains characteristic of classic PKC-ß and atypical PKC-ι within PKC-ζ(-PrC) provides a potential mechanism for this PRKCZ variant to modulate the malignant prostatic phenotype out-with normal cell-regulatory control.

High-density tissue microarrays from prostate needle biopsies.

BACKGROUND: Formalin-fixed prostate biopsies are frequently the only tissue collected at the time of prostate cancer diagnosis. There is therefore a requirement for techniques that allow the use of these prostate biopsy specimens in a high-throughput analysis of immunohistochemical and fluorescence-in-situ-hybridisation-detected biomarkers. METHODS: The authors have previously described methods that allow tissue microarray (TMA) construction from prostate biopsies. Here, we describe significant technical innovations that provide an easier and more robust system of biopsy-TMA construction. RESULTS AND DISCUSSION: The TMAs produced are of a high density (up to 104 cores each, 8 × 13) and allow a multiplex analysis of biomarkers in the context of clinical trials.

The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

Our previous work identified a chromosomal translocation t(4;6) in prostate cancer cell lines and primary tumors. Using probes located on 4q22 and 6q15, the breakpoints identified in LNCaP cells, we performed fluorescence in situ hybridization analysis to detect this translocation in a large series of clinical localized prostate cancer samples treated conservatively. We found that t(4;6)(q22;q15) occurred in 78 of 667 cases (11.7%). The t(4;6)(q22;q15) was not independently associated with patient outcome. However, it occurs more frequently in high clinical T stage, high tumor volume specimens and in those with high baseline PSA (P=0.001, 0.001 and 0.01, respectively). The t(4;6)(q22;q15) occurred more frequently in samples with two or more TMPRSS2:ERG fusion genes caused by internal deletion than in samples without these genomic alterations, but this correlation is not statistically significant (P=0.0628). The potential role of this translocation in the development of human prostate cancer is discussed.

Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.

BACKGROUND: The discovery of ERG/ETV1 gene rearrangements and PTEN gene loss warrants investigation in a mechanism-based prognostic classification of prostate cancer (PCa). The study objective was to evaluate the potential clinical significance and natural history of different disease categories by combining ERG/ETV1 gene rearrangements and PTEN gene loss status. METHODS: We utilised fluorescence in situ hybridisation (FISH) assays to detect PTEN gene loss and ERG/ETV1 gene rearrangements in 308 conservatively managed PCa patients with survival outcome data. RESULTS: ERG/ETV1 gene rearrangements alone and PTEN gene loss alone both failed to show a link to survival in multivariate analyses. However, there was a strong interaction between ERG/ETV1 gene rearrangements and PTEN gene loss (P<0.001). The largest subgroup of patients (54%), lacking both PTEN gene loss and ERG/ETV1 gene rearrangements comprised a 'good prognosis' population exhibiting favourable cancer-specific survival (85.5% alive at 11 years). The presence of PTEN gene loss in the absence of ERG/ETV1 gene rearrangements identified a patient population (6%) with poorer cancer-specific survival that was highly significant (HR=4.87, P<0.001 in multivariate analysis, 13.7% survival at 11 years) when compared with the 'good prognosis' group. ERG/ETV1 gene rearrangements and PTEN gene loss status should now prospectively be incorporated into a predictive model to establish whether predictive performance is improved. CONCLUSIONS: Our data suggest that FISH studies of PTEN gene loss and ERG/ETV1 gene rearrangements could be pursued for patient stratification, selection and hypothesis-generating subgroup analyses in future PCa clinical trials and potentially in patient management.

Hsp-27 expression at diagnosis predicts poor clinical outcome in prostate cancer independent of ETS-gene rearrangement.

BACKGROUND: This study was performed to test the hypothesis that expression of small heat shock protein Hsp-27 is, at diagnosis, a reliable predictive biomarker of clinically aggressive prostate cancer. METHODS: A panel of tissue microarrays constructed from a well-characterised cohort of 553 men with conservatively managed prostate cancer was stained immunohistochemically to detect Hsp-27 protein. Hsp-27 expression was compared with a series of pathological and clinical parameters, including outcome. RESULTS: Hsp-27 staining was indicative of higher Gleason score (P<0.001). In tissue cores having a Gleason score >7, the presence of Hsp-27 retained its power to independently predict poor clinical outcome (P<0.002). Higher levels of Hsp-27 staining were almost entirely restricted to cancers lacking ERG rearrangements (chi2 trend=31.4, P<0.001), although this distribution did not have prognostic significance. INTERPRETATION: This study has confirmed that, in prostate cancers managed conservatively over a period of more than 15 years, expression of Hsp-27 is an accurate and independent predictive biomarker of aggressive disease with poor clinical outcome (P<0.001). These findings suggest that apoptotic and cell-migration pathways modulated by Hsp-27 may contain targets susceptible to the development of biologically appropriate chemotherapeutic agents that are likely to prove effective in treating aggressive prostate cancers.