Properties of non-coding mutation hotspots as urinary biomarkers for bladder cancer detection.

Mutations at specific hotspots in non-coding regions of ADGRG6, PLEKHS1, WDR74, TBC1D12 and LEPROTL1 frequently occur in bladder cancer (BC). These mutations could function as biomarkers for the non-invasive detection of BC but this remains largely unexplored. Massively-parallel sequencing of non-coding hotspots was applied to 884 urine cell pellet DNAs: 591 from haematuria clinic patients (165 BCs, 426 non-BCs) and 293 from non-muscle invasive BC surveillance patients (29 with recurrence). Urine samples from 142 non-BC haematuria clinic patients were used to optimise variant calling. Non-coding mutations are readily detectable in the urine of BC patients and undetectable, or present at much lower frequencies, in the absence of BC. The mutations can be used to detect incident BC with 66% sensitivity (95% CI 58-75) at 92% specificity (95% CI 88-95) and recurrent disease with 55% sensitivity (95% CI 36-74) at 85% specificity (95% CI 80-89%) using a 2% variant allele frequency threshold. In the NMIBC surveillance setting, the detection of non-coding mutations in urine in the absence of clinically detectable disease was associated with an increased relative risk of future recurrence (RR = 4.62 (95% CI 3.75-5.48)). As urinary biomarkers, non-coding hotspot mutations behave similarly to driver mutations in BC-associated genes and could be included in biomarker panels for BC detection.

Prediction of histological stage based on cystoscopic appearances of newly diagnosed bladder tumours.

INTRODUCTION In the 75-80% of urothelial bladder cancers (UBC) presenting as non-muscle invasive bladder cancer (NMIBC), transurethral resection of bladder tumour (TURBT) is the key treatment and staging procedure. In the 20-25% of patients with muscle invasive bladder cancer (MIBC), further cross-sectional imaging is required to complete the staging process before considering radical treatment. Given the adverse effects of ionising radiation, clinicians identify patients believed to have MIBC, and so requiring further imaging pre-TURBT, at the tumour histology/stage based on the tumour's visual characteristics. There is minimal evidence describing the accuracy of such predictions in newly-diagnosed patients. METHODS Over a 6-year period, a database of patients undergoing resection of newly-diagnosed bladder lesions in a single UK centre was prospectively established. Predictions based on histology were simultaneously recorded, and the accuracy of these predictions of histology/stage subsequently assessed. RESULTS One hundred and twenty two (73.1%) patients with histologically confirmed NMIBC had predictions recorded versus 45 (26.9%) patients with MIBC. Visual assessment predictions of MIBC had a sensitivity of 88.9% (95% confidence interval [CI] 76.5%-95.2%) and a specificity of 91.0% (95% CI 84.6%-94.9%), giving a positive predictive value of 78.4% (95% CI 65.4%-87.5%) and a negative predictive value of 95.7% (95% CI 90.3%-98.1%). CONCLUSIONS We find that visual assessment is accurate in predicting the presence of MIBC. This supports the practice of stratifying patients at the time of initial cystoscopy for those requiring further radiological staging pre-TURBT.

Viscoelastic properties of human bladder tumours.

The urinary bladder is an organ which facilitates the storage and release of urine. The bladder can develop tumours and bladder cancer is a common malignancy throughout the world. There is a consensus that there are differences in the mechanical properties of normal and malignant tissues. However, the viscoelastic properties of human bladder tumours at the macro-scale have not been previously studied. This study investigated the viscoelastic properties of ten bladder tumours, which were tested using dynamic mechanical analysis at frequencies up to 30Hz. The storage modulus ranged between 0.052MPa and 0.085MPa while the loss modulus ranged between 0.019MPa and 0.043MPa. Both storage and loss moduli showed frequency dependent behaviour and the storage modulus was higher than the loss modulus for every frequency tested. Viscoelastic properties may be useful for the development of surgical trainers, surgical devices, computational models and diagnostic equipment.

Protein shedding in urothelial bladder cancer: prognostic implications of soluble urinary EGFR and EpCAM.

BACKGROUND: Better biomarkers must be found to develop clinically useful urine tests for bladder cancer. Proteomics can be used to identify the proteins released by cancer cell lines and generate candidate markers for developing such tests. METHODS: We used shotgun proteomics to identify proteins released into culture media by eight bladder cancer cell lines. These data were compared with protein expression data from the Human Protein Atlas. Epidermal growth factor receptor (EGFR) was identified as a candidate biomarker and measured by ELISA in urine from 60 noncancer control subjects and from 436 patients with bladder cancer and long-term clinical follow-up. RESULTS: Bladder cancer cell lines shed soluble EGFR ectodomain. Soluble EGFR is also detectable in urine and is highly elevated in some patients with high-grade bladder cancer. Urinary EGFR is an independent indicator of poor bladder cancer-specific survival with a hazard ratio of 2.89 (95% CI 1.81-4.62, P<0.001). In multivariable models including both urinary EGFR and EpCAM, both biomarkers are predictive of bladder cancer-specific survival and have prognostic value over and above that provided by standard clinical observations. CONCLUSIONS: Measuring urinary EGFR and EpCAM may represent a simple and useful approach for fast-tracking the investigation and treatment of patients with the most aggressive bladder cancers.

Frequency dependent viscoelastic properties of porcine bladder.

The aim of this study was to measure the viscoelastic properties of bladder tissue. Porcine bladders were dissected into rectangular strips and loops. Dynamic Mechanical Analysis was used to measure the viscoelastic properties of the bladder tissue (storage and loss stiffness) tested in a frequency range of up to 10 Hz. Storage stiffness was found to be consistently higher than loss stiffness. Average storage stiffness was found to be 1.89 N/mm and 0.74 N/mm for looped and rectangular samples, respectively. Average loss stiffness was found to be 0.24 N/mm and 0.11 N/mm for looped and rectangular samples, respectively. The results of this study are important for computational modelling of the bladder and for ensuring that engineered bladder tissues have physiological viscoelastic properties.

The effects and effectiveness of electromotive drug administration and chemohyperthermia for treating non-muscle invasive bladder cancer.

INTRODUCTION: Preliminary studies show that device assisted intravesical therapies appear more effective than passive diffusion intravesical therapy for the treatment of non-muscle invasive bladder cancer (NMIBC) in specific settings, and phase III studies are now being conducted. Consequently, we have undertaken a non-systematic review with the objective of describing the scientific basis and mechanisms of action of electromotive drug administration (EMDA) and chemohyperthermia (CHT). METHODS: PubMed, ClinicalTrials.gov and the Cochrane Library were searched to source evidence for this non-systematic review. Randomised controlled trials, systematic reviews and meta-analyses were evaluated. Publications regarding the scientific basis and mechanisms of action of EMDA and CHT were identified, as well as clinical studies to date. RESULTS: EMDA takes advantage of three phenomena: iontophoresis, electro-osmosis and electroporation. It has been found to reduce recurrence rates in NMIBC patients and has been proposed as an addition or alternative to bacillus Calmette-Guérin (BCG) therapy in the treatment of high risk NMIBC. CHT improves the efficacy of mitomycin C by three mechanisms: tumour cell cytotoxicity, altered tumour blood flow and localised immune responses. Fewer studies have been conducted with CHT than with EMDA but they have demonstrated utility for increasing disease-free survival, especially in patients who have previously failed BCG therapy. CONCLUSIONS: It is anticipated that EMDA and CHT will play important roles in the management of NMIBC in the future. Techniques of delivery should be standardised, and there is a need for more randomised controlled trials to evaluate the benefits of the treatments alongside quality of life and cost-effectiveness.

Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential.

BACKGROUND: Epithelial cell adhesion molecule is overexpressed in bladder tumours and released from bladder cancer cells in vitro. We test the hypotheses that urinary EpCAM could act as a biomarker for primary bladder cancer detection and risk stratification. METHODS: Epithelial cell adhesion molecule was measured by ELISA in urine from 607 patients with primary bladder tumours and in urine from 53 non-cancer controls. Mann-Whitney tests and ROC analyses were used to determine statistical significance and discrimination between non-cancer controls and different stages and grades of disease. Multivariable modelling and Kaplan-Meier analyses were used to determine prognostic significance. The structure of urinary EpCAM was investigated by western blotting and mass spectrometry. RESULTS: Urinary EpCAM levels increase with stage and grade of bladder cancer. Alongside grade and stage, elevated urinary EpCAM is an independent indicator of poor prognosis with a hazard ratio of 1.76 for bladder cancer-specific mortality. The soluble form of EpCAM in urine is the extracellular domain generated by cleavage between ala243 and gly244. Further studies are required to define the influence of other urinary tract malignancies and benign urological conditions on urinary EpCAM. CONCLUSION: The extracellular domain of EpCAM is shed into urine by bladder tumours. Urinary EpCAM is a strong indicator of bladder cancer-specific survival, and may be useful within a multi-marker panel for disease detection or as a stand-alone marker to prioritise the investigation and treatment of patients. The mechanisms and effects of EpCAM cleavage in bladder cancer are worthy of further investigation, and may identify novel therapeutic targets.

Combined proteome and transcriptome analyses for the discovery of urinary biomarkers for urothelial carcinoma.

BACKGROUND: Proteomic discovery of cancer biomarkers in body fluids is challenging because of their low abundance in a complex background. Altered gene expression in tumours may not reflect protein levels in body fluids. We have tested combining gene expression profiling of tumours with proteomic analysis of cancer cell line secretomes as a strategy to discover urinary biomarkers for bladder cancer. METHODS: We used shotgun proteomics to identify proteins secreted by three bladder cancer cell lines. Secreted proteins with high mRNA levels in bladder tumours relative to normal urothelium were assayed by ELISA in urine samples from 642 patients. RESULTS: Midkine and HAI-1 were significantly increased in bladder cancer patients, with the highest levels in invasive disease (area under the receiver operating characteristic curve 0.89 vs non-cancer). The urinary concentration of both proteins was too high to be explained by bladder cancer associated haematuria and most likely arises by direct tumour secretion. CONCLUSIONS: This 'dual-omic' strategy identified tumour secreted proteins whose urine concentrations are increased significantly by bladder cancer. Combined secretome-transcriptome analysis may be more useful than direct proteomic analysis of body fluids for biomarker discovery in both bladder cancer and other tumour types.

Cadherin switching dictates the biology of transitional cell carcinoma of the bladder: ex vivo and in vitro studies.

Bladder cancer is the fifth most common malignancy in the UK. Clinically, the most important process in determining prognosis is the development of invasion, initially of the lamina propria and then beyond as these transitional cell carcinomas (TCCs) progress from stage pT1 to stages T2+. Cadherins and catenins are the main mediators of cell-cell interactions in epithelial tissues, and loss of membranous E-cadherin immunoreactivity is strongly correlated with high grade, advanced stage and poor prognosis in bladder cancer and other malignancies. However, the role of P-cadherin is yet to be fully elucidated in bladder TCC. The objectives of this study were to establish how the expression of cadherins and catenins determines clinical and in vitro behaviour in bladder TCC. Utilizing immunohistochemistry, immunofluorescence and western blotting, we demonstrated a significant reduction in the expression of E-cadherin and beta-catenin as grade and stage of bladder TCC progress, accompanied by a significant increase in P-cadherin expression (all p < 0.05, Pearson's chi2 test). Increased P-cadherin expression was also associated with a significantly worse bladder cancer-specific survival (log rank p = 0.008), with Cox regression showing P-cadherin to be an independent prognostic factor. Utilizing a variety of tissue culture models in a range of functional studies, we demonstrated that P-cadherin mediates defective cell-cell adhesion and enhances anchorage-independent growth. The results provide evidence that increased P-cadherin expression promotes a more malignant and invasive phenotype of bladder cancer, and appears to have a novel role late in the disease.

The role of beta-catenin signaling in the malignant potential of cystitis glandularis.

PURPOSE: Chronic inflammation is a risk factor for malignant transformation in the bladder. The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) is a mediator of such inflammation that induces nuclear localization of the adherens junction component beta-catenin. This mechanism has a key role in the initiation and progression of the premalignant lesion Barrett's metaplasia of the esophagus. Cystitis glandularis is a metaplastic lesion of the bladder urothelium occurring in the presence of chronic inflammation and in up to 13% of asymptomatic bladders. Two subtypes are described (typical and intestinal/colonic) with uncertain malignant potential. Etiologically and histologically cystitis glandularis mimics Barrett's metaplasia. We investigated the roles of beta-catenin and TNFalpha in cystitis glandularis. MATERIALS AND METHODS: Immunohistochemistry and immunofluorescence were used to demonstrate the expression and localization of E-cadherin, beta-catenin and TNFalpha in 9 sections of typical cystitis glandularis and 4 of intestinal/colonic cystitis glandularis. Appropriate controls were used for all experiments. RESULTS: Immunohistochemistry demonstrated normal membranous expression of E-cadherin and beta-catenin in all cystitis glandularis sections with increased TNFalpha expression. Immunofluorescence showed nuclear localization of beta-catenin in the intestinal/colonic subtype only, which was not observed in typical cystitis glandularis. CONCLUSIONS: The presence of nuclear beta-catenin suggests that intestinal/colonic cystitis glandularis shares the same signaling pathway with the premalignant lesion Barrett's metaplasia of the esophagus and the intestinal/colonic subtype of cystitis glandularis may have the potential to progress to malignancy. This finding has important implications for the management of this lesion.

Axillary lipoblastoma--tumor recurrence in the right atrium.

This case report describes, for the first time, vascular invasion and recurrence of a lipoblastoma 6 months after the macroscopically complete excision of the initial cervico-axillary tumour. The importance of cytogenetics in the diagnosis of lipoblastoma is emphasized, as is the need to be wary of the diagnosis of lipoma in infancy.

Delay and survival in bladder cancer.

OBJECTIVE: To assess in detail and evaluate the effect on survival of delays in the diagnosis and treatment of cancer (which might lead to a worse prognosis), dividing the delay from onset of symptoms to first treatment into several components, comprising patient delay, general practitioner (GP) delay, and two or more periods of hospital delay. PATIENTS AND METHODS: Data were prospectively collected on 1537 new cases of urothelial cancer in the West Midlands from 1 January 1991 to 30 June 1992. Death information was obtained from the West Midlands Cancer Intelligence Unit and censored at 31 July 2000. The influence of delay times on survival was explored. RESULTS: The median delay from onset of symptoms to GP referral was 14 days (Delay 1), from GP referral to first hospital attendance was 28 days (Delay 2), and from first hospital attendance to first transurethral resection of bladder tumour was 20 days (Delay 3). The median hospital delay (Delay 2 + 3) was 68 days and the median total delay (Delay 1 + 2 + 3) was 110 days. Patients with a shorter Delay 1 had a lower tumour stage and a 5% better 5-year survival. Patients with a shorter hospital delay had worse survival; total delay had no effect on survival. CONCLUSIONS: There was significantly better survival for patients referred to hospital within 14 days of the onset of symptoms. The relationship between delay and survival in bladder cancer is complex. Hospital delays may be influenced more by comorbidity than by the characteristics of the tumour. However, the adverse effects of delay seem to be most pronounced for patients with pT1 tumours.

Laparoscopic peritoneal lavage in staging gastric and oesophageal cancer.

AIMS: Accurate staging of gastric, oesophageal and oesophagogastric cancer is essential to avoid unnecessary laparotomies in patients where only palliation is appropriate. This requires a multimodal approach utilizing endoscopy, computed tomography and laparoscopy. Previous authors have found that the presence of free peritoneal tumour cells (FPTCs) detected at laparoscopy or laparotomy confers a poorer prognosis. However, various methods of peritoneal lavage are described. The aim of this study was to evaluate the prognostic value of our technique of peritoneal lavage. MATERIALS AND METHODS: 88 staging laparoscopies with peritoneal lavage were carried out between March 1997 and February 1999 on patients eligible for attempted curative resection of a gastric, oesophageal or oesophagogastric cancer. During laparoscopy the pelvis was irrigated with 200 ml of normal saline, with 100 ml aspirated and examined cytologically. Patients were followed-up until September, 1999. RESULTS: 11 patients had FPTC-positive cytology with a median survival following laparoscopy of 122 days (95% CI 82-161) with only a single patient surviving more than one year. In the FPTC-negative group, median survival was 378 days (95% CI 256,-). Log-rank Chi(2)=16.7, P<0.001. CONCLUSIONS: The presence of FPTCs detected by our technique is a contraindication to attempted curative resection - palliation only (medical or surgical) is appropriate.

Infectious disease morbidity in the US region bordering Mexico, 1990-1998.

The United States and Mexico share an international boundary approximately 3000 km long. This border separates 2 nations with great differences in health status. The objective of this study was to assess morbidity due to infectious diseases in the US region bordering Mexico. The incidence between 1990 and 1998 of 22 nationally notifiable infectious diseases was compared between border and nonborder regions. Disease rates, reflected as rate ratios, were higher in the border region for botulism, brucellosis, diphtheria, hepatitis A, measles, mumps, rabies, rubella, salmonellosis, and shigellosis than in either of 2 nonborder comparison regions. These data indicate that incidence rates for a variety of infectious diseases of public health importance are significantly higher in the United States along the Mexican border than in nonborder regions. These results suggest that an inadequate public health infrastructure may contribute to excess morbidity due to infectious diseases in the border region.

Hantavirus pulmonary syndrome in pregnancy.

This comprehensive case review of hantavirus pulmonary syndrome (HPS) during pregnancy in 5 women characterizes the effect of Sin Nombre virus infection on maternal and fetal outcomes. Histopathologic, serological, and clinical information were evaluated for evidence of vertical transmission. Maternal ages ranged from 20 to 34 years and gestational ages from 13 to 29 weeks. Symptoms, physical findings, and laboratory values other than those related to pregnancy were not noticeably different from those of nonpregnant patients with HPS, although fevers were somewhat lower. One maternal death and 2 fetal losses occurred. Gross, microscopic, and immunohistochemical examination for hantavirus antigen were done on 2 fetal autopsies and 3 placentas showing no evidence of transplacental hantavirus transmission. There was no serological evidence of conversion in the 3 surviving children. Maternal and fetal outcomes of HPS appear similar to those of nonpregnant HPS patients and of pregnant patients with other causes of acute respiratory distress syndrome. No evidence of vertical transmission of Sin Nombre virus was found.

Primary sternal osteomyelitis in infants: a report of two cases.

Primary sternal osteomyelitis in infants, older children, and adults is rare. Secondary sternal osteomyelitis, however, is more common because of the increased frequency of cardiothoracic surgery and intravenous drug abuse. Primary sternal osteomyelitis is reviewed, two infants with further cases of primary sternal osteomyelitis are presented, and diagnosis and management are discussed.

Climatic and environmental patterns associated with hantavirus pulmonary syndrome, Four Corners region, United States.

To investigate climatic, spatial, temporal, and environmental patterns associated with hantavirus pulmonary syndrome (HPS) cases in the Four Corners region, we collected exposure site data for HPS cases that occurred in 1993 to 1995. Cases clustered seasonally and temporally by biome type and geographic location, and exposure sites were most often found in pinyon-juniper woodlands, grasslands, and Great Basin desert scrub lands, at elevations of 1,800 m to 2,500 m. Environmental factors (e.g., the dramatic increase in precipitation associated with the 1992 to 1993 El Niño) may indirectly increase the risk for Sin Nombre virus exposure and therefore may be of value in designing disease prevention campaigns.

Declining CD4+ T-lymphocyte counts are associated with increased risk of enteric parasitosis and chronic diarrhea: results of a 3-year longitudinal study.

From January 1991 through September 1994, we observed people who were infected with HIV to assess the impact of enteric parasite-associated diarrhea. Respondents answered comprehensive questionnaires covering clinical and epidemiologic information and provided stool specimens monthly, which were examined unstained as well as stained with trichrome, chromotrope 2R, and with Kinyoun carbol-fuchsin, and with indirect immunofluorescence for Cryptosporidium. In all, 602 participants, who were interviewed, provided stool specimens at 3254 monthly visits. Parasites were associated with 50 of 354 (14.1%) acute diarrheal episodes (lasting < or = 28 days) and with 97 of 279 (34.8%) chronic episodes (lasting > 28 days). A parasite was associated with 31 of 222 (14.0%) episodes that occurred when CD4+ counts were > or = 200 cells/microl and with 150 of 566 (26.5%) episodes that occurred when CD4+ counts were < 200 cells/microl. The most commonly identified parasite was C. parvum, which was associated with 18 of 354 (5.1%) acute episodes and 36 (12.9%) of the 279 chronic episodes of diarrhea. In this patient population, enteric protozoan parasites were commonly associated with illness, particularly as immunosuppression worsened, and were more likely to be associated with chronic rather than acute diarrhea.