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.

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.

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.

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.

Atovaquone is effective treatment for the symptoms of gastrointestinal microsporidiosis in HIV-1-infected patients.

OBJECTIVE: To report the clinical response to atovaquone in HIV-1-infected patients with symptomatic intestinal microsporidiosis. DESIGN: A retrospective review of a cohort of AIDS patients with symptomatic intestinal microsporidiosis who received atovaquone. SETTING: Infectious Disease Program of the Grady Memorial Hospital, Veterans Affairs Medical Center and private physicians' offices in Atlanta, Georgia. PATIENTS AND METHODS: HIV-1-infected patients (n = 371) were offered a complete stool evaluation and monthly follow-up. Among them, 22 were diagnosed with intestinal microsporidial infection using stool smears stained with modified trichrome stain. Species confirmation was made by light microscopy or electron microscopy on small intestinal biopsy specimens in some patients. MAIN OUTCOME MEASURE: Differences in symptoms, number of stools, and body weight were compared before and after a minimum of 1 month of atovaquone therapy. RESULTS: Eight patients received atovaquone treatment. The mean onset of clinical improvement after beginning treatment was 13 days (SEM, +/- 2). The mean number of stools per day decreased from 10 +/- 2.5 to 2 +/- 1 (P = 0.02, paired t test). The mean weight gain was 3 +/- 2 kg. The parasite was continuously present in the repeated stool specimens. However, semiquantitative analysis performed on two patients' stool specimens showed a decreased parasite burden. Four patients underwent small intestinal endoscopy was consistent with Enterocytozoon bieneusi in all four patients. Only one out of these four patients demonstrated a decrease in parasite burden in the biopsy specimen. Ultrastructural analysis performed in another of these four patients following treatment demonstrated the presence of electron-dense granules in spores, suggestive of toxic effects. CONCLUSION: Atovaquone demonstrates promise as a symptomatic treatment for intestinal microsporidiosis. A double-blind and placebo-controlled clinical trial is currently in progress.

Pathology of symptomatic microsporidial (Encephalitozoon hellem) bronchiolitis in the acquired immunodeficiency syndrome: a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture.

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.

Emerging infectious diseases in the United States, Improved surveillance, a requisite for prevention.

Emerging infectious diseases such as prolonged diarrheal illness due to water-borne Cryptosporidium, hemorrhagic colitis and renal failure from food-borne E. coli O157:H7, and rodent-borne hantavirus pulmonary syndrome as well as reemerging infections such as tuberculosis, pertussis, and cholera vividly illustrate that we remain highly vulnerable to the microorganisms with which we share our environment. Prompt detection of new and resurgent infectious disease threats depends on careful monitoring by modern surveillance systems. This article focuses on five important elements of improved surveillance for emerging infections: 1) strengthening the national notifiable disease system, 2) establishing sentinel surveillance networks, 3) establishing population-based emerging infections programs, 4) developing a system for enhanced global surveillance, and 5) applying new tools and novel approaches to surveillance.

Viral hemorrhagic fevers and hantavirus infections in the Americas.

Several arenaviruses and hantaviruses have been isolated in the Americas during the last 4 decades. These are rodent-borne viruses responsible for the South American hemorrhagic fevers (SAHF) and hantavirus pulmonary syndrome (HPS). Although rare, SAHF and HPS are serious illnesses with high mortality rates. Most viral isolates found in the Americas represent New World lineages of their respective viral families. Their presence in the Western hemisphere is likely ancient, their relationship with their rodent hosts is likely coevolutionary, and their recent detection forebodes the likelihood of detecting additional arena- and hantaviral species in the Americas.

Community participation in vector control: lessons from Chagas' disease.

As applied to vector-borne disease control, the term community participation has been broadly interpreted. Community-based vector control projects have been described as having both active and passive components. Recently, community participation in organized efforts to control Chagas' disease has become more dynamic, with increasingly active involvement by local community members. Chagas' disease is a particularly significant vector-borne disease problem in the South American countries of Brazil, Venezuela, and Bolivia, and health officials there are beginning to emphasize horizontal or decentralized approaches to control of triatomine vectors. Experience suggests that vector control programs using community participation have significant and sustainable impact on vector density, appear to be more cost-effective than purely vertically structured programs, are readily integrated with other health or development programs, promote an enduring sense of pride in home and community, and are politically viable vector control strategies. Community participation per se has inherent value because of its positive effect on social relationships and community solidarity. Moreover, it is a dynamic process that results in accrued benefits for public health that exceed most vector control program goals and persist well beyond program termination.

Corneal microsporidiosis in a patient with AIDS.

Microsporidia are obligate intracellular protozoan parasites that are becoming increasingly recognized as opportunistic pathogens in patients with AIDS. They have been associated with enteritis, hepatitis, and peritonitis and recently keratoconjunctivitis. Gram stain demonstrates the presence of these organisms on light microscopic sections. The specific diagnostic features that distinguish microsporidia from other small nonspore-forming organisms are best demonstrated by electron microscopy, which is also used to characterize the members of Microsporea. In this study, salient histopathologic features of microsporidia in corneal epithelium obtained from an HIV-seropositive individual who developed AIDS are illustrated and discussed.

Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.

A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in filarial antigen-specific immunologic reactivity following treatment with ivermectin and diethylcarbamazine.

The presence of circulating microfilariae has been associated with alterations in B and T cell functions. In this study, we compared the influence of diethylcarbamazine (DEC) and ivermectin on filarial antigen-specific immune responses in a Haitian population. Both drugs were effective at reducing microfilaremia levels to less than 10% of pretreatment levels for up to one year. This reduction in microfilaremia was associated with two phases of altered cellular responsiveness monitored with in vitro assays. Five days post-treatment, cellular proliferation in response to both filarial and nonfilarial antigens was significantly increased, as was the background response in the absence of any antigen. At both nine months and one year post-treatment, the filarial antigen-specific reactivity of both DEC- and ivermectin-treated patients was significantly increased over baseline levels. No differences were observed between the two treatment groups in terms of humoral or cellular reactivity to filarial antigens, despite evidence suggesting a role for DEC in adult worm killing. These results provide additional evidence that microfilariae modulate antifilarial immune reactivity.

Application of the enzyme-linked immunoelectrotransfer blot to filter paper blood spots to estimate seroprevalence of cysticercosis in Bolivia.

An enzyme-linked immunoelectrotransfer blot (EITB) assay was used to study the prevalence of cysticercosis in rural Bolivia. Dried blood spots on filter paper from fingersticks were used as assay samples. Before the serosurvey, experiments were performed to show that samples eluted from dried whole blood on filter paper exhibited no decrease in sensitivity when compared with the more traditional serum samples used in the EITB. Fingerstick blood dried on filter paper is a convenient, economical way of transporting and storing field samples for epidemiologic surveys of cysticercosis in developing countries. This report shows the utility of this sample collection method in underdeveloped countries where refrigeration is not possible and where venipuncture is a problem. Blood was obtained from randomly selected residents in three rural regions of Bolivia: Chuquisaca (n = 1,859), Cochabamba (n = 1,516), and Tarija (n = 1,010). The estimated seroprevalence on 10% of the sample collected for the three regions were 9%, 4.5%, and 2%, respectively.

Comparison of high dose ivermectin and diethylcarbamazine for activity against bancroftian filariasis in Haiti.

This three-phase study was designed to compare high dose ivermectin with a standard diethylcarbamazine (DEC) regimen for patient tolerability, potential to kill adult filaria, and duration of microfilarial suppression in 30 Haitian subjects with Wuchereria bancrofti microfilaremia. All were first given a 1-mg oral dose of ivermectin (phase 1) to reduce microfilaria densities. Participants were randomized into three groups: Group 1 received DEC (6mg/kg per day for 12 days), Group 2 received 200 mcg/kg of ivermectin, and Group 3 received 400 mcg/kg of ivermectin (200 mcg/kg per day for 2 days). All drug regimens were well tolerated with few adverse reactions. Most reactions occurred during phase I and consisted primarily of headache, fever, and myalgia. At the end of phase 1, 27 of 30 (90%) patients were microfilaria negative. During phase 2, four of the six men receiving DEC developed scrotal reactions suggesting killing adult worms; no such reactions were noted in 10 men receiving ivermectin (p less than 0.05). At one-year follow up (phase 3), all treatment groups had less than 10% return to pretreatment microfilaria levels. The mean percent of baseline microfilaria counts were for Group 1, 0.9% (range 0-5%); Group 2, 8.2% (range 0-31%); and Group 3, 3.8% (range 0-25%). Seven individuals in Group 1 were microfilaria-negative, while only one and three individuals were microfilaria-negative in Groups 2 and 3, respectively. These results suggest that DEC causes more damage to the adult worms and greater reduction in microfilaria densities than ivermectin, but that high doses of ivermectin may suppress microfilaremia in lymphatic filariasis for periods much longer than previously reported.

An outbreak of hantavirus pulmonary syndrome in western Paraguay.

During an investigation of hantavirus pulmonary syndrome (HPS) in Paraguay in 1995, sera from persons with HPS-like illness, houshold contacts of confirmed HPS case-patients, and a sample of the area residents were analyzed by ELISA for antibodies to Sin Nombre virus (SNV). Rodent serosurveys and analysis of precipitation records were also conducted. Twenty-three of 24 available probable cases were SNV antibody-positive, 17 of whom were ill between July 1995 and January 1996. Four (14.8%) of 27 case-contacts and 44 (12.8%) of 345 community residents were also seropositive. Calomys laucha (vesper mouse) was the most common rodent species captured and the most frequently SNV-seropositive. Rainfall in May 1995 was 10-fold greater than that seen in May over the preceding 11 years. This 17 case-cluster represents the largest documented outbreak since HPS was first recognized in 1993. Calomys laucha is the likely primary rodent reservoir for a SNV-like hantavirus in western Paraguay. Fluctuations in monthly precipitation rates may have contributed to increased risk for HPS in this region.