Diagnostic value of cytology of voided urine

Cytologic examination of the sediment of voided urine is the only noninvasive method of detection, diagnosis and follow-up of tumors of the bladder and other anatomic components of the lower urinary tract. In order to assess the value of cytology of voided urine, we analyzed the diagnostic yield in 203 episodes, each composed of three sediments of voided urine obtained on consecutive days. For each one of these episodes, histologic material was available and was reviewed. Of special interest were 181 instances of primary or recurrent bladder tumors; in 37 of these patients, random biopsies of the bladder were also available for review. The concept of intraurothelial neoplasia (IUN), graded I, II or III, was introduced to describe degrees of atypia in flat urothelium, with IUN grade III corresponding to nonpapillary carcinoma in situ. The results documented that cytology of voided urine is highly reliable in the diagnosis of high-grade tumors, with a sensitivity of 94.2%. In primary flat carcinoma in situ (IUN III), the sensitivity was 100%. The method failed in the recognition of grade I papillary tumors and in about one-third of grade II tumors. There were no false-positive results in this study. In the 151 positive cases, the cytologic diagnosis was established on the first specimen in 79%, on the second specimen in an additional 14% and on the third specimen in 7% of cases. These results justify the use of three consecutive daily urine specimens for optimal diagnostic results. There is a remarkable similarity between the presence of cancer cells in voided urine and the DNA ploidy of bladder tumors, as established by Tribukait. The observations reported herein suggest that positive urine cytologies may correspond to aneuploid tumors and hence be not only of diagnostic but also of prognostic value. A direct proof of this hypothesis is under investigation; the results of this study justify the need for a field trial of an automated image analysis diagnostic system that was developed in this department.     

Protocols for disease classification from mass spectrometry data

We report our results in classifying protein matrix-assisted laser desorption/ionization-time of flight mass spectra obtained from serum samples into diseased and healthy groups. We discuss in detail five of the steps in preprocessing the mass spectral data for biomarker discovery, as well as our criterion for choosing a small set of peaks for classifying the samples. Cross-validation studies with four selected proteins yielded misclassification rates in the 10-15% range for all the classification methods. Three of these proteins or protein fragments are down-regulated and one up-regulated in lung cancer, the disease under consideration in this data set. When cross-validation studies are performed, care must be taken to ensure that the test set does not influence the choice of the peaks used in the classification. Misclassification rates are lower when both the training and test sets are used to select the peaks used in classification versus when only the training set is used. This expectation was validated for various statistical discrimination methods when thirteen peaks were used in cross-validation studies. One particular classification method, a linear support vector machine, exhibited especially robust performance when the number of peaks was varied from four to thirteen, and when the peaks were selected from the training set alone. Experiments with the samples randomly assigned to the two classes confirmed that misclassification rates were significantly higher in such cases than those observed with the true data. This indicates that our findings are indeed significant. We found closely matching masses in a database for protein expression in lung cancer for three of the four proteins we used to classify lung cancer. Data from additional samples, increased experience with the performance of various preprocessing techniques, and affirmation of the biological roles of the proteins that help in classification, will strengthen our conclusions in the future.     

Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling

Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including alpha-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.     

Size-Based Enrichment of Exfoliated Tumor Cells in Urine Increases the Sensitivity for DNA-Based Detection of Bladder Cancer

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.     

The use of hierarchic classification in the image analysis of a complex cell population. Experience with the sediment of voided urine

Microscopic analysis of cells in the sediment of voided urine is the principal noninvasive method of diagnosing and detecting cancer of the lower urinary tract, mainly the bladder. The sediments contain several populations of cells of unequal diagnostic value. By applying a system of hierarchic classification to the computer analysis of digitized cell images, we were able to eliminate from diagnostic consideration cells that are difficult to classify, such as degenerated cells, multinucleated cells, cell clusters, renal tubular cells and cells infected by the human polyomavirus. When this method of triage was applied to the images of sequentially encountered epithelial cells and clusters, the cell images accepted for final analysis by the computer were sufficient in number and quality to automatically construct cytologic profiles of documented diagnostic value in 15 patients with bladder cancer. The method proved to be applicable to smears and quantitative cytocentrifuge preparations processed by methods developed in our laboratory. This work clearly documents the feasibility of automated analysis of cells in voided urine for the purpose of diagnosing bladder cancer. It also confirms prior observations suggesting that a relatively small sample of sequential images of epithelial cells (200 to 300) is sufficient to establish a diagnostic profile of clinical value on patients with high-grade cancer of the urinary bladder.     

CCL18 in a multiplex urine-based assay for the detection of bladder cancer

The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak?) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p?=?0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to 0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.     

Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells.

OBJECTIVE--To investigate non-invasive detection of cancer by testing for unusual CD44 gene activity in a clinical sample as an indicator of exfoliated tumour cells. DESIGN--Case-control study. SUBJECTS--44 unselected, consecutive patients with bladder cancer and 46 people with no evidence of neoplasia. MAIN OUTCOME MEASURE--Presence or absence of large CD44 gene products containing exon 6 derivatives in urine samples. RESULTS--Novel abnormalities in the pattern of expression of this gene, seen specifically in tumour tissue, led to cloning of a newly recognised coding region in it (exon 6). This was tested as a probe for detection of exfoliated malignant cells in naturally voided urine. CD44 gene products extracted from the urine and amplified with polymerase chain reaction contained predicted electrophoretic band of 735 base pairs in 40 of the 44 patients with bladder cancer (sensitivity 91%). Products from 38 of the 46 people with no evidence of neoplasia showed no such band (specificity 83%). CONCLUSIONS--Unusual activity of the CD44 locus in neoplasia and malignancy is confirmed, and techniques for the analysis of such activity can enable non-invasive investigation of patients for primary or recurrent bladder cancer or for other tumours that shed neoplastic cells into body fluids.     

Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.     

Free DNA in urine: a new marker for bladder cancer? Preliminary data

The aim of the present preliminary study was to investigate the presence of free DNA (FDNA) in urine as a possible marker for the diagnosis of bladder cancer. Naturally voided morning urine specimens were collected from 57 patients with suspected bladder cancer before cystoscopy. A standard urine test was performed; the specimens were then processed in order to obtain a quantitative evaluation of the presence of free DNA in the urine. Twenty-two patients were excluded from the study because they had leukocyturia and/or bacteriuria. Free DNA concentrations higher than 250 ng/mL were found in all 16 patients showing bladder cancer at cystoscopy and in seven (36.8%) of the 19 patients with negative cystoscopy. Urinary FDNA seems to have an excellent sensitivity: we observed no false negative cases and 36.8% false positive cases. By contrast, only 6.25% of the bladder cancer patients had positive urine cytology. Our results seem promising, although further studies and larger numbers are needed to define urinary free DNA as a reliable marker of bladder cancer.     

Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.     

Enhancement of intravesical delivery with Syn3 potentiates interferon-alpha2b gene therapy for superficial bladder cancer

Intravesical administration of interferon alpha-2b protein (IFN) has been successfully used in the treatment of patients with superficial bladder tumors. Local dosing of IFN minimizes well-known systemic side effects of the drug, but exposure to bladder tumors is limited by the duration of instillation and transient concentrations achieved in the urothelium. Intravesical delivery of the gene encoding interferon results in an alternative strategy for IFN-based therapy of the disease, enabling sustained exposure of IFN protein that results from production by tumor and non-tumor cells in the urothelium. Efficient gene delivery and expression of IFN has been achieved using a recombinant adenovirus gene delivery system (rAd-IFN) in conjunction with the novel small molecule excipient Syn3. Studies with rAd-IFN/Syn3 in animal models result in urine concentrations of IFN that persisted for weeks and correlated with potent anti-tumor effects. The objective of this review is to communicate the rationale and preclinical findings that support ongoing clinical investigation of intravesical rAd-IFN/Syn3 in superficial bladder cancer.     

miR-126 在膀胱癌患者尿液中的表达及临床意义

目的:探讨miR-126在膀胱癌患者尿液中的表达与临床病理特征的关系,评估miR-126的肿瘤标志物诊断价值。方法:收集48例初发膀胱尿路上皮癌患者与32例健康对照者晨尿,提取尿液总RNA,通过实时荧光定量PCR技术检测各样本中的miR-126的表达水平,并经受试者工作曲线(ROC)分析其诊断价值。结果:膀胱癌患者尿液中的miR-126表达水平相对健康对照组明显上调(P0.01),其表达水平在不同病理级别之间存在显著差异(P均0.05),且低级别组表达水平略高于高级别组,与肿瘤大小、数目以及淋巴转移也有一定的相关性(P0.05),而与患者的年龄、性别、TNM分期等均无相关性(P0.05)。通过ROC曲线分析尿液中miR-126诊断膀胱肿瘤的曲线下面积(AUC)为0.861,当最佳切点定在7.475时,miR-126诊断膀胱肿瘤的敏感性和特异性分别为75.0%、81.2%。结论:膀胱癌患者尿液中miR-126的表达差异能够反映病情进展程度,其表达水平对膀胱肿瘤的早期诊断及病情评估具有一定的价值。     

Enhancement of intravesical delivery with Syn3 potentiates interferon-α2b gene therapy for superficial bladder cancer

Intravesical administration of interferon alpha-2b protein (IFN) has been successfully used in the treatment of patients with superficial bladder tumors. Local dosing of IFN minimizes well-known systemic side effects of the drug, but exposure to bladder tumors is limited by the duration of instillation and transient concentrations achieved in the urothelium. Intravesical delivery of the gene encoding interferon results in an alternative strategy for IFN-based therapy of the disease, enabling sustained exposure of IFN protein that results from production by tumor and non-tumor cells in the urothelium. Efficient gene delivery and expression of IFN has been achieved using a recombinant adenovirus gene delivery system (rAd-IFN) in conjunction with the novel small molecule excipient Syn3. Studies with rAd-IFN/Syn3 in animal models result in urine concentrations of IFN that persisted for weeks and correlated with potent anti-tumor effects. The objective of this review is to communicate the rationale and preclinical findings that support ongoing clinical investigation of intravesical rAd-IFN/Syn3 in superficial bladder cancer.     

A prognostic 11 long noncoding RNA expression signature for breast invasive carcinoma

Breast cancer, the most common cancer in women worldwide, is associated with high mortality. The long non-coding RNAs (lncRNAs) with a little capacity of coding proteins is playing an increasingly important role in the cancer paradigm. Accumulating evidences demonstrate that lncRNAs have crucial connections with breast cancer prognosis while the studies of lncRNAs in breast cancer are still in its primary stage. In this study, we collected 1052 clinical patient samples, a comparatively large sample size, including 13 159 lncRNA expression profiles of breast invasive carcinoma (BRCA) from The Cancer Genome Atlas database to identify prognosis-related lncRNAs. We randomly separated all of these clinical patient samples into training and testing sets. In the training set, we performed univariable Cox regression analysis for primary screening and played the model for Robust likelihood-based survival for 1000 times. Then 11 lncRNAs with a frequency more than 600 were selected for prediction of the prognosis of BRCA. Using the analysis of multivariate Cox regression, we established a signature risk-score formula for 11 lncRNA to identify the relationship between lncRNA signatures and overall survival. The 11 lncRNA signature was validated both in the testing and the complete set and could effectively classify the high-/low-risk group with different OS. We also verified our results in different stages. Moreover, we analyzed the connection between the 11 lncRNAs and the genes of ESR1, PGR, and Her2, of which protein products (ESR, PGR, and HER2) were used to classify the breast cancer subtypes widely. The results indicated correlations between 11 lncRNAs and the gene of PGR and ESR1. Thus, a prognostic model for 11 lncRNA expression was developed to classify the BRAC clinical patient samples, providing new avenues in understanding the potential therapeutic methods of breast cancer.     

Selection of Microsatellite Markers for Bladder Cancer Diagnosis without the Need for Corresponding Blood

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.     

Aquaporin Expression Contributes to Human Transurothelial Permeability In Vitro and Is Modulated by NaCl

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm²), with mean diffusive water and urea permeability coefficients of 6.33×10⁻⁵ and 2.45×10⁻⁵ cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.     

Epithelial cells transdifferentiation into bladder urothelium in experiments in vivo

Atoplastic surgery using intestinal tissue has been used for the reconstructive therapy of the urinary tract since the mid-20th century; however, cell mechanisms of the urothelium engraftment are still unclear. Intestinal stem cells possess plasticity and, after autoplastic surgery, are presumably able to transdifferentiate into mature cells of the urinary tract. Using the preliminarily developed model for evaluating of the transdifferentiaion of somatic cells into urothelium in vivo, we found that, in syngeneic C57BL mice, epithelial Gfp-producing intestinal cells transdifferentiate into the cryoinjured bladder urothelium. Gfp was detected in the bladder tissue of recipient mice using reverse polymerase chain reaction, fluorescence and immunofluorescence. Colocalization of Her-4 protein revealed by common urothelium expression pattern and Gfp was demonstrated in few urothelial cells by double immunohistochemical staining of the bladder tissue with specific antibodies. The results obtained suggest that epithelial intestinal cells are able to transdifferentiate into bladder urothelium; however, the level of transdifferentiation is low and, presumably, cannot ensure the full functional urothelium engraftment in the case of autoplastic bladder surgery using intestinal tissue.     

Classifying Ten Types of Major Cancers Based on Reverse Phase Protein Array Profiles

Gathering vast data sets of cancer genomes requires more efficient and autonomous procedures to classify cancer types and to discover a few essential genes to distinguish different cancers. Because protein expression is more stable than gene expression, we chose reverse phase protein array (RPPA) data, a powerful and robust antibody-based high-throughput approach for targeted proteomics, to perform our research. In this study, we proposed a computational framework to classify the patient samples into ten major cancer types based on the RPPA data using the SMO (Sequential minimal optimization) method. A careful feature selection procedure was employed to select 23 important proteins from the total of 187 proteins by mRMR (minimum Redundancy Maximum Relevance Feature Selection) and IFS (Incremental Feature Selection) on the training set. By using the 23 proteins, we successfully classified the ten cancer types with an MCC (Matthews Correlation Coefficient) of 0.904 on the training set, evaluated by 10-fold cross-validation, and an MCC of 0.936 on an independent test set. Further analysis of these 23 proteins was performed. Most of these proteins can present the hallmarks of cancer; Chk2, for example, plays an important role in the proliferation of cancer cells. Our analysis of these 23 proteins lends credence to the importance of these genes as indicators of cancer classification. We also believe our methods and findings may shed light on the discoveries of specific biomarkers of different types of cancers.     

Human alpha defensin 5 expression in the human kidney and urinary tract

Background

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.

Methodology/Principal Findings

Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.

Conclusions/Significance

DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.