Diagnosis of Bladder Cancer Recurrence Based on Urinary Levels of EOMES,HOXA9, POU4F2, TWIST1, VIM,and ZNF154 Hypermethylation

Background

Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine.

Methodology/Principal Findings

The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%–89% and specificity in the range 94%–100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88–94% and specificities of 43–67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P≤0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR = 7.8, P = 0.006), POU4F2 (HR = 8.5, P = 0.001), TWIST1 (HR = 12.0, P = 0.015), VIM (HR = 8.0, P = 0.001), and ZNF154 (HR = 13.9, P<0.001). Interestingly, for one group of patients (n = 15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect.

Conclusion/Significance

Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.     

TCF21 and PCDH17 methylation: An innovative panel of biomarkers for a simultaneous detection of urological cancers

The three main types of urological cancers are mostly curable by surgical resection, if early detected. We aimed to identify novel DNA methylation biomarkers common to these three urological cancers, potentially suitable for non-invasive testing. From a candidate list of markers created after gene expression assessment of pharmacologically treated cell lines and tissue samples, two genes were selected for further validation. Methylation levels of these genes were quantified in a total of 12 cancer cell lines and 318 clinical samples. PCDH17 and TCF21 methylation levels provided a sensitivity rate of 92% for bladder cancer, 67% for renal cell tumors and 96% for prostate cancer. Methylation levels were significantly different from those of cancer free individuals (n = 37) for all tumor types (p < 0.001), providing 83% sensitivity and 100% specificity for cancer detection. Although in urine samples the sensitivity was 60%, 32% and 26% for bladder, renal, and prostate tumors, respectively (39% overall), absolute specificity was retained. We identified novel and highly specific methylation markers common to the three main urological cancers. However, additional efforts are required to increase the assay’s sensitivity, enabling the simultaneous non-invasive screening of urological tumors in a single voided urine analysis.     

Detection of bladder cancer using proteomic profiling of urine sediments

We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.     

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.     

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.     

Identification of Methylated Genes Associated with Aggressive Bladder Cancer

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.     

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.     

A Multi-Analyte Assay for the Non-Invasive Detection of Bladder Cancer

Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.     

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.     

Ras oncogene mutations in urine sediments of patients with bladder cancer

Early detection of bladder cancer is particularly important since it dramatically affects the survival rates. However, neither urinary cytology nor tumor markers that are currently used are sensitive enough for the early detection of bladder cancer or recurrent disease. The ras genes are frequently mutated in cancer. In this study, we investigated the diagnostic potential of ras mutation analysis in urinary sediments of patients with bladder cancer using a single-strand conformation polymorphism analysis and polymerase chain reaction. Mutation in codon 12 of the H-ras gene was observed in 39% of the patients. Our results indicate that this approach may significantly improve diagnostic sensitivity in detecting bladder tumors.     

Detection of MicroRNAs in Archival Cytology Urine Smears

MicroRNAs’ dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06–4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope = -3.4084; R-squared = 0.99; efficiency = 1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.     

Proteomic analysis of urinary fibrinogen degradation products in patients with urothelial carcinomas

Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.     

Hypermethylation of Genes Detected in Urine from Ghanaian Adults with Bladder Pathology Associated with Schistosoma haematobium Infection

Purpose

Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens.

Materials and Methods

A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARβ2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection.

Results

RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023).

Conclusions

In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.     

Platelet-Derived Growth Factor Receptor Beta: A Novel Urinary Biomarker for Recurrence of Non-Muscle-Invasive Bladder Cancer

Non-muscle-invasive bladder cancer (NMIBC) is one of the most common malignant tumors in the urological system with a high risk of recurrence, and effective non-invasive biomarkers for NMIBC relapse are still needed. The human urinary proteome can reflect the status of the microenvironment of the urinary system and is an ideal source for clinical diagnosis of urinary system diseases. Our previous work used proteomics to identify 1643 high-confidence urinary proteins in the urine from a healthy population. Here, we used bioinformatics to construct a cancer-associated protein-protein interaction (PPI) network comprising 16 high-abundance urinary proteins based on the urinary proteome database. As a result, platelet-derived growth factor receptor beta (PDGFRB) was selected for further validation as a candidate biomarker for NMIBC diagnosis and prognosis. Although the levels of urinary PDGFRB showed no significant difference between patients pre- and post-surgery (n = 185, P>0.05), over 3 years of follow-up, urinary PDGFRB was shown to be significantly higher in relapsed patients (n = 68) than in relapse-free patients (n = 117, P<0.001). The levels of urinary PDGFRB were significantly correlated with the risk of 3-year recurrence of NMIBC, and these levels improved the accuracy of a NMIBC recurrence risk prediction model that included age, tumor size, and tumor number (area under the curve, 0.862; 95% CI, 0.809 to 0.914) compared to PDGFR alone. Therefore, we surmise that urinary PDGFRB could serve as a non-invasive biomarker for predicting NMIBC recurrence.     

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.     

Alterations of DNA methylome in human bladder cancer

Bladder cancer is the fourth most common cancer in men in the United States, and its recurrence rate is highest among all malignancies. The unmet need for improved strategies for early detection, treatment, and monitoring of the progression of this disease continues to translate into high mortality and morbidity. The quest for advanced diagnostic, therapeutic, and prognostic approaches for bladder cancer is a high priority, which can be achieved by understanding the molecular mechanisms of the initiation and progression of this malignancy. Aberrant DNA methylation in single or multiple cancer-related genes/loci has been found in human bladder tumors and cancer cell lines, and urine sediments, and correlated with many clinicopathological features of this disease, including tumor relapse, muscle-invasiveness, and survival. The present review summarizes the published research on aberrant DNA methylation in connection with human bladder cancer. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in this prime epigenetic field of research. Identifying the potentially reversible and ‘drugable’ aberrant DNA methylation events that initiate and promote bladder cancer development can highlight biological markers for early diagnosis, effective therapy and accurate prognosis of this malignancy.     

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in this study. Cells were isolated from urine before surgical therapy. For FISH analysis, a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 9p21 and 17, was used. Twenty-five cells were analyzed in each case by two observers. A FISH result was obtained in 121 cases. Overall, sensitivity was 60% and specificity reached 82.6%. Sensitivity and specificity by cytology were 24.1% and 90.5%, respectively. Analyzing results concerning T-category, sensitivity of FISH and cytology was 36.1% and 15% in pTa, 65.2 and 25.7% in pT1, 100% and 66.7% in pT2-3 tumors, respectively. Concerning tumor grade, similar results were obtained: sensitivity was 37% and 14% in G1, 65.4% and 40% in G2, 91.7% and 50% in G3 tumors, for FISH and cytology, respectively. In conclusion, FISH on cells from urine has been shown in all studies to be highly sensitive and specific for detection of bladder cancer. Sensitivity of FISH is higher than conventional cytology and can be used in routine diagnosis additionally to conventional cytology especially in doubtful or negative cases. FISH can detect recurrence earlier than other methods like cytology, cystoscopy or biopsy histological examination.     

DNA cytophotometry of voided urine sediment. Comparison with results of cytologic diagnosis and image analysis

Measurements of nuclear DNA were performed in urothelial cells in 54 Feulgen-restained cytocentrifuge preparations of voided urine previously studied visually and with an image analysis system. The study included 30 patients with bladder tumors of various grades, 9 patients with prostatic disease and 15 control samples from normal donors. A number of additional control measurements were performed, including measurements in tissue samples of the 30 bladder tumors corresponding to the cytologic samples. It was documented that DNA can be measured in most urinary sediments. The diagnostic performance of the image analysis system reflected the DNA patterns in 47 of the 54 cases. In several instances, particularly in cases of prostatic disease, the image analysis system recognized abnormal DNA patterns in the absence of significant morphologic abnormalities in the urothelial cells. In seven cases, the image analysis findings failed to conform with the DNA patterns. The reasons for these surprising results are discussed, and future modifications of the image analysis system are proposed.     

Critical evaluation of urinary markers for bladder cancer detection and monitoring

Bladder cancer is currently diagnosed using cystoscopy and cytology in patients with suspicious signs and symptoms. These tests are also used to monitor patients with a history of bladder cancer. The recurrence rate for bladder cancer is high, thus necessitating long-term follow-up. Urine cytology has high specificity but low sensitivity for low-grade bladder tumors. Recently, multiple noninvasive urine-based bladder cancer tests have been developed. Although several markers have been approved by the US Food and Drug Administration for bladder cancer surveillance, only a few are approved for detection of bladder cancer in high-risk patients.     

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.