Detection Of Urothelial Carcinoma Research Articles

Cytohistological correlation of urine cytology in a tertiary centre with application of the Paris system.

Urine cytology has a high sensitivity in morphological recognition of high-grade urothelial carcinoma (HGUC) as compared to low-grade urothelial neoplasms. The Paris system (TPS) was designed to standardise the reporting of HGUC on urine cytology. We evaluated the diagnostic efficacy of urine cytology in routine practice with the cytohistological correlation and application of TPS. A retrospective review of urine cytology reported from 2014 to 2015 was carried out with cytohistological correlation, wherever available. The cases were also recategorised according to TPS with the calculation of malignancy risk of all categories. More than 4000 specimens from 1396 patients were evaluated. Histopathology was available in 244 cases. Urine cytology had a sensitivity of 70.5%, specificity of 78.4%, with an overall diagnostic accuracy of 71.7%. The urine cytology when categorised by TPS had 0.9% unsatisfactory cases, 75.9% negative for HGUC, 8.5% atypical urothelial cells, 0.2% suspicious for HGUC, 14.1% HGUC and 0.4% cases of other malignancies. The risk of HGUC with each category of negative for HGUC, atypical, suspicious for HGUC, HGUC and other malignancy was 11.6%, 12.3%, 33.3%, 58.8% and 80%, respectively. Urine cytology continues to show high diagnostic accuracy for the detection of urothelial carcinoma with a relatively high sensitivity for HGUC. TPS for reporting urine cytology defines specific criteria for diagnosing atypical and HGUC but increases the cases in the atypical category.

The preliminary study for safety of acrinol, as a novel dye, which can be used to visualize cancer nuclei of bladder urothelial carcinoma, for probe-based confocal laser endomicroscopy.

e16510Background: Recently, the utility of probe-based confocal laser endomicroscopy (pCLE) using fluorescein for detection of urothelial carcinoma (UC) was reported. Fluorescein revealed only tiss...

Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study

IntroductionHematuria is the most common symptom of urothelial carcinomas (UC) but is often idiopathic. Cystoscopy is expensive which involves considerable patient discomfort, and conventional urine cytology for noninvasive UC detection and disease monitoring suffers from poor sensitivity. We aim to evaluate the performance of genes selected from a previous study in detecting UC, especially among patients with gross hematuria, as well as upper tract urothelial carcinoma (UTUC) and bladder carcinoma separately, in voided urine samples. MethodsUsing methylation-specific polymerase chain reaction, we examined the promoter methylation status of 10 genes in voided urine samples among 473 patients at our institution, including 217 UC patients and 256 control subjects. ResultsThe final combination of VIM, CDH1, SALL3, TMEFF2, RASSF1A, BRCA1, GDF15, and ABCC6 identified UC with a sensitivity of 0.83 and a specificity of 0.60. Additionally, a panel of selected genes (CDH1, HSPA2, RASSF1A, TMEFF2, VIM, and GDF15) identified UTUC with a sensitivity of 0.82 and a specificity of 0.68, while a panel of selected genes (VIM, RASSF1A, GDF15, and TMEFF2) identified bladder carcinoma with a sensitivity of 0.82 and a specificity of 0.53. Remarkably, a different panel (CDH1, SALL3, THBS1, TMEFF2, VIM, and GDF15) identified UC in patients with gross hematuria with 0.89 sensitivity and 0.74 specificity, and sensitivity (0.91) and specificity (0.92) could be achieved when cytology was included. ConclusionsThe selected urine-DNA methylation biomarkers are reliable, noninvasive, and cost-effective diagnostic tools for bladder carcinoma and UTUC, especially among patients with gross hematuria.

MP58-02 ABERRANT N-GLYCOSYLATION PROFILE OF SERUM IMMUNOGLOBULINS IS A DIAGNOSTIC BIOMARKER OF UROTHELIAL CARCINOMAS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2018MP58-02 ABERRANT N-GLYCOSYLATION PROFILE OF SERUM IMMUNOGLOBULINS IS A DIAGNOSTIC BIOMARKER OF UROTHELIAL CARCINOMAS Toshikazu Tanaka, Tohru Yoneyama, Daisuke Noro, Yuta Kojima, Kengo Imanishi, Hayato Yamamoto, Yuki Tobisawa, Kazuyuki Mori, Atsushi Imai, Shingo Hatakeyama, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Masakazu Tanaka, Shin-Ichiro Nishimura, Shizuka Kurauchi, Ippei Takahashi, and Chikara Ohyama Toshikazu TanakaToshikazu Tanaka More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Daisuke NoroDaisuke Noro More articles by this author , Yuta KojimaYuta Kojima More articles by this author , Kengo ImanishiKengo Imanishi More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Kazuyuki MoriKazuyuki Mori More articles by this author , Atsushi ImaiAtsushi Imai More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Takahiro YoneyamaTakahiro Yoneyama More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , Takuya KoieTakuya Koie More articles by this author , Masakazu TanakaMasakazu Tanaka More articles by this author , Shin-Ichiro NishimuraShin-Ichiro Nishimura More articles by this author , Shizuka KurauchiShizuka Kurauchi More articles by this author , Ippei TakahashiIppei Takahashi More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1834AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The clinical diagnosis of urothelial carcinoma (UC) relies on invasive methods in patients with hematuria. Although more sensitive and noninvasive screening methods are required, a specific serum biomarker for UC is lacking. To determine whether the aberrant N-glycosylated serum immunoglobulins (Igs) can be applied as a diagnostic marker of urothelial carcinoma (UC). METHODS Between 2009 and 2016, N-glycan levels on Igs were retrospectively examined in the sera of 60 upper urinary tract UC (UTUC), 177 UC of the bladder (UCB), 317 healthy volunteer (HV) controls and 96 prostate cancer (PC) patients by high-throughput N-glycomics. The diagnostic N-glycan score (dNGScore) for detection of UCs was established by multiplying five N-glycan levels on Igs by each function value that calculated by multivariable discriminant analysis. The diagnostic performance of dNGScore was evaluated by receiver operating characteristic curves and compare the diagnostic performance of classic urine cytology and hematuria. RESULTS Total 32 types of N-glycans were identified from Igs fraction of all samples. Of those 5 N-glycans (biantannary and core fucosylated bisecting GlcNAc type N-glycans) on Igs were selected as UC associated aberrant N-glycosylated Igs and we found asialo bisecting GlcNAc type N-glycan carrying Igs were accumulated in UC patients.The dNGScore established by combination of 5 N-glycans on Igs discriminated UC patients from HV and PC patient with 92.8% sensitivity, 97.2% specificity. The area under the curve (AUC) of dNGScore was 0.969 for UC detection that was much superior to that of urine cytology (AUC, 0.707) and hematuria (AUC, 0.892). Furthermore, dNGScore can detect hematuria and urine cytology negative patients. CONCLUSIONS dNGscore based on aberrant N-glycosylation signatures of Igs were found to be promising diagnostic biomarkers of UCs. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e773 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Toshikazu Tanaka More articles by this author Tohru Yoneyama More articles by this author Daisuke Noro More articles by this author Yuta Kojima More articles by this author Kengo Imanishi More articles by this author Hayato Yamamoto More articles by this author Yuki Tobisawa More articles by this author Kazuyuki Mori More articles by this author Atsushi Imai More articles by this author Shingo Hatakeyama More articles by this author Takahiro Yoneyama More articles by this author Yasuhiro Hashimoto More articles by this author Takuya Koie More articles by this author Masakazu Tanaka More articles by this author Shin-Ichiro Nishimura More articles by this author Shizuka Kurauchi More articles by this author Ippei Takahashi More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1014 - Detection of urothelial carcinoma using selected urine-DNA methylation biomarkers in patients with gross hematuria

1014 - Detection of urothelial carcinoma using selected urine-DNA methylation biomarkers in patients with gross hematuria

Aberrant N-Glycosylation Profile of Serum Immunoglobulins is a Diagnostic Biomarker of Urothelial Carcinomas.

The aim of this study to determine whether the aberrant N-glycosylated serum immunoglobulins (Igs) can be applied as a diagnostic marker of urothelial carcinoma (UC). Between 2009 and 2016, we randomly obtained serum available from 237 UC and also 96 prostate cancer as other cancer controls from our serum bank and also obtained—from 339 healthy volunteers (HV)—controls obtained from community-dwelling volunteers in Iwaki Health Promotion Project. A total of 32 types of N-glycan levels on Igs were determined by high-throughput N-glycomics and analyzed by multivariable discriminant analysis. We found five UC-associated aberrant N-glycans changes on Igs and also found that asialo-bisecting GlcNAc type N-glycan on Igs were significantly accumulated in UC patients. The diagnostic N-glycan Score (dNGScore) established by combination of five N-glycans on Igs discriminated UC patients from HV and prostate cancer (PC) patients with 92.8% sensitivity and 97.2% specificity. The area under the curve (AUC) for of the dNGScore was 0.969 for UC detection that was much superior to that of urine cytology (AUC, 0.707) and hematuria (AUC, 0.892). Furthermore, dNGScore can detect hematuria and urine cytology negative patients. The dNGscore based on aberrant N-glycosylation signatures of Igs were found to be promising diagnostic biomarkers of UCs.

Voided urine versus bladder washing cytology for detection of urothelial carcinoma: which is better?

Objective: Cytology is recommended as part of the follow-up of high-grade non-muscle-invasive bladder cancer (NMIBC). However, currently there are no solid guideline recommendations regarding the use of voided urine versus bladder washing for cytology as part of the diagnosis or follow-up of NMIBC. The aim of this study was to investigate whether the cytological outcome was equal regarding the two techniques.Materials and methods: The authors reviewed all outpatient flexible cystoscopies carried out in their department in 2013. Patient records in the registry of pathology were examined and those with simultaneous urine and bladder washing cytology were included. Previous urothelial disease and positive histology within 3 months after the cystoscopy were registered.Results: A total of 1458 patients had both voided urine and bladder washing cytology and were included in the study, of whom 643 (44%) had a history of urothelial disease. An equal outcome of urine and bladder washing cytology was found in 1447 patients (99.2%). For the remaining 11 patients, only four patients underwent further examinations based on cytology findings in addition to what had already been planned after cystoscopy. Of the included patients, 100 (6.9%) had a positive histological outcome within 3 months.Conclusions: In most patients, no relevant difference between voided urine and bladder washing cytology was observed. Therefore, if cytology is indicated, it is recommended to use the test that is most readily available locally. The additional gain in using both urine and bladder wash is minimal, and can therefore be discarded.

MP44-04 SIGNIFICANCE OF SERUM N-GLYCAN PROFILING AS A DIAGNOSTIC BIOMARKER IN UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2017MP44-04 SIGNIFICANCE OF SERUM N-GLYCAN PROFILING AS A DIAGNOSTIC BIOMARKER IN UROTHELIAL CARCINOMA Masaaki Oikawa, Shingo Hatakeyama, Tohru Yoneyma, Yuki Tobisawa, Takuma Narita, Hayato Yamamoto, Yasuhiro Hashimoto, Takuya Koie, Shintaro Narita, Atsushi Sasaki, Norihiko Tsuchiya, Tomonori Habuchi, Ippei Takahashi, Shigeyuki Nakaji, and Chikara Ohyama Masaaki OikawaMasaaki Oikawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Tohru YoneymaTohru Yoneyma More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Takuma NaritaTakuma Narita More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , Takuya KoieTakuya Koie More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Atsushi SasakiAtsushi Sasaki More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , Tomonori HabuchiTomonori Habuchi More articles by this author , Ippei TakahashiIppei Takahashi More articles by this author , Shigeyuki NakajiShigeyuki Nakaji More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1335AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The clinical diagnosis of urothelial carcinoma (UC) relies on invasive methods in patients with hematuria. Although more sensitive and noninvasive screening methods are required, a specific serum biomarker for UC is lacking.To examine whether the serum glycan-based biomarkers can be applied to detecting UC. METHODS Between April 1994 and June 2016, serum N-glycan concentrations were retrospectively measured in 212 patients with UC before treatment (UC group) and 212 pair-matched controls (VLT group) using glycoblotting and mass spectrometry.N-glycan levels were compared between the groups using receiver operating characteristic (ROC) curves to select candidate N-glycans. We created an N-glycan score based on the combination of candidate N-glycans. The specificity and sensitivity of the candidate N-glycan score were evaluated using ROC curves. RESULTS The N-glycan score was calculated using six N-glycans (m/z 1566, 1687, 1769, 1871, 2011, 2337) that were significantly associated with UC. The median N-glycan score was significantly higher in the UC group than in the VLT group (5.0 vs. 1.0, P < 0.001). The predictive value of the N-glycan score for UC detection was significant, with a sensitivity, specificity, and area under the curve of 93%, 81%, and 0.95%, respectively. CONCLUSIONS Serum N-glycan content has the potential to be a specific and sensitive novel serum biomarker that may improve the accuracy of detection for UC and reduce unnecessary invasive screening. Validation of this test in a large-scale prospective study is needed. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e564-e565 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Masaaki Oikawa More articles by this author Shingo Hatakeyama More articles by this author Tohru Yoneyma More articles by this author Yuki Tobisawa More articles by this author Takuma Narita More articles by this author Hayato Yamamoto More articles by this author Yasuhiro Hashimoto More articles by this author Takuya Koie More articles by this author Shintaro Narita More articles by this author Atsushi Sasaki More articles by this author Norihiko Tsuchiya More articles by this author Tomonori Habuchi More articles by this author Ippei Takahashi More articles by this author Shigeyuki Nakaji More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNA promoter methylation: a new tool for accurate detection of urothelial carcinoma

Background:Urothelial carcinoma (UC) is the most common cancer affecting the urinary system, worldwide. Lack of accurate early detection tools entails delayed diagnosis, precluding more efficient and timely treatment. In a previous study, we found that miR-129-2 and miR-663a were differentially methylated in UC compared with other genitourinary tract malignancies. Here, we evaluated the diagnostic performance of those microRNAs in urine.Methods:Promoter methylation levels of miR-129-2 and miR-663a were assessed, using real-time quantitative methylation-specific PCR, in UC tissue samples (using normal urothelium as control) and, subsequently, in urine samples from UC and other genitourinary malignancies. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.Results:Promoter methylation levels of both microRNAs were significantly higher in UC tissue samples compared with normal urothelium. In urine, the assay was able to distinguish UC from other genitourinary tract carcinomas with 87.7% sensitivity and 84% specificity, resulting in 85.85% overall accuracy.Conclusions:This panel of miRNAs promoter methylation accurately detects UC in urine, comparing well with other promising epigenetic-based biomarkers. This may constitute the basis for a non-invasive assay to detect UC.

ProEx C as Diagnostic Marker for Detection of Urothelial Carcinoma in Urinary Samples: A Review.

The gold standard for the detection of urothelial carcinoma is represented by urethro-cystoscopy and biopsy. Both procedures are invasive and expensive and therefore cytology is often used as first approach to investigate on a possible neoplasia, being a safe and cost-effective diagnostic modality of evaluation. Because cytology alone is not highly sensitive for detection of low grade urothelial carcinoma and recurrence of the disease, several adjunct markers and urine based tests for urothelial carcinoma have been developed, which can help in the final diagnosis. In particular, ProEx C is an immunohistochemical cocktail containing antibodies direct against topoisomerase IIα (TOP2A) and minichromosome maintenance 2 (MCM2) proteins. It proved to be a valid biomarker especially in detecting squamous intraepithelial lesions in cervical liquid-based samples and in discerning these lesions from their mimickers, as well as in ovarian, endometrial, vulvar, primary and metastatic melanomas, breast, pancreatic and renal cell carcinomas. This brief review covers the effective utility of ProEx C as adjunct tool in assessing the urothelial lesions in urine cytology, also providing prognostic and therapeutic information to help in clinical decisions.

Significance of Serum N-glycan Profiling as a Diagnostic Biomarker in Urothelial Carcinoma

BackgroundThe clinical diagnosis of urothelial carcinoma (UC) relies on invasive methods in patients with hematuria. Although more sensitive and noninvasive screening methods are required, a specific serum biomarker for UC is lacking. ObjectiveTo examine whether serum glycan-based biomarkers can be applied to UC detection. Design, setting, and participantsBetween April 1994 and June 2016, serum N-glycan concentrations were retrospectively measured in 212 patients with UC before treatment (UC group) and 212 pair-matched controls using glycoblotting and mass spectrometry. Outcome measurements and statistical analysisN-glycan levels were compared between the groups using receiver operating characteristic curves to select candidate N-glycans. We created an N-glycan score based on the combination of candidate N-glycans. The specificity and sensitivity of the candidate N-glycan score were evaluated using receiver operating characteristic curves. Results and limitationsThe N-glycan score was calculated using six N-glycans (m/z 1566, m/z 1687, m/z 1769, m/z 1871, m/z 2011, and m/z 2337) that were significantly associated with UC. The median N-glycan score was significantly higher in the UC group than in the pair-matched control group (5.0 vs 1.0, p<0.001). The N-glycan score correctly classified UC patients with a sensitivity, specificity, and area under the curve of 93%, 81%, and 0.95, respectively. The limitations of our study included its retrospective nature and nonclinical setting. ConclusionsSerum N-glycan content has the potential to be a specific and sensitive novel serum biomarker that may improve the accuracy of the detection for UC and reduce unnecessary invasive screening. Validation of this test in a large-scale prospective study is needed. Patient summaryCombination of serum N-glycan (N-glycan score) is a novel serum marker for urothelial carcinoma that is expressed by 93% of patients and thus is far more sensitive than classic urine cytology. Validation in a large patient cohort is needed.

Abstract 1946: Urinary cell-free microRNA panel in detection of urothelial carcinoma of the urinary bladder

Abstract Urothelial carcinoma of the urinary bladder (UCUB) is the most common malignancy of the urinary system. Although about 80% of cases is a non-muscle invasive form of UCUB a high rate of local recurrence and progression to invasive form is observed. Early-stage tumors have very good prognosis, but current diagnostic methods (cystoscopy and urine cytology) suffer from low sensitivity. This reflects a large number of relapse, which occurs in almost 70% of superficial UCUB. This has led to the development of multiple molecular urinary biomarkers, but none are sufficiently robust to enter clinical practice. In this study we aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling early detection of UCUB and prediction of risk of progression to muscle-invasive form. In the first phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 UCUB patients (6 invasive, 5 high-grade non-invasive, 5 low-grade non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. MicroRNAs able distinguish between UCUB and control groups were further validated using specific TaqMan assays and qRT-PCR method on independent cohort of 100 UCUB patients, 40 controls and 25 RCC patients (training phase - 40 UCUB, 15 controls, 10 RCC; validation phase - 60 UCUB, 25 controls, 15 RCC, 20 UTI). Global expression profiling revealed set of 76 miRNAs significantly differentially expressed in urine of UCUB patients (P &amp;lt; 0,01) compared to healthy controls, thereof 64 highly up-regulated and 12 down-regulated. These miRNAs showed specificity for UCUB even when compared to other examined cohorts (RCC, UTI). Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P &amp;lt; 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p &amp;lt; 0,01). Set of 12 miRNAs with highest specificity and expression level was validated in training phase of study. Based on training phase results the panel of 3 miRNAs was profiled. Validation phase confirmed diagnostic potential and ability of this urine miRNA-based panel to differentiate between UCUB patients and controls with high sensitivity and specificity (AUC = 0,857, sensitivity = 82%, specificity = 80%). Our data have shown that urinary microRNAs could serve as sensitive and specific biomarkers of UCUB and could be useful tool to increase sensitivity of standard cytological examination and to decrease high costs for long-term follow-up of UCUB patients. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved. Citation Format: Jaroslav Juracek, Barbora Peltanova, Hana Mlcochova, Michal Stanik, Tana Machackova, Michal Fedorko, Robert Iliev, Jitka Mlcochova, Jiri Sana, Zuzana Ozanova, Jan Dolezel, Ondrej Slaby. Urinary cell-free microRNA panel in detection of urothelial carcinoma of the urinary bladder. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1946.

Role of Tetrasomy for the Diagnosis of Urothelial Carcinoma Using UroVysion Fluorescent In Situ Hybridization.

-UroVysion fluorescent in situ hybridization (FISH) is routinely used to detect urothelial carcinoma (UC). A positive threshold is defined as chromosome polysomy in 4 or more cells, which also includes tetrasomy, a natural product of cell division. -To evaluate tetrasomy for UC detection and explore the relation to the surgical diagnosis or patient history. -The FISH was performed on 1532 urine samples from patients with cytology results and 4 or more years of follow-up. We created separate polysomy and tetrasomy categories and constructed receiver operating curves to determine appropriate thresholds using biopsy (n = 194) as the gold standard. Standard FISH and a novel assay integrating cytomorphology and FISH (Target-FISH) were compared. Matching tissue biopsies of urine samples with 10 or more tetrasomy cells were analyzed. -No significant threshold was found for tetrasomy cells. Exclusion of tetrasomy from the polysomy category changed the threshold from 8.5 to 4.5 cells, increased specificity (59.2% to 78.9%), but reduced sensitivity (78.9% to 65.9%). In Target-FISH, the same approach yielded a specificity of 93.7% and sensitivity of 65.2%. Similarly, specificity improved significantly for low- and high-grade UC, but sensitivity decreased for low-grade UC. No evidence of UC was observed in 95% (52 of 55) of the patients referred for screening who had 10 or more tetrasomy cells by FISH. Matching biopsies for urines containing 10 or more tetrasomy cells showed few or no tetrasomy cells. -Tetrasomy is a nonspecific finding frequently encountered in urine FISH and should be excluded from the polysomy classification. Target-FISH is an optimal approach, offering the ability to detect rare tetrasomy tumors.

Urinary mRNA biomarker panel for the detection of urothelial carcinoma

The early detection of bladder cancer is important as the disease has a high rate of recurrence and progression. The development of accurate, non-invasive urinary assays would greatly facilitate detection. In previous studies, we have reported the discovery and initial validation of mRNA biomarkers that may be applicable in this context. In this study, we evaluated the diagnostic performance of proposed molecular signatures in an independent cohort.Forty-four mRNA transcripts were monitored blindly in urine samples obtained from a cohort of 196 subjects with known bladder disease status (89 with active BCa) using quantitative real-time PCR (RT-PCR). Statistical analyses defined associations of individual biomarkers with clinical data and the performance of predictive multivariate models was assessed using ROC curves. The majority of the candidate mRNA targets were confirmed as being associated with the presence of BCa over other clinical variables. Multivariate models identified an optimal 18-gene diagnostic signature that predicted the presence of BCa with a sensitivity of 85% and a specificity of 88% (AUC 0.935). Analysis of mRNA signatures in naturally micturated urine samples can provide valuable information for the evaluation of patients under investigation for BCa. Additional refinement and validation of promising multi-target signatures will support the development of accurate assays for the non-invasive detection and monitoring of BCa.

AB051. Comparison of urine cytology and UroVysion FISH for upper tract urothelial carcinoma detection in Chinese patients

ObjectiveUroVysion FISH has been used for screening bladder cancer in western countries, but still limited in China. This non-invasive assay is also practical for detecting upper tract urothelial carcinoma. The present study evaluates the effectiveness of UroVysion and urine cytology in detecting upper tract urothelial carcinoma (UTUC) in 216 urine samples from 75 Chinese patients.MethodsA total of 75 cases from two centers (Chengde medical college affiliated hospital and Beijing Friendship hospital) were selected with the following criteria: valid results of both UroVysion and cytology from same urine sample; histological and/or cystoscopic follow up within at least one year. The results of the UroVysion and cytology were correlated with clinical outcome that was derived from combination of histological, cystoscopic and clinical follow up information.ResultsOverall sensitivity, specificity, PPV and NPV in detecting UTUC were 69.3%, 83.3%, 91.2% and 52.1%, respectively for UroVysion, and 50.7%, 86.7%, 86.4% and 41.3%, respectively for cytology. The performance of both UroVysion and cytology was generally better in the surveillance population and in samples with high grade UTUC, but UroVysion manifests more efficiently than cytology in samples with low grade UTUC. In 57 of 216 samples from patients treated by endoscope or partial resection of ureter, the sensitivity of UroVysion and cytology are 70.1% and 61.3%.ConclusionsComparing with urine cytology, UroVysion is an efficient assay for upper tract urothelial carcinoma (UTUC) diagnosis and monitoring, with excellent clinical performance and utility in Chinese patients.

Detection of Urothelial Carcinoma: Comparison of Reduced-Dose Iterative Reconstruction with Standard-Dose Filtered Back Projection

To prospectively assess radiation dose, image quality, and diagnostic performance of computed tomography (CT) urography for detection of urothelial carcinomas by performing reduced-dose scanning with iterative reconstruction (IR) compared with standard-dose scanning with filtered back projection (FBP). The institutional review board approved the study with written informed patient consent. In total, 2163 patients at high risk for urothelial carcinomas randomly underwent standard-dose scanning with FBP (protocol A, 120 kVp for >80 kg body weight; protocol B, 100 kVp for 50-80 kg body weight) or reduced-dose scanning with IR (protocol C, 100 kVp for >80 kg body weight; protocol D, 80 kVp for 50-80 kg body weight). Objective image quality (signal-to-noise ratio and contrast-to-noise ratio) between the two groups with same weight range was measured for various regions of interest. Subjective image quality (visual image noise, artifact, ureter depiction, and overall image quality) and diagnostic accuracy (per lesion and per patient) were assessed with three- and five-point scores, respectively. Size-specific dose estimate (protocol A vs protocol C, 24.2 mGy vs 19.2 mGy, respectively; protocol B vs protocol D,13.9 mGy vs 8.8 mGy, respectively) was significantly lower in reduced-dose scanning (P < .001 for both). There were significantly higher signal-to-noise and contrast-to-noise ratios in reduced-dose scanning, except for the abdominal aorta (P < .05 for all). There was no significant difference in subjective image quality, except for artifacts in protocols B and D (range, 4-5 vs 3-4; P < .05). Per-lesion diagnostic accuracy was 90.8% (89 of 98, protocol A), 91.3% (105 of 115, protocol B), 92.9% (79 of 85, protocol C), and 88.8% (111 of 125, protocol D). Reduced-dose scanning with IR showed dose reduction and no significant difference of image quality in detection of urothelial carcinomas, except for some artifacts in 80-kVp scanning.

Identification of circulating microRNA signatures for upper tract urothelial carcinoma detection.

The involvement of circulating microRNAs (miRNAs) in cancer and their potential as diagnostic and prognostic biomarkers are becoming increasingly known. However, the significance of circulating miRNAs in upper tract urothelial carcinoma (UTUC) has remained to be investigated. The present study performed a genome wide serum miRNA analysis using a deep sequencing platform for initial screening. Subsequently, serum samples of 46 UTUC patients and 30 cancer free individuals with hematuria were subjected to a quantitative reverse transcription polymerase chain reaction analysis. The expression of thirteen miRNAs (miR-664a-3p, miR-423-5p, miR-431-5p, miR-191-5p, miR-92a-3p, miR-22-3p, miR-26a-5p, miR-33b-3p, miR-16-5p, let-7a 5p, let-7b-5p, let-7f-5p and let-7c) was significantly different in serum from UTUC patients compared with that in control samples. Receiver operator characteristic analysis showed that 10 miRNAs (miR-664a-3p, miR-431-5p, miR-423-5p, miR-191-5p, miR-33b-3p, miR-26a-5p, miR-22-3p, miR-16-5p, let-7b-5p and let-7c) had the potential to distinguish individuals with UTUC from the controls (areas under the curve >0.8). The present study provided the first evidence for the potential use of circulating miRNAs as biomarkers for UTUC diagnosis, which remains to be verified by further studies.

Ubiquilin2 as a novel marker for detection of urothelial carcinoma cells in urine.

Ubiquilin 2 (UBQLN2), an ubiquitin-related protein, is strongly expressed in urothelial carcinoma cells, in contrast to no or less expression in non-neoplastic cells; it protects cancer cells from reactive oxygen species (ROS)-induced cytotoxicity. In this study, we investigated whether UBQLN2 immunostaining, using liquid-based cytology sample could improve the accuracy of cytological urine diagnosis. Two-hundred and forty-five urinary samples, including 143 negative controls and 102 urothelial carcinomas, consisting of 42 low-grade and 60 high-grade urothelial carcinomas, were used for immunocytochemical analysis of UBQLN2. Urothelial carcinoma cells were positive for UBQLN2-staining, while non-neoplastic cells, including renal tubular cells and degenerative atypical cells, were negative. Interestingly, percentage of nuclear stain immunopositive for UBQLN2 was significantly higher in carcinoma cells with high grade/invasive phenotype than in those with low grade/noninvasive phenotype. UBQLN2 immunostaining had an overall sensitivity of 87.6%, specificity of 98.6%, positive predictive value of 97.8% and negative predictive value of 92.8% for the detection of urothelial carcinoma. UBQLN2 immunostaining is a practical test for urine cytology, even in samples with few cells, with slight atypia or severe degenerative changes. In addition, it allows prediction of tumor grade and stage by examining the cellular localization of UBQLN2.

Abstract 4943: Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma

Abstract GULP1 functions as a cytoplasmic adaptor protein in the engulfment of apoptotic corpses and cells. It was also reported to be an adapter protein of LRP1 which is known to be a key regulator of TGF-β signaling. TGF- β signaling pathway is known to contribute to urothelial carcinoma (UC) development by increasing invasiveness and metastasis, and inducing epithelial to mescenchymal (EMT) transition. By an integrated genetic approach, we recently identified GULP1 as a potential tumor suppressor gene and epigenetically silenced in ovarian cancer. In the present study we evaluated the promoter methylation of GULP1 in urothelial carcinoma (UC) and functionally characterize GULP1 in urothelial carcinogenesis. We first determined GULP1 protein expression by immunohistochemistry on a customized muscle-invasive UC (MIUC) tissue-microarray. The mechanism of gene silencing was probed using quantitative methylation-specific PCR (QMSP). The association between GULP1 expression and promoter methylation was analyzed by using a subset of primary UC and UC cell lines. Effects of forced overexpression and siRNA-mediated GULP1-knockdown were measured by cell proliferation and colony forming assays. Western blot analysis was performed for several key molecules to determine the effect of GULP1 modulation in the respective pathways. Complete loss of GULP1 expression was observed in 89 out of 104 MIUC (85.6%). In primary tumor tissues and cell lines, promoter methylation of GULP1 were inversely correlated with GULP1 expression and reactivation of GULP1 was observed by 5-aza-dC treatment of UC cell lines. Additionally QMSP assay of GULP1 was performed for 98 primary UC tissues of different cell types and 21 normal urothelial samples. Our initial analysis by using an empiric cut off value revealed GULP1 promoter methylation was significantly associated with UC (P = 0.007, Fisher's exact test). To determine the feasibility of GULP1 QMSP assay for non-invasive detection of UC using urine DNA, we tested urine from 58 UC patients and 46 healthy people. As expected, 33 out of 58(56.9%) UC cases were methylation positive while very low level of methylation were detected in 4 out of 46 urine control samples (8.7%) (P&amp;lt;0.001, Fisher's exact test). Effect of in vitro modulation of GULP1 was determined by MTT assay, BrdU assay and colony formation assay. The results consistently revealed that GULP1 had anti-proliferative activities. In order to detect the potential pathway related to GULP1, RNA from T24 and shGULP1 transfected T24 cells were applied to Cancer pathway finder array (Qiagen). Heme oxygenase 1 (HMOX1) solely showed over 3 fold increase in T24shGULP1 cells. Other associated molecules altered due to GULP1 knock-down include TGF-β RI, p-Smad3C, p15 and LRP1. In conclusion, GULP1 is a candidate tumor suppressor in UC carcinogenesis and it may have a potential for non-invasive UC detection and targeting therapy. Citation Format: Masamichi Hayashi, Leonardo O. Reis, Alexander Baras, Leonel Maldonado, Eliza Guida, Evgeny Izumchenko, Mariana Brait, Trinity Bivalacqua, George J. Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Engulfment gene GULP1 is a functional tumor suppressor through influencing TGF-β pathway and is silenced by promoter methylation in urothelial carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2015-4943

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.