Dataset Information

Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence In Situ Hybridization (Quartet) Test.

ABSTRACT: BACKGROUND:Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations. OBJECTIVE:We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine. DESIGN, SETTING, AND PARTICIPANTS:We performed genome-wide copy number variation analysis on cohorts from the University of Texas MD Anderson Cancer Center (n=40) and The Cancer Genome Atlas (n=129), and identified the most frequently amplified chromosomal regions. These data were used to select four of the amplified regions to design a multicolor FISH test, referred to as the quartet test. Assay validation was performed on urine samples from 98 patients with bladder cancer: 56 with low-grade papillary, 42 with high-grade invasive disease, and 48 benign controls. INTERVENTION:The quartet test can be used in clinical practice for noninvasive detection of bladder cancer. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:We initially analyzed samples using a fraction of abnormal cell scores and then by the quantitative score, which included not only the proportion of cells with abnormal copy numbers, but also the proportion of cells with numbers of altered copies and degree of amplification. We used receiver operator characteristic (ROC) curves to identify cutoff values for the scores at which performances of sensitivity and specificity were maximized. RESULTS AND LIMITATIONS:The copy number status assessed by probes detected in voided urine reflected the amplification status of the primary tumor. An ROC curve summarizing the proportion of assayed cells with any abnormal copy numbers gave specificity of 93.8% and sensitivity of 78.6% using the proportion of cells with abnormal copy numbers. The quantitative score giving extra weight to cells with multiple simultaneous amplifications provided 95.8% specificity and 76.8% sensitivity. Both percentage of abnormal cells and quantitative scores were highly effective for assessing the grade of the tumor. The full spectrum of potential clinical applications was not explored in the current study, and further validation studies are needed. CONCLUSIONS:The quartet test shows promising specificity and sensitivity results, but it requires validation on a larger multi-institutional cohort of samples. PATIENT SUMMARY:The quartet test can be used for noninvasive detection of bladder cancer in voided urine. It can also be used to assess the grade of the tumor and tumor recurrence as well as post-treatment effects.

SUBMITTER: Zhang S

PROVIDER: S-EPMC6081265 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence In Situ Hybridization (Quartet) Test.

Zhang Shizhen S Wang Yan Y Bondaruk Jolanta J Majewski Tadeusz T Yao Hui H Lee Sangkyou S Lee June Goo JG Cogdell David D Lotan Yair Y Dinney Colin C Wei Peng P Baggerly Keith K Czerniak Bogdan B

European urology focus 20180207 4

<h4>Background</h4>Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations.<h4>Objective</h4>We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine.<h4>Design, setting, and participants</h4>We performed genome-wide copy number variation analysis on cohorts from ...[more]

PMID: 29428551

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Project description:BACKGROUND: Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. METHODS: Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. RESULTS: The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. CONCLUSION: Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.

Project description:Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Dataset Information

Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence In Situ Hybridization (Quartet) Test.

Publications

Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence In Situ Hybridization (Quartet) Test.

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