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The detection of chromosomal aneusomy by fluorescence in situ hybridization in sputum predicts lung cancer incidence.

ABSTRACT: Lung cancer usually is disseminated (advanced) and has a poor prognosis at diagnosis. Current and former smokers are at a high risk for lung cancer and are candidates for prevention and early detection strategies. Sputum is a potential source of biomarkers that might determine either lung cancer risk or the presence of early lung cancer, but no current sputum test is sufficiently sensitive and specific for effective screening. We used fluorescence in situ hybridization (FISH) to measure chromosomal aneusomy (CA) in sputum samples collected prospectively from 100 incident lung cancer cases and 96 controls (matched on age, gender, and date of collection) nested within an ongoing high-risk cohort. The CA-FISH assay was aimed at four DNA targets: epidermal growth factor receptor, MYC, 5p15, and CEP 6. The sensitivity of a positive CA-FISH assay (abnormal for two or more of the four markers) for lung cancer was substantially higher for samples collected within 18 months (76% sensitivity) than for samples collected more than 18 months (31%) before lung cancer diagnosis. Sensitivity was higher for squamous cell cancers (94%) than for other histologic types (69%). CA-FISH specificity based on samples collected within 18 months before diagnosis was 88%. The adjusted odds ratio (OR) of lung cancer for specimens collected within 18 months before a cancer diagnosis was higher for the CA-FISH assay [OR, 29.9; 95% confidence interval (95% CI), 9.5-94.1] than for previously studied ORs of cytologic atypia (OR, 1.8; 95% CI, 1.3-2.6) and gene promoter methylation (OR, 6.5; 95% CI, 1.2-35.5). Whether CA-FISH is an indicator of extreme risk for incident lung cancer or detects exfoliated cancer cells is unknown. The apparent promise of CA-FISH in sputum for assessing lung cancer risk and/or for lung cancer early detection now needs to be validated in a clinical screening trial.

SUBMITTER: Varella-Garcia M

PROVIDER: S-EPMC2939746 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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: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.

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The detection of chromosomal aneusomy by fluorescence in situ hybridization in sputum predicts lung cancer incidence.

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