The Development of a Multitarget, Multicolor Fluorescence in Situ Hybridization Assay for the Detection of Urothelial Carcinoma in Urine

Abstract

The purpose of this study was to develop a multitarget, multicolor fluorescence in situ hybridization (FISH) assay for the detection of urothelial carcinoma (UC) in urine specimens. Urinary cells obtained from voided urine specimens of 21 patients with UC and 9 normal donors were analyzed with nine different centromere enumeration probes and a single locus-specific indicator probe to determine an optimal set of FISH probes for UC detection. The four probes with the greatest sensitivity for UC detection were then labeled with a unique fluorophore and combined into a single probe set. The probes with the greatest combined sensitivity for UC detection were CEP3, CEP7, CEP17, and the 9p21 (P16) LSI. This probe set was used to evaluate urine specimens acquired from 179 patients for prospective testing (46 with biopsy-proven UC). FISH slides were evaluated by scanning the slide for cells with nuclear features suggestive of malignancy and assessing the FISH signal pattern of these cells for polysomy (ie, gains of two or more different chromosomes). A receiver operator characteristic curve revealed that a cutoff of 5 cells with polysomy as the positive criterion for cancer resulted in an overall sensitivity of 84.2% for patients with biopsy-proven UC and a specificity of 91.8% among patients with genitourinary disorders but no evidence of UC. This study demonstrates that a multitarget, multicolor FISH assay containing centromeric probes to chromosomes 3, 7, and 17 and a locus-specific probe to band 9p21 has high sensitivity and specificity for the detection of UC in voided urine specimens.

Fluorescence in situ hybridization assay detects upper urinary tract transitional cell carcinoma in patients with asymptomatic hematuria and negative urine cytology

“…This UroVysion probe mixture consists of centromeric enumeration probes (CEPs) for chromosomes 3, 7, and 17, and a locus-specific indicator (LSI) probe to the 9p21 band that are labeled with red, green, aqua, and gold fluorophores, respectively [12,13]. The UroVysion™ FISH assay has been increasingly used as a valuable adjunct in detecting and monitoring bladder cancer, not only in cytologically negative, but also in cytologically inconclusive urine samples [5].…”

“…Although a number of probes have been investigated to determine which combination yields the highest sensitivity and specificity for TCC detection, the optimal combination was found to combine CEPs for chromosomes 3, 7, and 17, with a LSI probe to the 9p21 band. This combination significantly increased both the sensitivity and specificity of the assay compared with single probe analysis [5,6,12,13].…”

Chromosomal gains and genomic loss of p53 and p16 genes in Barrett's esophagus detected by fluorescence in situ hybridization of cytology specimens

“…The scoring approach used in this study is analogous to that previously developed and validated for the detection of exfoliated malignant transitional cells in urine cytology specimens by multicolor FISH. 23,24 Using this approach, 25 cells are assessed initially focusing on the atypical/metaplastic cells marked by the cytologist in conjunction with their DAPI staining pattern (large, pale, and/or nonhomogeneous staining is typical for dysplastic/carcinoma cells, elongated nuclei with homogeneous DAPI staining are the usual appearance of metaplastic columnar cells). In cases where less than the diagnostic number of cells with FISH abnormalities were identified among the initial 25 cells, additional cells were analyzed and, unless sufficient cells to establish a positive FISH diagnosis were found, all cells on the entire slide were analyzed to ensure that no abnormal population was missed.…”

The use of Urovysion™ fluorescence in situ hybridization in the diagnosis and surveillance of non-urothelial carcinoma of the bladder

“…However, this was later extended to include its use as a screening tool in patients with hematuria and risk factors for urothelial carcinoma. 17,18 Urovysiont FISH has a reported sensitivity of 73-92%, a specificity of 89-96% for urothelial carcinoma detection and several studies have confirmed its usefulness in the diagnosis and surveillance of these tumors. 8,14,[19][20][21][22] The test is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus in malignant urothelial cells that are shed in the urine of persons with urothelial carcinoma.…”