Unlabeled blocking DNA is also included with the probes to suppress sequences contained within the target loci that are common to other chromosomes. When hybridized and visualized, these probes provide information on chromosome copy number for chromosome ploidy enumeration. Cells recovered from urine pellets were fixed on slides. The DNA was denatured to its single stranded form and subsequently allowed to hybridize with the UroVysion probes. Following hybridization, the unbound probe was removed by a series of washes, and the nuclei were counterstained with DAPI 4, 6 diamidinophenylindole and viewed using a fluorescent microscope.
At least 25 morphologically abnormal cells were scored. Two independent observers blinded to the clinical information evaluated all samples. In case of disagreement, both observers evaluated the sample again at the same time. A sample was considered positive for UC if at least one of the following criteria was met: 1 identification of four or more cells with gains in two or more different chromosomes 3, 7 or 17 , 2 observation of homozygous deletion of 9p21 in 12 or more cells.
Pathologists performed the histopathology evaluation on the patients who underwent TUR or biopsy. The sensitivity of FISH and cytology was determined for the patients with pathology-proven UC, stratified for tumor stage and grade. The specificity of FISH and cytology was calculated for the patients with no histological confirmation of UC or negative cystoscopy findings. The difference between the two methods was determined by the McNemar test.
SPSS There were patients with abnormal lesions on cystoscopy, and all of them underwent TUR or biopsy. One hundred and sixty-six patients who did not undergo TUR or biopsy were negative on cystoscopy. In all, patients had confirmed BUC based on the pathology reports.
The tumors were pTis in six patients, pTa in 55, pT1 in 41, and pT2 in 20; 78 were low-grade and 44 high-grade. Among the patients with histologically confirmed UC, 30 were FISH-positive with the concurrent cytology results suggestive of malignancy or positive Fig.
FISH shows four copies of chromosome 3 red , three copies of chromosome 7 green and six copies of chromosome 17 aqua B.
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Click for larger image Fig. FISH shows four copies of chromosome 3 red and 7 green , and three copies of chromosome 17 aqua B. Thirty-six patients who had no visible tumor with flat, erythematous mucosa suspicious lesions underwent TUR or biopsies of the bladder lesion. Among them, FISH were positive in 20 There were 61 patients with a history of UC in this population. Fifty-three of them had documented recurrences and 28 In the analysis of the patients evaluated for suspicious symptoms without a history of UC, 69 had histologically confirmed UC. Among patients with no histological evidence for UC or had negative cystoscopy findings, FISH was negative in The use of genetic analysis of urine cells has demonstrated specificity equivalent to that of cytology 9 , FISH is used for the detection of cytogenetic abnormalities in malignant cells.
Several prior studies 10 - 12 have reported that FISH was significantly more sensitive than cytology for the detection of UC and that the specificity of FISH and cytology were not significantly different. However, since the confirmation of higher sensitivity of FISH than that of cytology, few investigations for usefulness of FISH have been performed in clinical practice. The collected volume of urine for FISH or cytology was more than 40 mL, respectively in this study, thus avoiding problems caused by insufficient sample. Nevertheless, the sensitivity of FISH and cytology, in the present study, was unexpectedly lower than in several prior reports 6 , 11 , 13 , The possible explanations for these findings are as follows: 1 Different tumor sizes, grade and stage in each study; it is known that tumor size, grade and stage influence sensitivity and specificity of urine testing for bladder tumor 15 , The population of this study included Based on the tumor size, there were 83 patients with tumors 1 cm or less, 23 with 3 cm or less and 16 with tumors more than 3 cm.
The tumor characteristics of our population may have resulted in the FISH and cytology sensitivity being lower than that of other studies. According to the study of Bhuiyan et al. Urine specimens were exclusively collected from voided urine in this study. All participants in this study were cases under surveillance for BUC in the clinical practice setting. According to several prior reports, a variety of criteria has been applied for FISH positive results, nevertheless, the optimal criteria to define FISH-positive results are not absolutely clear 7 , 8 , 18 , In some investigations, a specimen was considered FISH positive for BC if ten or more cells with gain of a single chromosome or if ten or more cells with homozygous loss of the 9p21 locus 18 , In this study, fifteen patients with false-negative FISH had at least one cell with an abnormal signal pattern consistent with polysomy of chromosome 3, 7, 17, and 9p We regard our criteria for FISH positive results as more strict than those of previous studies.
Among 59 patients who had had a history of BUC with or without upper tract UC, 19 underwent intravesical immunotherapy.
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Because intravesical therapy might cause false-positive result of urine test for detecting BUC 20 and chromosomal integrity was not affected by intravesical therapy 21 , 22 , it is not likely that intravesical therapy affected FISH result to show lower sensitivity of FISH than other study. Among 36 patients who had suspicious lesions without visible tumor on cystoscopy, 15 were FISH-positive with discordant results atypical or negative on cytology.
Thirteen In these patients, seven patients without visible tumor on cystoscopy were atypical or negative in cytology and 6 of them had the positive results of FISH. However, none of the patients with negative results of FISH had the results suggestive of malignancy or positive in cytology. The majority of them 51 were negative or atypical on cytology, while only three had results suggestive of malignancy or positive by cytology. Overall, 49 There were 18 false-positive results by FISH.
Among them, four patients had results suggestive of malignancy or positive by cytology; TUR or biopsy was performed in two patients with abnormal lesions on cystoscopy. The histopathology of these cases was chronic inflammation and reactive urothelium without malignancy. According to the report of Sarosdy et al. Another investigation reported that multi-target FISH might help to stratify the risk of UC recurrence, at the time of a negative cystoscopy, by using optimal criteria for FISH positive results 8.
Although two patients with false-positive FISH had BUC diagnosed during follow-up, it is hard to assess the clinical implication of the false-positive FISH results because of the limited follow-up duration of this study. The limitations of this study include that it was a retrospective study and that the histopathology results were not available in all patients because TUR or biopsy was determined by cystoscopy finding.
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However, cystoscopy, considered as the gold standard for the detection of BUC, was performed to identify the presence or absence of bladder lesions in all patients. Moreover, to exclude the influence of upper tract UC on the analysis, the study population was restricted to only patients being evaluated for BUC. Despite these limitations, this study is one of few studies that compared the performance characteristics of FISH and cytology in clinical practice setting, unlike other studies which included normal healthy volunteers and the patients with BPH as controls.
In conclusion, the sensitivity of the FISH assay using chromosomes 3, 7, 17, and 9p21 for the detection of BUC was superior to that of urine cytology, regardless of the tumor stage and grade, although FISH was slightly less specific than cytology.
Even if taking its high cost into consideration, FISH may be a significant additional and complementary method for the detection of BUC, especially in patients who have no visible tumor but flat, erythematous mucosa suspicious lesion on cystoscopy. J Korean Med Sci. Published online Nov 09, Address for correspondence: Hyun Moo Lee, M. Go to:. Patients and samples Voided urine specimens from patients were obtained for FISH analysis and cytology examination between April and July Cytology Cytology samples were centrifuged 1, rpm for 5 min and stained using Papanicolaou's technique.
Histopathology examination Pathologists performed the histopathology evaluation on the patients who underwent TUR or biopsy. Statistical analysis The sensitivity of FISH and cytology was determined for the patients with pathology-proven UC, stratified for tumor stage and grade.
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Biology and management of bladder cancer. N Engl J Med ;— The clinical value of urinary cytology: 12 years of experience with patients. J Clin Pathol ;— Urine based markers of urological malignancy. Raab correctly place urinary cytology in the backwater of the?
Materials and Methods
For a variety of reasons, these specimens are saved for the end of the day, cause the most trouble and frustration, and are the least successful from the standpoint of the pathologist, the urologist, or the patient. This book represents, in keeping with the philosophy behind the series, Essentials in Cytopathology, a systematic description of - croscopic?
Drawing upon their personal collections and the diagnostic resources of s- eral major cytologic laboratories, they have assembled examples of the common diagnostic entities in the? Handy tables accompany the p- tographs, offering help where needed. This is particularly relevant because the subtlety of urinary cytology de?
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