| BACKGROUND1. The incidence of urothelial carcinoma (UC) is highest in urinary system tumor. Most of them can be treated by operation, but its high recurrent rate often depresses us. Therefore, the important problem for urologist, which is how to diagnose it early and prevent its recurrence, must be solved urgently.2. The common clinical diagnosis methods are endoscopy, including cystoscopy and ureteroscopy, and cytology at present, but the sensitivity of cytology is lower and endoscopy is invasive, painful and hard to accept for patients.3. With the further development of bladder UC genetics study, research workers have discovered that most common genetic aberration is the partial or entire deletion of chromosome 9 (such as p16 gene locus), and this is closely related to development of bladder UC, and the aneuploidy of 3,7,17 chromosome is closely related to progression of bladder UC.OBJECTIVE1. To evaluate the clinical value of FISH assay in voided urine specimens for detection of UC (including bladder UC and upper urinary tract UC), and to compare the results with those afforded by urinary cytology analysis.2. To evaluate the clinical value of FISH assay in voided urine specimens of follow-up patients after TURBT, and to compare the results with cystoscopy at the same period, and analyze the concordance of the two.3. To account aberration rate of 3,7,17 chromosome and p16 gene locus in chinese UC patients. And to offer clinical evidence for Chinese UC patients to establish noninvasive, painless, and high specificity and sensitivity early diagnosis method.4. To found standard specimen management, quality control and normal operational procedure for FISH and establish base for clinical Utility.METHODS1. PatientsBetween October 2007 and Dec 2009, voided urines from 99 patients with bladder UC,23 hematuria patients without UC,26 consecutive patients with suspect upper urinary tract UC,20 urologic nonmalignant diseases or controls (for setting up threshold value) and 56 patients with bladder UC at 3 months,6months,9 months and 12 months after TURBT.2. FISH AnalysisCollection of urine samples from patients mentioned above were done in the morning (first urination after night) for FISH analysis. FISH assay was performed using a mixture of fluorescent labeled DNA probes for the centromeric regions of chromosomes 3,7,17, and p16 (9p21) locus. Two DNA probes were mixed together as a set double-target FISH and paired as follows:chromosome 3 (rhodamine) and chromosome 7 (FITC), chromosome 17 (FITC) and p16 (rhodamine). Visualization of the signals was done using a computer applied imaging system. Normal or abnormal was based signals number and abnormal cells percentage.3. Evaluation Methods100 ml urine samples in the three consecutive morning from 99 patients with bladder UC,23 hematuria patients without UC,26 consecutive patients with suspect upper urinary tract UC,20 urologic nonmalignant diseases or healthy persons were performed for urine cytology, the results were compared with FISH’s.56 patients with bladder UC at 3 months,6 months,9 months and 12 months after TURBT must be performed with cystoscopy, if new tumor was discovered, and biopsy must be done. And their results were compared with FISH’s too. The concordance of FISH with cystoscopy is analyzed.4. Statistical AnalysisAll data were analyzed by statistics software (SPSS13.0), and a 95% confidence interval was considered. All evaluation index of FISH assay and cytology were numeration data, and were used by chi-square test. When number was less than 40, Fisher’s exact test was used. The result of cystoscopy and FISH assay from 56 patients with bladder UC after TURBT was analyzed with Kappa test. When Kappa value was more 0.75, the two’s concordance was better, when it was less than 0.4, the two’s concordance was worse, and when it was between 0.4 and 0.75, the two’s concordance was middling.RESULTS1. In the bladder UC, the overall sensitivity and specificity of FISH assay was 81.8% and 95.3%, sensitivity of G,, G2 and G3 was 54.8%,89.5% and 100% respectively, and sensitivity of Ta, Tis, T, and T2-4 was 35.7%,100%,85.7% and 97.9%. The overall sensitivity and specificity of cytology analysis was 39.4% and 93.0%, sensitivity of G1, G2 and G3 was 3.2%,34.2% and 83.3%, and sensitivity of Ta, Tis, T1 and T2-4 was 0,100%,22.9% and 60.4%. Sensitivity for FISH assay was very high in patients with higher stage and higher grade, and sensitivity of FISH assay was higher in lower stage and grade than cytology (P<0.05), but specificity was similar. The genetically aberration rate of 3,7,17 chromosome centromeres and P16 gene was 70.7%(70/99),68.7%(68/99),80.8%(80/99), and 57.6%(57/99) respectively.2. In the upper urinary tract UC, the sensitivity of FISH and urine cytology was 90.9 %(20/22) and 59.1%(13/22) respectively. The sensitivity of FISH was significant higher (P<0.05). The specificity of FISH and urine cytology was 100%(24/24) and 95.8%(23/24) respectively. No significant difference between them were observed(P >0.05). The genetically aberration rate of 3,7,17 chromosome centromeres and p16 gene was 90.9%(20/22),86.4%(19/22),63.6%(14/22) and 68.2%(15/22) respectively.3. There were 8 positve effects in 56 patients with Bladder UC at 3 months,6 months,9 months and 12 months after TURBT, but there were only 5 recurring by cystoscopy. By statistics analysis, the concordance of FISH with cystoscopy was middling, and Kappa value is 0.604 (P<0.001).CONCLUSION1. FISH assay compared with urine cytology improves the sensitivity to diagnose bladder UC, and offers similar specificity. FISH raises accuracy to detect bladder UC patient with lower grade, lower stage and superficial badder UC, and detects most higher grade-stage, especially in some patients with invasive bladder UC. Compared with cytology, FISH assay is a efficient diagnosis tool for bladder UC, but cannot substitute cystoscopy completely.2. FISH assay has higher sensitivity and specificity for diagnosis of upper urinary tract UC, and has greater sensitivity than cytology while maintaining a similar specificity. FISH assay is better than cytology for diagnosis of upper urinary tract UC, and will be considered a effective and noninvasive methods.3. In our study, genetically aberration rate of 3,7,17 chromosome centromeres and P16 (9p21)is high, development of UC is related with gene mutation, and diagnosis of UC by FISH assay is feasible.4. FISH assay has good clinical value in postoperative monitoring for bladder UC, it might imply recurrence of bladder UC earlier than cystoscopy, but cannot replace cystoscopy now.FUNDATION1. This study is granted by national department of health first (number is WKJ 2007-3-001), multicentre clinical utility research of fluorescence in situ hybridization (FISH) in antepartum hereditary disease and partial cancer detection, one of branches, clinical utility study of FISH in bladder urothelial carcinoma (UC).2. The other grant is medical research fundation from Guang Dong province (number is B2009199), clinical utility research of FISH for the surveillance of bladder urothelial carcinoma patients treated with transurethral resection of bladder tumor (TURBT). |
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