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FISH Analysis in Urothelial Cancer

FISH Analysis in Urothelial Cancer. Michael Neat, Dr M Mason and Dr A Chandra. Interphase FISH in urothelial carcinoma. Low sensitivity of urine cytology esp in low grade lesions Need for additional tests for detection and monitoring In conjunction with not in lieu of routine procedures

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FISH Analysis in Urothelial Cancer

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  1. FISH Analysis in Urothelial Cancer Michael Neat, Dr M Mason and Dr A Chandra

  2. Interphase FISH in urothelial carcinoma • Low sensitivity of urine cytology esp in low grade lesions • Need for additional tests for detection and monitoring • In conjunction with not in lieu of routine procedures • The UroVysion FISH assay • First published 2000; 4 loci with best combined sensitivity from 10 candidates • 2 FDA trials • 2001 FDA approved for detection of recurrence • 2005 • Pts with haematuria • No prev Hx Ca bladder • Ca bladder histologically Dx in 50/497 (10.2%) • FISH detected 69% of these, cytology 38% • When TaG1 tumours excluded; FISH 83%, cytology 50% • FDA approval 2005 pts with haematuria

  3. The FISH assay Abbott Molecular Mix of 4 probes labelled with 4 different fluorochromes Unstained ThinPrep slides

  4. Analysis/scoring criteria • Initially select morphologically abnormal cells • Large nuclear size/irregular shape • Patchy DAPI stain • Cell clusters (non-overlapping) • If no morphologically abnormal cells present, scan all cells • Minimum analysis of 25 cells • FISH positive if: • ≥4 cells showing gain of at least 2 of #3, #7 & #17 • ≥12 cells showing homozygous deletion of p16 i.e. no p16 signals

  5. Examples of abnormal signal patterns Increased copy no. of #3, #7 & #17 Homozygous deletion of p16 ICN & homozygous deletion of p16

  6. Success rate • Analysis successful 58/59 (98%) cases • 1/59 – post treatment, probe hyb failed - ? DNA degraded • 14/58 (24%) FISH positive • Highly reproducible assay when samples adequate • 12/71 (17%) samples received insufficient material • Caraway et al 65/1006 (6%) insufficient (cytospin)

  7. Performance of the assay • Halling & Kipp Eur Ren Genotourinary Dis.2006;2:51-54 • Mean sensitivity of FISH cf. cytology in 12 studies • Cytology specificity higher than FISH (93% vs. 85%) • Hajdinjak Urol Oncol. 2008;26:646-651 • Meta-analysis (2477 FISH tests in 14 studies, cytology from 12) • Cytology specificity higher than FISH (96% vs. 83%)

  8. Conflicting data • May et al. Urology 2007;70(3):449-53 • Conventional cytology can be better than FISH in experienced hands • Sensitivity 71% vs. 53.2% • Specificity 83% vs. 74% • Moonen et al. Eur Urol 2007;51(5):1275-80 • No improvement over cytology in detection of recurrence • Sensitivity 40.6% vs. 39.1% • Specificity 89.7% vs. 89.7%

  9. Clinical applications • Detection of recurrence • Gross or microscopic haematuria • Anticipatory positive results • FISH can detect tumour before clinically detectable by cytoscopy or cytology • Helpful for clarifying equivocal cytology in patients with equivocal or negative cytoscopy • ? detection of non-UC bladder tumours • Histological variants detected on FFPE’s • ? Exfoliating tumours

  10. Clinical applications (cont.) • Follow-up post intravesical therapy • BCG-associated inflammation makes cytoscopic & cytologic interpretation difficult • Savic et al • 68 pts; NMIBC, post BCG • Both positive cytology and positive FISH predict failure of BCG • FISH superior when cytology non-definitive, i.e. equivocal, mild or moderate atypia • Whitson et al • Positive FISH after IVT significant predictor of recurrence in multivariate analysis • Detection of upper tract UC

  11. Disadvantages • Cost • Technical & interpretive difficulties • Training • equipment • False positives • BK polyoma virus (rare) • Tetraploidy • Reactive urothelial cells • Cells in S or G2 phase • ? Less specific predictor of malignancy • False negatives • low-grade neoplasms if representative cells are not shed into the urine sample • Lack of atypical cells on the slide used for FISH

  12. Conclusions • Useful adjunctive assay to increase sensitivity in targeted patient populations • In routine use in many countries • Developing assay • Does earlier detection translate into decreased mortality? • Is negative predictive value sufficient to decrease the need for or frequency of cytoscopic follow-up?

  13. Total FISH tests undertaken Total tests = 56 (41 patients) 13 43

  14. Follow up data

  15. Correlation

  16. Sensitivity and Specificity of FISH with histology

  17. Cytology categories • C1: Unsuitable for diagnosis • C2: Benign • C3: Atypia, probably reactive (expected outcome – 10-15% malignant) • C4: Atypia, probably malignant • C5: Malignant

  18. Sensitivity and Specificity of cytology with histology (C3=NEGATIVE)

  19. C3 cytology and FISH Total tests = 21 4 17

  20. Future applications • Emerging evidence that persistent positive FISH following BCG treatment is predictive of stage progression of bladder cancer • Cystectomy may be offered to these patients following a course of BCG and positive FISH test

  21. Acknowledgments • The UroCyt vials used in this study were provided by Hologic.

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