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

4. Analysis/scoring criteria Initially select morphologically abnormal cells 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 Minimum analysis of 25 cells FISH positive if: 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 Homozygous deletion of p16 Increased copy no. of #3, #7 & #17 ICN & homozygous deletion of p16

6. Success rate Analysis successful 58/59 (98%) cases 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 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 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%) Stage/grade FISH (%) Cytology (%) Ta6728 Tis9773 T19067 T2-T49274 Grade 1 5018 Grade 2 7545 Grade 3 9069 Stage/grade FISH (%) Cytology (%) All72(69-75)42(38-45) Excluding Ta 86(82-89)61(56-66) Hajdinjak 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 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 Detection of recurrence Gross or microscopic haematuria Gross or microscopic haematuria Anticipatory positive results 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 Helpful for clarifying equivocal cytology in patients with equivocal or negative cytoscopy ? detection of non-UC bladder tumours ? detection of non-UC bladder tumours –Histological variants detected on FFPE’s –? Exfoliating tumours

10. Clinical applications (cont.) Follow-up post intravesical therapy 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 Detection of upper tract UC Author FISH (%) Cytology (%) Marin-Aguilera et al 76.736 Akkad et al 87.560

11. Disadvantages Cost Cost Technical & interpretive difficulties Technical & interpretive difficulties –Training –equipment False positives False positives –BK polyoma virus (rare) –Tetraploidy  Reactive urothelial cells  Cells in S or G2 phase  ? Less specific predictor of malignancy False negatives 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 Useful adjunctive assay to increase sensitivity in targeted patient populations In routine use in many countries In routine use in many countries Developing assay Developing assay Does earlier detection translate into decreased mortality? Does earlier detection translate into decreased mortality? Is negative predictive value sufficient to decrease the need for or frequency of cytoscopic follow-up? Is negative predictive value sufficient to decrease the need for or frequency of cytoscopic follow-up?

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

14. Follow up data

15. Correlation

16. Sensitivity and Specificity of FISH with histology FISH NEGATIVE FISH POSITIVE HISTOLOGY NEGATIVE 142 HISTOLOGY POSITIVE 24 SENSITIVITY66.7%SPECIFICITY87.5%

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

18. Sensitivity and Specificity of cytology with histology (C3=NEGATIVE) CYTOLOGY NEGATIVE (C 1,2,3) CYTOLOGY POSITIVE (C4,5) HISTOLOGY NEGATIVE 63 HISTOLOGY POSITIVE 15 SENSITIVITY62.5% SPECIFICITY85.7%

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

20. Future applications Emerging evidence that persistent positive FISH following BCG treatment is predictive of stage progression of bladder cancer 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 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. The UroCyt vials used in this study were provided by Hologic.