1. UROVYSION® FISH Urine Cytology Assessment: Principles and Concepts
AHDI-FL 2017 ANNUAL MEETING
UROVYSION® FISH Urine Cytology Assessment: Principles and Concepts
Charanjeet Singh, MD
Asst. Prof. Pathology, Univ. of Central Florida
Staff Pathologist, Florida Hospital, Orlando

2. Objectives Background Urinary system anatomy and histology
Bladder cancer stages and management
Urine Cytology Principles and Paris System
FISH principles and UROVYSION FISH

3. Urinary System Anatomy
Image: kidneystoners.org

4. Urinary System Histology
Image: yale.edu

5. Urothelial Carcinoma
Tumor arising from the urothelial mucosa that lines the urinary bladder, ureters, and renal pelvis
Most common malignancy of the bladder and upper urinary tract

6. Types of tumor growths
Image: cancer.org

7. Urothelial Carcinoma Grade
Low grade
Papillary
High grade
Flat
Image: 123rf.com

8. Urothelial Cancer Stage How deep does the tumor invade?
Image: Cityofhope.org

9. Cystoscopy
Image: drtimnathan-urology.com.au

10. Rule out infections, stone
Symptoms
or mass on radiology
History
Rule out infections, stone
CYSTOSCOPY
Visible tumor,
Resection
or biopsy
HIGH GRADE AND DEEP TUMOR
RESECTION OF TUMOR OR ORGAN
LOW GRADE OR SUPERFICIAL TUMOR
RESECT AND FOLLOW-UP (LOW GRADE)
No visible tumor,
Check cytology
CYTOLOGY
No visible tumor on cystoscopy, POSITIVE
CIS
BCG therapy
No visible tumor on cystoscopy, ATYPICAL
UROVYSION
Post treatment for CIS or Visible tumor
POSITIVE
RESECTION OF BLADDER OR KIDNEY UROVYSION
ATYPICAL
Follow-up or UROVYSION

11. Urine Cytology Indications
Establish diagnosis in patients with hematuria (5%)
Superior than biopsy and cystoscopy for CIS
Screening in high risk patients
Paint industry, heavy metal industry, tobacco
Follow-up patients after treatment for urothelial cancer

12. Urine Cytology Urine types
Voided, clean catch is most common but poor preservation and genital epithelium contamination
Washing or brushing, better preservation and cellularity but localized
Catheterized, not contaminated by genital epithelium but poor preservation

13. Urine Cytology Diagnostic Accuracy
Voided urine on three consecutive days (75% vs. 50%)
Demographic better in high risk patients
Tumor grade (90% high grade vs. 40% low grade)

14. Urine Cytology Diagnostic Principle LOW GRADE HIGH GRADE
Contact of cells with urine
YES
Degree of cell shedding
(cell adhesion)
FEW
MANY
Degree of difference of cells from normal
MINIMAL
MARKED

15. Urothelial Carcinoma Grade
Low grade
Papillary
High grade
Flat
Image: 123rf.com

16. Urothelial Carcinoma Grade How bad does the tumor look?

17. Paris System of Reporting
Makes reporting uniform
Easier for clinicians between different institutions to understand reports
Makes future studies easier because same criteria are used by everyone
Adopting models from gynecologic and thyroid cytology

18. Paris System of Reporting
HIGH GRADE CARCINOMA
EVERYTHING ELSE
HIGH GRADE ATYPICAL NEGATIVE

19. Paris System of Reporting
Negative for high grade urothelial carcinoma
Caveat: Cannot exclude low grade urothelial carcinoma
Atypical urothelial cells
Favor reactive or favor neoplastic
Suspicious for high grade urothelial carcinoma
Positive for high grade urothelial carcinoma
Others
Low grade urothelial carcinoma

20. Visible lesion clinically Positive for high grade carcinoma
URINE CYTOLOGY
ATYPIA NO
No visible lesion
Negative
Visible lesion clinically
Fibrovascular cores
Low grade carcinoma
YES
MILD
History of treatment?
No history of treatment
ATYPICAL
SEVERE
LESS THAN 10 CELLS
SUSCPICIOUS
MANY CELLS
Positive for high grade carcinoma
RED ALL CAPS
INDICATIONS FOR FISH

21. FISH: What are we looking at?

22. Definitions
Gene: A part of double stranded DNA that codes for a protein
Denaturation: The process of separating two complementary strands of DNA, typically using heat.
Hybridization: Inducing a DNA probe sequence to bind to its complementary sequence on a chromosome.

23. Definitions
FISH - Fluorescence In Situ Hybridization: A molecular detection technique that uses fluorescently-labeled DNA to hybridize to complimentary regions within the specimen DNA.
CEP- Chromosome Enumerating Probe: binds repetitive sequences of DNA. Enumeration of chromosome copies.
LSI- Locus Specific Identifier: Probe designed to bind to the loci of a specific target region or gene. loss or gain of loci.
DAPI II- (4'-6-Diamidino-2-phenylindole): Fluorescent counterstain that binds to background double-stranded DNA.

24. Image: Wikimedia.org

25. What does FISH detect?
Aneuploidy (change in normal copy number) of entire chromosomes (CEP)
Gain of specific genes or portions of chromosomes (LSI)
Deletion of specific genes or portions of chromosomes (LSI)
Translocations- rearrangements of parts of chromosomes

26. Urothelial Carcinoma Genetic changes
Sporadic changes most commonly associated with tumor initiation:
Homozygous deletion of the p16 gene at 9p21
Aneuploidy (copy number increase) in chromosomes 3, 7, 8, 9, 11, 15, 17

27. Benefits of “FISH” Objective and highly reproducible
Narrows down from atypical to negative or positive
Highly Sensitive (70-90%)
Specific (50%) but less than urine (85%)
Tastes good and is a good source of omega fats!!!

28. UROVYSION FDA approved Marketed by Abbott Molecular Bladder Cancer
Detects:
Aneuploidies of chromosomes 3, 7, 17
Deletions of 9p21

29. UROVYSION Sensitivity
Scenario
Sensitivity
Hematuria surveillance
FISH Positive / Cytology Negative
30% patients
60% urothelial carcinoma
History of Carcinoma
FISH Positive / Cytology and Cystoscopy Negative
65% chance of recurrence in 3 years
FISH Negative / Cytology and Cystoscopy Negative
13% chance of recurrence
Post BCG therapy
FISH Positive
10x more risk of high stage

30. UROVYSION FISH Probes

31. Specimen Transport and Collection

32. Specimen Handling

33. Special microscope DAPI Single Filter Green/Red Dual Filter
Aqua Single Filter
Gold Single Filter
Epifluorescence microscope equipped with 100-watt mercury lamp.
Magnification of 400X for scanning 600X to 1000X for analysis.

34. Which cells to scan?

36. UROVYSION Cartoons
Negative
Loss of gold, positive
Aneuploidy

37. ANEUPLOIDY

38. POSITIVE CRITERIA
Gains (more than 2 signals) in two or more chromosome enumeration probes (3-red, 7-green, 17-aqua)
POSITIVE
(4 RED, 2 GREEN, 3 AQUA)
NEGATIVE
(2 RED, 2 GREEN, 3 AQUA)

39. Homozygous deletion of 9p21: NO gold signals in nucleus
POSITIVE CRITERIA
Homozygous deletion of 9p21: NO gold signals in nucleus

40. IT’S LIKELY CANCER WHEN:
Four nuclei with gains in multiple chromosomes OR Twelve nuclei with zero gold signals

41. Image: Arch. Pathol. Lab Med. 2016

42. Urovysion FISH Worksheet

43. Sample Negative report
Does not rule out the possibility of LOW GRADE NON-INVASIVE UROTHELIAL CARCINOMA
Sample report
Sample Negative report

44. Sample POSITIVE report
“CONSISTENT” with HIGH GRADE UROTHELIAL OR OTHER TUMOR of urinary tract. If no visible tumor then follow-up (65%)
Sample POSITIVE report

45. Objectives Background Urinary system anatomy and histology
Bladder cancer stages and management
Urine Cytology Principles and Paris System
FISH principles and UROVYSION FISH

46. AHDI-FL 2017 ANNUAL MEETING
Thank you!
AHDI-FL 2017 ANNUAL MEETING