1 UROVYSION® FISH Urine Cytology Assessment: Principles and ConceptsAHDI-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 histologyBladder cancer stages and management Urine Cytology Principles and Paris System FISH principles and UROVYSION FISH

3 Urinary System AnatomyImage: kidneystoners.org

4 Urinary System HistologyImage: 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 GradeLow grade Papillary High grade Flat Image: 123rf.com

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

10 Rule out infections, stoneSymptoms 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 IndicationsEstablish 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 typesVoided, 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 AccuracyVoided 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 GRADEContact 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 GradeLow grade Papillary High grade Flat Image: 123rf.com

16 Urothelial Carcinoma Grade How bad does the tumor look?

17 Paris System of ReportingMakes 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 ReportingHIGH GRADE CARCINOMA EVERYTHING ELSE HIGH GRADE ATYPICAL NEGATIVE

19 Paris System of ReportingNegative 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 carcinomaURINE 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 changesSporadic 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 reproducibleNarrows 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 CancerDetects: Aneuploidies of chromosomes 3, 7, 17 Deletions of 9p21

29 UROVYSION SensitivityScenario 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 FilterAqua 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?

35

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 nucleusPOSITIVE 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 reportDoes 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 histologyBladder cancer stages and management Urine Cytology Principles and Paris System FISH principles and UROVYSION FISH

46 AHDI-FL 2017 ANNUAL MEETINGThank you! AHDI-FL 2017 ANNUAL MEETING