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

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

Urinary System Anatomy Image: kidneystoners.org

Urinary System Histology Image: yale.edu

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

Types of tumor growths Image: cancer.org

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

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

Cystoscopy Image: drtimnathan-urology.com.au

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

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

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

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)

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

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

Urothelial Carcinoma Grade How bad does the tumor look?

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

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

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

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

FISH: What are we looking at?

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.

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.

Image: Wikimedia.org

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

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

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!!!

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

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

UROVYSION FISH Probes

Specimen Transport and Collection

Specimen Handling

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.

Which cells to scan?

UROVYSION Cartoons Negative Loss of gold, positive Aneuploidy

ANEUPLOIDY

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)

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

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

Image: Arch. Pathol. Lab Med. 2016

Urovysion FISH Worksheet

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

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

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

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