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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
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Objectives Background Urinary system anatomy and histology
Bladder cancer stages and management Urine Cytology Principles and Paris System FISH principles and UROVYSION FISH
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Urinary System Anatomy
Image: kidneystoners.org
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Urinary System Histology
Image: yale.edu
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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
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Types of tumor growths Image: cancer.org
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Urothelial Carcinoma Grade
Low grade Papillary High grade Flat Image: 123rf.com
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Urothelial Cancer Stage How deep does the tumor invade?
Image: Cityofhope.org
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Cystoscopy Image: drtimnathan-urology.com.au
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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
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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
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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
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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)
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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
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Urothelial Carcinoma Grade
Low grade Papillary High grade Flat Image: 123rf.com
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Urothelial Carcinoma Grade How bad does the tumor look?
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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
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Paris System of Reporting
HIGH GRADE CARCINOMA EVERYTHING ELSE HIGH GRADE ATYPICAL NEGATIVE
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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
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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
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FISH: What are we looking at?
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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.
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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.
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Image: Wikimedia.org
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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
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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
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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!!!
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UROVYSION FDA approved Marketed by Abbott Molecular Bladder Cancer
Detects: Aneuploidies of chromosomes 3, 7, 17 Deletions of 9p21
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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
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UROVYSION FISH Probes
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Specimen Transport and Collection
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Specimen Handling
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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.
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Which cells to scan?
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UROVYSION Cartoons Negative Loss of gold, positive Aneuploidy
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ANEUPLOIDY
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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)
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Homozygous deletion of 9p21: NO gold signals in nucleus
POSITIVE CRITERIA Homozygous deletion of 9p21: NO gold signals in nucleus
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IT’S LIKELY CANCER WHEN:
Four nuclei with gains in multiple chromosomes OR Twelve nuclei with zero gold signals
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Image: Arch. Pathol. Lab Med. 2016
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Urovysion FISH Worksheet
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Sample Negative report
Does not rule out the possibility of LOW GRADE NON-INVASIVE UROTHELIAL CARCINOMA Sample report Sample Negative report
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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
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Objectives Background Urinary system anatomy and histology
Bladder cancer stages and management Urine Cytology Principles and Paris System FISH principles and UROVYSION FISH
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AHDI-FL 2017 ANNUAL MEETING
Thank you! AHDI-FL 2017 ANNUAL MEETING
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