fish

1. Presenter
Rohini Singh
Moderator
Prof. Kamal Agarwal

2. ADVANTAGES OF URINE CYTOLOGY FOR
UROTHELIAL CARCINOMA
 Urinary cytology have a well established role in the
detection and monitoring of urothelial carcinoma,
the main strength being high specificity for high
grade urothelial carcinoma and carcinoma in situ.
 Combined cystoscopy and cytology are the gold
standard for diagnosis and followup of urothelial
carcinoma

3. LIMITATIONS OF URINARY CYTOLOGY
 Low sensitivity for low grade urothelial carcinoma and non
invasive tumors, due to overlap of cytological features with
benign lesions.
 Early diagnostic difficulty in lesions of upper urinary tract.
 Difficulty in interpretation of results after radiotherapy and
intravesical therapy of Bacillus Calmette Guerin(BCG).
 Commonly used category of poorly defined atypical
cytology of uncertain significance.

4. Review of Fluorescence in situ Hybridization

5. Fluorescence in situ Hybridization (FISH)
FISH - a process which vividly paints
chromosomes or portions of chromosomes
with fluorescent molecules

6. Fluorescence in situ Hybridization (FISH)
 Identifies chromosomal abnormalities.
 Aids in gene mapping, toxicological studies,
analysis of chromosome structural
aberrations, and ploidy determination

7. Fluorescence in situ Hybridization (FISH)
 Used to identify the presence and location of
a region of DNA or RNA within
morphologically preserved chromosome
preparations, fixed cells or tissue sections

8. Fluorescence in situ Hybridization (FISH)
 This means you can view a segment or entire
chromosome with your own eyes.
 Was often used during M phase but is now
used on I phase chromosomes as well.

9. Fluorescence in situ Hybridization (FISH)
Advantage: less labor-intensive method
for confirming the presence of a DNA
segment within an entire genome than
other conventional methods like
Southern blotting.

10. FISH Procedure
 Denature the chromosomes
 Denature the probe
 Hybridization
 Fluorescence staining
 Examine slides or store in the dark

11. FISH PROCEDURE

12. FISH Uses
 Detection of high concentrations of base
pairing.
 Also used in germ cell or prenatal diagnosis
of conditions such as aneuploidy.

13. FISH - Uses
 FISH can be used in the study of transgenic
animals (eg: Polly)
 Selective markers show if the human DNA
was inserted successfully and pinpoint where
the human DNA is
 Transgenic Mouse

14. WHY FISH….?
 Development of cancer are driven by genetic
aberrations such as mutations and
chromosomes/gene copy abnormalities.
 FISH is a robust technique for detection of
chromosome copy number gains, amplification and
deletion of DNA loci and translocation.
 FISH analysis of cytological specimen have become
a standard method to identify chromosomal
aberrations for diagnostic, prognostic or predictive
purpose.

15. FISH IN URINE CYTOLOGY
INDICATIONS OF FISH ANALYSIS IN URINARY
CYTOLOGY
 Hematuria in patients with an increased risk of UC
 Atypical urine cytology
 Control after BCG treatment
 Upper urinary tract cytology
 Surveillance after transurethral resection

16. APPLICATION
FISH is applicable to almost all types of
cytological specimen such as :
 air dried smears
 alcohol fixed smears
 Liquid based specimen
 Unstained or papanicolaou stained smears

17. SAMPLE FOR FISH TECHNIQUE
 VOIDED URINE
 CATHETERIZED URINE
 BLADDER WASHING
 BLADDER BRUSHING

18. CHROMOSOMAL ABERRATIONS IN
UROTHELIAL CARCINOMA
 Increased number of copy of chromosome 3,7 and
17.
 Deletion of 9p21,the site of the tumour suppressor
gene p16,is also common and occurs early in the
development of both papillary and flat urothelial
neoplasia

19. PROCEDURE AND INTERPRETATION OF
RESULT
Four probes are selected –
 Chromosome 3
 Chromosome 7
 Chromosome 17
 Locus specific identifier for the region 9p21, the site
of P16 tumor suppressor gene.

20. SCORING OF FISH SIGNALS-
 Few number of abnormal cells are preferred over
high number of random cells, basic principle being
chromosomal polysomy-strongly associated with
morphological features of the nuclei like nuclear
enlargement, irregular borders.
 25 cells are examined.

21. POSITIVE FISH RESULT-
 Presence of >or= 4 morphologically abnormal cells
with multiple gains i.e >2 signals of atleast 2 of 3
chromosomes 3,7,17
OR
 A loss of both copies(homozygous deletion) of 9p21
in >or= 12/25 cells.
But if diagnostic criteria are kept so high, sensitivity
decreases as few low grade urothelial carcinoma
show heterozygous deletion-so the recommended
rate of 9p21 deletion is >10/25 cells

25. ADVANTAGE OVER URINE CYTOLOGY
 Sensitivity and specificity of FISH is 72 to 83% as
compared to 42 to 96% for urine cytology.
 In urothelial carcinoma of upper urinary tract FISH
sensitivity and specificity is 54-86 to 78-100% as
compared to cytology which range from 24 to 40% .
 Diagnostic difficulty in atypical or equivocal urinary
cytology is over come by FISH.

26. ● In BCG treatment of urothelial carcinoma FISH result are
superior to cytology as a marker of relapse. FISH in BCG
treated patient is an independent indicator of cytology
and cystoscopy result.
● Negative test results indicate the presence of benign
cytological changes and helps to avoid unnecessary
investigations such as cystoscopy or other invasive
procedures.

27. Pitfalls of FISH
 False positive results are seen in few benign lessions.
But the distinguishing being tetraploidy with a
balanced duplication of the whole genome which
prevail in non-neoplastic conditions of the bladder.
 False negative results are seen in few cases(<30%)
of low grade urothelial neoplasia.

28. CONCLUSION
Considering the limitations and diagnostic
pitfall, FISH is a promising tool in urothelial
carcinoma for early diagnosis, prognosis and
follow up cases,being the minimal invasive
procedure.

29. THANK YOU