FISH Analysis in Urothelial
Cancer
Michael Neat, Dr M Mason
and Dr A Chandra
Interphase FISH in urothelial
carcinoma

Low sensitivity of urine cytology esp in low grade lesions
– Need for additional tests for detection and monitoring
– In conjunction with not in lieu of routine procedures

The UroVysion FISH assay
– First published 2000; 4 loci with best combined sensitivity from 10
candidates
– 2 FDA trials
 2001 FDA approved for detection of recurrence
 2005
–
–
–
–
–
–
Pts with haematuria
No prev Hx Ca bladder
Ca bladder histologically Dx in 50/497 (10.2%)
FISH detected 69% of these, cytology 38%
When TaG1 tumours excluded; FISH 83%, cytology 50%
FDA approval 2005 pts with haematuria
The FISH assay
Abbott Molecular
Mix of 4 probes labelled with 4 different fluorochromes
Unstained ThinPrep slides
Analysis/scoring criteria

Initially select morphologically abnormal cells
–
–
–
–
Large nuclear size/irregular shape
Patchy DAPI stain
Cell clusters (non-overlapping)
If no morphologically abnormal cells present, scan all
cells
Minimum analysis of 25 cells
 FISH positive if:

– ≥4 cells showing gain of at least 2 of #3, #7 & #17
– ≥12 cells showing homozygous deletion of p16 i.e. no
p16 signals
Examples of abnormal signal
patterns
Increased copy no.
of #3, #7 & #17
Homozygous
deletion of p16
ICN & homozygous
deletion of p16
Success rate

Analysis successful 58/59 (98%) cases
– 1/59 – post treatment, probe hyb failed - ? DNA
degraded
– 14/58 (24%) FISH positive

Highly reproducible assay when samples adequate
– 12/71 (17%) samples received insufficient material
– Caraway et al 65/1006 (6%) insufficient (cytospin)
Performance of the assay

Halling & Kipp
Eur Ren Genotourinary Dis.
2006;2:51-54
– Mean sensitivity of FISH cf.
cytology in 12 studies
– Cytology specificity higher than
FISH (93% vs. 85%)

Stage/grade
FISH (%)
Cytology (%)
Ta
67
28
Tis
97
73
T1
90
67
T2-T4
92
74
Grade 1
50
18
Grade 2
75
45
Grade 3
90
69
Stage/grade
FISH (%)
Cytology (%)
All
72
(69-75)
42
(38-45)
Excluding
Ta
86
(82-89)
61
(56-66)
Hajdinjak
Urol Oncol. 2008;26:646-651
– Meta-analysis (2477 FISH tests
in 14 studies, cytology from 12)
– Cytology specificity higher than
FISH (96% vs. 83%)

Conflicting data
– May et al. Urology 2007;70(3):449-53
 Conventional cytology can be better than FISH in experienced
hands
 Sensitivity 71% vs. 53.2%
 Specificity 83% vs. 74%
– Moonen et al. Eur Urol 2007;51(5):1275-80
 No improvement over cytology in detection of recurrence
 Sensitivity 40.6% vs. 39.1%
 Specificity 89.7% vs. 89.7%
Clinical applications





Detection of recurrence
Gross or microscopic haematuria
Anticipatory positive results
– FISH can detect tumour before clinically
detectable by cytoscopy or cytology
Helpful for clarifying equivocal cytology in
patients with equivocal or negative cytoscopy
? detection of non-UC bladder tumours
– Histological variants detected on FFPE’s
– ? Exfoliating tumours
Clinical applications (cont.)


Follow-up post intravesical therapy
– BCG-associated inflammation makes cytoscopic & cytologic
interpretation difficult
– Savic et al
 68 pts; NMIBC, post BCG
 Both positive cytology and positive FISH predict failure of BCG
 FISH superior when cytology non-definitive, i.e. equivocal, mild
or moderate atypia
– Whitson et al
 Positive FISH after IVT significant predictor of recurrence in
multivariate analysis
Detection of upper tract UC
Author
FISH (%)
Cytology (%)
Marin-Aguilera et al
76.7
36
Akkad et al
87.5
60
Disadvantages


Cost
Technical & interpretive difficulties
– Training
– equipment

False positives
– BK polyoma virus (rare)
– Tetraploidy
 Reactive urothelial cells
 Cells in S or G2 phase
 ? Less specific predictor of malignancy

False negatives
– low-grade neoplasms if representative cells are not shed into the
urine sample
– Lack of atypical cells on the slide used for FISH
Conclusions
Useful adjunctive assay to increase
sensitivity in targeted patient populations
 In routine use in many countries
 Developing assay
 Does earlier detection translate into
decreased mortality?
 Is negative predictive value sufficient to
decrease the need for or frequency of
cytoscopic follow-up?

Total FISH tests undertaken
Total tests =
56
13
(41 patients)
Negative
Positive
43
Follow up data
41
45
40
35
30
25
20
15
10
5
0
lo
gy
15
N
o
hi
st
o
to
lo
gy
H
is
pe
rfo
rm
ed
os
co
py
al
p
at
ie
nt
s
22
Cy
st
To
t
37
Total number
Cytology- Cytology- FISHHistology FISH Histology
Correlation
18
11
0
5
10
4
12
4
15
Match
Mis-match
4
20
25
Sensitivity and Specificity of FISH with
histology
FISH NEGATIVE
HISTOLOGY
NEGATIVE
HISTOLOGY
POSITIVE
FISH POSITIVE
14
2
2
4
SENSITIVITY
66.7%
SPECIFICITY
87.5%
Cytology categories


C1: Unsuitable for diagnosis
C2: Benign

C3: Atypia, probably reactive (expected
outcome – 10-15% malignant)

C4: Atypia, probably malignant
C5: Malignant

Sensitivity and Specificity of cytology
with histology (C3=NEGATIVE)
CYTOLOGY
NEGATIVE (C 1,2,3)
HISTOLOGY
NEGATIVE
HISTOLOGY
POSITIVE
6
1
SENSITIVITY
62.5%
SPECIFICITY
85.7%
CYTOLOGY
POSITIVE
(C4,5)
3
5
C3 cytology and FISH
Total tests = 21
4
C3 FISH
negative
C3 FISH positive
17
Future applications


Emerging evidence that persistent positive FISH
following BCG treatment is predictive of stage
progression of bladder cancer
Cystectomy may be offered to these patients
following a course of BCG and positive FISH
test
Acknowledgments

The UroCyt vials used in this study were
provided by Hologic.