Molecular Markers in Diagnosis and Prognostication of Bladder Cancer Farhat Abbas M.D.

Molecular Markers in Diagnosis and
Prognostication of Bladder Cancer
Farhat Abbas M.D.
Associate Professor
Department of Surgery (Urology)
The Aga Khan University, Karachi, PAKISTAN
Bladder cancer
!
!
!
!
!
2nd most common GU tumor
15 per 100,000 persons
USA: 53,200 new cases; 12,200
deaths / year
90% TCC
70% Superficial, 30% muscle-invasive
Superficial bladder cancer
!
!
!
!
!
70% will recur
10 -15% will progress
Grade 1:
< 10% risk of recurrence and
progression
Grade 3:
70 - 80% recurrence
33-45% progression
Recurrence and progression may occur with > 5 year
interval
Therefore there is a strong need for long-term surveillance
Situation in underdeveloped world
!
!
Most common urological tumor
30 – 65% present with invasive disease:
!
!
!
!
!
!
inadequate diagnostic facilities
Lack of screening of high-risk individuals
lack of community awareness
High bladder cancer-related morbidity &
mortality
Critical goal: to enhance early detection
A case-control study of urinary molecular
markers
Objectives
!
To compare the telomerase activity with
other molecular markers in voided urine
samples and urine cytology for the
detection of bladder cancer
!
To compare the telomerase activity in
bladder tumor versus normal urothelium
and correlate it to tumor grade
Collaborators
Farhat Abbas: Urology (P.I.)
Jamsheer Talati: Urology
Amanullah Memon: Urology
Anwar Siddiqui: Biochemistry
Naila Kiyani: Pathology
Saeed Akhtar: Community Health Sciences
Raziuddin Biyabani: Urology
Hammad Ather: Urology
Aga Khan University, Karachi
Telomeres
&
Telomerase
W hat are Telomeres ?
Telomeres (TTAG G G) act as protective
caps at end ofthe chromoso mes
"To protect from exonuclease digestion
"To prevent aberrant recombination
"To prevent apoptosis
• Each time a cell divides itloses 50100 base pairs atthe end ofits
telomers
• Once a certain amount oftelomeres
are lost,the cellstops dividing and
goes into apoptosis – senescence
process
Cell have evolved different mechanisms to
prevent this progressive telomere loss
"Co mplex recombination
"Retrotransposition schemes
"Telo merase: a specialized reverse
transcriptase that adds back telo mere
sequences that are lost during
replication
Telomerase
!
a specialized reverse
transcriptase that adds
back telomere
sequences that are lost
during replication.
!
Germ cells and cancer
cells produce
telomerase whereas
normal somatic cells
lack its activity
3’
5’
G T T AG GG T T AG G G T T AG GG
C CCAA T C CC
Telomerase contains:
"R NA molecule
"Reverse transcriptase
"Telomerase binds to 3’ end of DNA and
extends them by copying R NA template
in multiples of hexamer repeat sequences
" Telomerase
activityis turned offin cells
from most tissues as they differentiate.
" Itis reactivated in most human cancers.
Type of cancer
Type of sample/ Cancer
No. of
telomerase
positive
sa mples
Head & Neck
Oral rinse/ Squamous Cancer
14/44
Lung
Bronchiallavage/ lung Cancer
29/37
Lung
Pleuralfluid/lung Cancer
64/70
Pancrease
Pancreaticjuice/ ductal Cancer
9/12
Colon
Colonic wash/ colon Cancer
9/15
Thyroid
FNA/ Papillary Cancer
9/14
Breast
FNA/ Invasive Cancer
52/71
Prostate
Prostate biopsy/ Prostate Cancer
17/19
Bladder
Urine/ Bladder Cancer
CIS
Cystitis
Normal
88/104
5/5
16/47
0/35
Ovaries
Peritoneal fluid/ Ovarian Cancer
37/42
Cervix
Pap smear/ Cervical Cancer
15/17
Normal cell
Telomere
shortening
Telomerase
Telomere maintenance
p53
Rb
Immortalization
Ras
Oncogenic transformation
Tumor cell
Apoptosis
(Senescence)
Markers
Diagnostic
Telo merase
N M P22
FDP
BTA(stat)
He moglobin
dipstick
Urine
Cytology
Prognostic
Telo merase
Ki 67
P53, MD M,
p16
Selection criteria for the patients
Exclusion
Inclusion
# Patients with known
bladder tum or
#
Urinary instrumentation
within 2 preceding weeks
# Patients with suspected
bladder tum or
#
Frank urinary tract
infection
# Patients on surveillance
with prior resection of
bladder tum or
#
Patients not consenting
for additional tests to be
performed on urine/
tissue sam ples
Selection criteria for controls
Inclusion
#
Rando mly selected
patients undergoing
cystoscopy for non
m alignant cause
Exclusion
#
#
#
#
Current or prior history of
urinary tract cancer
Urinary instrumentation
within 2 preceding weeks
Frank urinary tract
infection
Patients not consenting
for additional tests to be
performed on urine/
tissue sam ples
Urine sa mple collection and handling
Voided urine/ Bladder washings
He moglobin
dipstick
2-3mls at roo m temp within3 hrs..
BTA
STAT
5 drops at roo m temp within3 hrs..
N M P22
Telo merase
FDP
Cytology
20-30 mls in urine stabilizers at
roo m temp. within 3 hrs.
50-100 mls im m ediately transferred on ice
1 ml within2 hrs. at roo m temp.
50 - 100 mls within 2 hrs at roo m temp.
Tissue Sample Handling
Bladder tum or
patient
Grossly norm al
Bladder mucosa
Control Patient
Bladder tumor
10 % Formalin
For Histopathology &
Im munostaining
Grossly norm al
Bladder mucosa
Im mediate storage
in liquid nitrogen & transport
to lab for storage
at –80°° C
Tissue Sa mple Collection
Patient
Control
Bladder tumor
Nor mal Bladder
M ucosa
Histopathology
p53, MD M, p16
Ki-67
Telo merase
activity
Extraction from cells (voided Urine
and bladder washings)
Add PBS
Cells were pelleted at 3000xg and PBS
was added
Add Lysis
reagent
W ashed 3X in PBS
Resuspended in Lysis reagent and
incubated on ice for 30 min.
Centrifuged at 16000xg
Supernatant was collected and stored
At –70ºC
Keep on ice
Extraction from Tissues
cryostat sections were prepared
and ho mogenized
Resuspended in lysis reagent &
incubated on ice for 30 minutes
Centrifuged at 16000xg
Supernatant collected and stored
at –70ºC
Amplification
To the reaction mixture
PCR Program
Step1: 10 min, 25ºC
Te mplate was added
nuclease free water
Step2: 5 min, 94 º C
30s, 94 º C
Add sa mple, negative control &
Positive control
30s, 50 º C
30s, 72 º C
Start PCR progra m m e
Denaturation
Add denaturation and
incubate
Step3: 10min, 72 º C
Step4: hold, 4 º C
Hybridization/ELISA
Add hybridization buffer T
Transfer to MTP wells and incubate
Wash & add anti DIG-HRP and
incubate
Wash and add TMB substrate and
incubate
Add stop reagent and read Abs. at
450nm.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
RESULTS
Voided Urine Samples
Urine
Telomerase NMP22 BTA FDP Urine
N=32
N=32 N=33 N=33 Dipstic Cytology
k
N=33
N=33
Positive
26
22
17
14
24
16
Sensitivity
81%
69%
52%
42%
73%
48%
TRAP ASSAY
! 29
/ 32 +ve in TCC samples – 92% sensitivity
! Mean Telomerase activity:
- Voided urine in TCC cases = 52.9 + 25.3
- Normal urothelium
= 20.7 + 7.8
- Bladder tumor
= 561 + 231.5
(p=0.001)
Telomerase activity in urine samples in patients with bladder carcinoma
References
Sample Size (n)
telomerase activity (sensitivity)
Muller et al 1996
30
0%
Kinoshita et al 1997
42
55%
Yoshida et al 1997
26
62%
Linn et al 1997
12
0%
Dalbagni et al 1997
63
35%
Kavaler et al 1998
104
85%
Yokota et al 1998
29
86%
Muller et al 1998
30
7%
Ramakumar et al 1999
57
70%
Kitsukawa et al 1999
26
85%
Rahat et al 1999
21
81%
Gelimi et al 2000
33
94%
Arai et al 2000
19
21%
Bailkowska – Hobrozanka
et al 2000
35
49%
Muller M, Oncogene, 2002
Telomerase activity in tissue samples in patients with bladder carcinoma
References
Sample Size (n)
telomerase activity (sensitivity)
Muller et al 1996
75
96%
Lin et al 1996
40
97%
Kamata et al 1996
13
100%
Kinoshita et al 1997
42
98%
Yoshida et al 1997
56
86%
Linn et al 1997
12
92%
Kyo et al 1997
13
100%
Lee et al 1998
23
96%
Yokota et al 1998
26
100%
Ito et al 1998a
22
100%
Mayfield et al 1998
52
81%
Rahat et al 1999
29
90%
Gelimi et al 2000
33
94%
Lancelin et al 2000
14
78%
Muller M, Oncogene, 2002
Commercial Assays (FDA Approved)
Sensitivity
Overall
Grade
Specificity
Stage
Cytology
26%
G1 - 6%
G2 - 36%
G3 - 79%
PTis - 67%
PTa - 26%
PT1-4 64%
> 93%
BTAstat
65-79%
G1 - 57%
G2 - 81%
G3 - 80%
PTis - 100%
PTa - 53%
PT1 - 70%
PT2-4 88%
60 – 64 %
BTAtrak
66%
G1 - 48%
G2 - 59%
G3 - 88%
PTis - 60%
PTa - 51%
PT1 - 88%
PT2-4 88%
62 – 69%
NMP22
73-100%
G1 - 57 %
G2 - 81 %
G3 - 80%
PTis – 66%
PTa-1 71%
PT2-4 92%
85 – 97 %
AccuDx
(FDPs)
68%
G1 - 63%
G2 - 88%
G3 - 95%
PTis - 100%
PTa – 2 75-85%
PT3 - 100%
96 %
ImmunoCyt
86%
G1 - 84%
G2 - 84%
G3 - 89%
PTis - 100%
PTa - 86%
PT1 - 85%
PT2 - 3 83%
79%
Saad A, Eur Urol, 2001
Research – Based Assays
Sensitivity
Overall
Specificity
Grade
Stage
TRAP
62%
G1 - 79%
G2 - 84%
G3 - 87%
PTis - 100%
PTa -1 75%
PT2-3 84%
96%
CD44
91%
NA
NA
83%
HA
83%
G1 - 80%
G2 - 80%
G3 - 85%
PTis - 87%
PTa - 76%
PT1 - 87%
PT2-3 92%
90%
HAase
81%
G1 - 22%
G2 - 82%
G3 - 81%
PTis - 77%
PTa - 42%
PT1 - 87%
PT2-3 86%
84%
HA-HAase
92%
G1 - 86 %
G2 - 95 %
G3 - 93%
PTis – 93%
PTa-1 87%
PT1 - 93%
PT2-3 92-100%
85%
Cadherine-E
35%
NA
PTa – T1 - 53%
PT2-3 – 25%
NA
Lewis X
85%
NA
PTis – 100%
85%
Microsatellite
assay
83%
G1 - 50%
G3 - 81%
G3 - 81%
G2 - 50%
G2 - 50%
PTa – T1 60%
72%
PT2 -4 72%
PT2-4
10%
Saad A, Eur Urol, 2001
Cytokeratins
Sensitivity
Overall
Specificity
Grade
Stage
UBC-EISA Test
64%
G1 - 50%
G2 - 50%
G3 - 68%
PTis - 100%
PTa - 62%
PT1- 53%
PT2 – 80%
UBC-Rapid Test
66%
G1 - 44%
G2 - 78%
G3 - 75%
PTis - 50%
PT1- 69%
PTa - 60%
PT2 - 80%
83%
83-96%
G1 - 75%
G2 - 90%
G3 - 92%
PTis - 100% PTa - 75%
PT1 - 86%
PT2 - 72%
PT3-4 100%
90%
CYFRA-21-1
96%
TPS
64%
NA
NA
84%
CK20
86%
G1 - 71 % G2 - 80
%
G3-4 100%
NA
85%
TPA
80%
G1 - 75 %
G2 - 87 %
G3 - 80%
Ptis - 66%
PTa -75%
PT1 - 84%
PT2 - 54%
NA
486 P 3/12
89%
NA
NA
85%
AN43 & BB369
47%
NA
NA
10%
DD23
85%
NA
NA
Saad A, 2001
95%
Conclusions
!
Our interim results and the literature
substantiates the potential usefulness of
urinary telomerase in detection of bladder
cancer.
!
Further studies on the specificity of these
markers, prognostic markers, and sub
units of telomerase are being done.
`