Bladder carcinoma- urinary biomarkers diagnosis and staging

URINARY BIOMARKERS,
DIAGNOSIS AND STAGING
OF BLADDER CANCER
Dept of Urology
Govt Royapettah Hospital and Kilpauk Medical College
Chennai 1

Moderators:
Professors:
• Prof. Dr. G. Sivasankar, M.S., M.Ch.,
• Prof. Dr. A. Senthilvel, M.S., M.Ch.,
Asst Professors:
• Dr. J. Sivabalan, M.S., M.Ch.,
• Dr. R. Bhargavi, M.S., M.Ch.,
• Dr. S. Raju, M.S., M.Ch.,
• Dr. K. Muthurathinam, M.S., M.Ch.,
• Dr. D. Tamilselvan, M.S., M.Ch.,
• Dr. K. Senthilkumar, M.S., M.Ch.
Dept of Urology, GRH and KMC, Chennai. 2

CLINICAL DIAGNOSIS
Signs and Symptoms
• painless gross hematuria – m/c (85%)
• Microscopic hematuria – nearly all patient
• hematuria - intermittent can be related to Valsalva maneuver…1
• Thus, if a patient in the bladder cancer age range has unexplained hematuria
on a urine specimen (microscopic or gross) and a “confirmatory” second
specimen is free of any hematuria, cystoscopic examination is still
warranted…2
3
Dept of Urology, GRH and KMC, Chennai.

• Irritative voiding symptoms (frequency, urgency, and dysuria) - usually a/w
diffuse carcinoma in situ or invasive bladder cancer.
• Other signs and symptoms of bladder cancer include flank pain from
ureteral obstruction, lower extremity edema, and pelvic mass.
• Very rarely, patients present with symptoms of advanced disease - weight
loss and abdominal or bone pain.
4

CYSTOSCOPY
• GOLD STANDARD…1
• SOC –WLC…2
• sensitivity = 62-84% , specificity = 43-98%...
• Less reliable - small papillary bladder tumors & CIS
• This is expressed in the high early recurrence rate after TUR because
"what the eyes cannot see, the hands cannot resect.“
• In order to decrease the number of tumors that are overlooked or not
completely resected during TUR, it is important to improve the
endoscopic visualization of bladder tumors. 5

• on WLC growths are seen as Papillary, flat or diffuse red velvety
lesions
• three relatively new optical techniques that have been introduced to
advance endoscopy either by improving visualization of bladder
tumors or by providing real-time pathological information
6

FLOURESENT CYSTOSCOPY
• Aim - to improve the visualization of bladder cancer based on
cystoscopic detection of fluorescent signals from neoplastic tissue.
• This fluorescence is accomplished by the intravesical administration
of photosensitizing agents (5-aminolevulenic acid (5-ALA)- or its
derivative hexaminolevulinate (HAL- FDA APPROVED ) which cause
selective accumulation of photoactive porphyrins in rapidly
proliferating cells and emit red fluorescence under blue wavelength
light with a wavelength of 360-450 nm
• The solution containing the photosensitizing agents is instilled in the
bladder via a transurethral catheter prior to surgery.
7

• The timing of instillation depends on the type of agent used:
1 hour – HAL
2-3 hours - 5-ALA.
• By illuminating the mucosa with blue-violet light, the neoplastic cells
appear red or pink against a blue background
8

9

COMPARISON (WLC VS BLC)
• Photodynamic diagnosis, in addition to white light cystoscopy,
improves the detection of bladder cancer, for both papillary lesions
and CIS.
• BLC detected 14.7% more Ta tumours and 40.8% more CIS lesions
compared with WLC
• residual tumor rate – Low (15% vs 35%)
• A longer recurrence-free survival (15.8-27% at 12 months vs 12-15% at
24 months)
10

• FP -Higher (8.8-62.5% vs. 7.1-47%, respectively).
- Inflammation
- recent TUR
- tangential illumination of the bladder mucosa
- intravesical therapy, especially bacillus Calmette-Guérin.
➢Photodynamic diagnosis has a good safety profile
11

NARROW BAND IMAGING
Endoscopic optical image enhancement technique designed for
endoscopy to enhance contrast b/w mucosal surfaces & microvascular
surfaces
• Mechanism - filtering of white light into two narrow bandwidths of
light, 415 nm (blue light) and 540 nm (green light), which penetrate
tissue only superficially and are specifically absorbed by hemoglobin.
• Because bladder tumors tend to be well vascularised, NBI will
increase the contrast between these lesions and normal bladder
12

Vascular structurs appear dark brown or green
against pink or white mucosal background
13

• 22-56% of NMIBC patients narrow-band imaging detected additional
tumors.
• FP - 32% to 36%.
• limitation - severe hematuria, visualization is suboptimal because of
the absorption of the narrow-band imaging light by circulating
erythrocytes.
14

COMPARISON
• 12 month recurrence rate – similar (27% - WLC TURBT VS 25.4% -
NBI Assisted TURBT)
15

OPTICAL COHERENCE TOMOGRAPHY
Optical coherence tomography is a noninvasive optical technique that can provide cross-
sectional images containing subsurface tissue information
• It is the optical equivalent of B-mode ultrasound imaging, except that it is based on depth-
resolved detection of backscattered light instead of reflected sound waves.
• This results in images with a high resolution of up to 2 μm (approaching the resolution of
microscopy) and a maximum imaging depth of 2-3 mm.
• Because optical coherence tomography does not require a medium or direct contact with the
tissue under investigation; it is well suitable for endoscopic applications (by inserting a
(flexible) optical coherence tomography probe through the working channel of a cystoscope)
16

• it is feasible to distinguish the different bladder wall layers including
lamina propria and muscularis propria with OCT
• OCT can differentiate urothelial carcinoma (UC) from normal bladder
mucosa with a sensitivity and specificity ranging from 84% to 100%
and from 78% to 90%, respectively
17

• the discrimination of normal urothelium versus UC is based on
qualitative analysis of the OCT images:
➢ normal tissue - the urothelium is uniform and the bladder wall layers
are clearly delineated based on their different backscattering capacities
➢UC - increased backscattering, heterogeneity, and broadened
urothelium in case of papillary tumors.
18

• OCT could discriminate Ta, T1, and T2 tumors with a sensitivity of
90%, 75%, and 100%, respectively and a specificity of 89%, 97%, and
90%, respectively.
• With OCT during TUR, discrimination between muscle-invasive and
nonmuscle-invasive tumors could be done with a sensitivity of 100%
and specificity of 77%
19

LIMITATIONS OCT
• False-positives - scarring or inflammation of the mucosa.
• Large tumors with extensive broadened urothelium imaging depth
will be impaired, thus compromising the staging ability of OCT
>>>>>> overcome by applying optical coherence tomography to the
tumor base.
• limited field of view >>>> not very suitable for bladder mapping
20

VIRTUAL CYSTOSCOPY
• This technique is based on 3D digital reconstruction of CT
or MR images of the contrast or air-filled bladder and
could possibly serve as a noninvasive technique to evaluate
the bladder mucosa…
• Limitations
- lack of possible therapeutic intervention
- inability to detect flat lesions or changes in color of the
mucosa 21

SUMMARY
• Photodynamic diagnosis and NBI both focus on improvement of the
visualization of bladder tumors, OCT focuses on real-time
pathological diagnosis.
• NBI and photodynamic diagnosis both can aid in reducing the high
rate of early recurrences, because more radical resection can be
performed due to better visualization of UC.
• In addition, in case of positive cytology and negative white light
cystoscopy, photodynamic diagnosis and NBI can improve detection
of CIS 22

• OCT may possibly assist in differentiation between inflammation and
CIS, which both can present as red lesions.
• It may also aid in accomplishing a more complete resection, by
measuring the resection margins with OCT and extending resection if
vital tumor is still present.
• In addition, optical coherence tomography may provide pathological
diagnosis for the patients with recurrent tumors under follow-up…
23

24

INVESTIGATIONS
❖CYTOLOGY
• high specificity (94-100%) but has low sensitivity (31-62%) especially in
low-grade, low-stage tumors.
• Advantage of being cheap, office based but is highly dependent on the
expertise of the cyto-pathologist
• Malignant urothelial cells can be observed on microscopic
examination of the urinary sediment or bladder washings
25

• Characteristically, tumor cells have large nuclei with irregular, coarsely
textured chromatin
• The limitations of microscopic cytology are due to the cytologically normal
appearance of cells from well-differentiated tumors and because well-
differentiated cancer cells are more cohesive, they are not readily shed into
the urine.
• Therefore, microscopic cytology is more sensitive in patients with high-grade
tumors or carcinoma in situ
• Even in patients with high-grade tumors, however, urinary cytology may be
falsely negative in 20%
26

image
27

FLOURESCENT CYTOLOGY
• by performing a bladder lavage on patients who had 5-aminolevulinic
acid (ALA) instilled into the bladder which improves sensitivity.
• A bladder lavage specimen was centrifuged and then promptly
examined using blue-violet light (390 to 430 nm wavelength) to induce
fluorescence.
• The red fluorescence of the malignant epithelial cells improved the
sensitivity over white light standard cytology (with counterstaining
and fixation) from 79% to 86%
• This gain was exclusively for well-differentiated cancers, a lesion that
is routinely overlooked by standard cytology.
28

• …Drawbacks
- very rapid fading of fluorescence from malignant cells…1
- loss of nuclear detail …2
• Thus, the same slide could not be examined for classic cytological
nuclear abnormalities, and the standard cytology specimen had to be
prepared separately.
29

FLOW CYTOMETRY
• Flow cytometry measures the DNA content of cells whose nuclei have
been stained with a DNA-binding fluorescent dye.
• Quantitate the aneuploid cell populations and the proliferative activity
(percentage of S phase cells) in a tumor
• DNA diploid tumors tend to be of low grade and low stage > favorable
prognosis
• Triploid to tetraploid chromosome number have unfavorable
pathologic characteristics > patients have a poor prognosis.
30

• Low-grade superficial tumors, which are usually diploid, often
produce false-negative results.
• Aneuploidy is a common feature of high-grade tumors, and thus flow
cytometry is especially accurate in patients with carcinoma in situ or
high-grade malignancies.
• Flow cytometry has not supplanted conventional cytology in
managing bladder cancer patients.
31

IMAGE ANALYSIS
• Quantitative fluorescent image analysis is an automated cytologic technique
that analyzes smears of cells on a microscope slide and quantitatively
measures DNA content in each cell
• It combines quantitative biochemical analysis and more subjective visual
evaluation of individual cells (cytometry can analyze only a population of
cells).
• This technique uses a computer-controlled fluorescent microscope that
automatically scans and images the nucleus of each cell on a slide
32

• The computer quantitates the amount of emitted fluorescence, which
is directly proportional to the nucleic acid content, and identifies each
cell that contains an abnormal amount of DNA.
• Thus, a cytotechnologist can focus on each abnormal cell identified
by an automatic analyzer for morphologic evaluation.
• Because individual cells can be examined by image analysis, this
technique can more easily use voided urine specimens than can flow
cytometry, which requires large cell populations for its analysis
33

• multiparameter image analysis can also be performed. Labeled
monoclonal antibodies to various tumor markers used in conjunction
with DNA fluorescence can increase the specificity of image analysis
for bladder cancer detection and monitoring of treatment response
• This technique is more sensitive than either standard cytology or flow
cytometry for detecting low-grade bladder tumors without reduced
specificity
• image analysis can be performed using fluorescently labeled DNA
probes to specific chromosomes of interest in conjunction with in situ
hybridization, that demonstrate tumors with trisomy of the
centromeric region of chromosome 7, loss of various regions (or all)
of chromosome 9 or deletions of 17p.
34

URINARY BIOMARKERS
developed as an adjunct to standard diagnostic modalities for bladder
cancer diagnosis and monitoring
• Noninvasive testing with improved sensitivity over urine cytology has
been proposed as a desirable alternative to cystoscopy which costly
and uncomfortable
• Many urine-based markers are being used in clinical practice, but
none of them have shown better specificity than urine cytology, but
have shown definitely a better sensitivity than urine cytology.
35

• Commercially available biomarkers:-
- NMP22
- BTA stat
- immunocyt/uCyt+
- uroVysion
- CxBladder
36

• An ideal tumor marker or a test in detecting bladder cancer should be
a one that is non-invasive, has highest specificity and sensitivity, easy
to perform, rapid, reproducible, office based and cost-effective and
should detect bladder cancer even before it becomes visible on
cystoscopy.
• UroVysion and telomerase have shown a great promise in sensitivity
and specificity.
37

38

NMP 22
• Important role in structural framework of nucleus.
• Involved in mitosis by enabling correct distribution of chromatin to daughter
cell.
• 20 times more prevalent in malignant urothelial cells compared with normal
cells
• 2 assay- 1) NMP22 BC test kit-ELISA test
2) NMP22 bladder check test – immunochromatin assay i,e
point of care ( POC) test.
39

• Quantitative test – Sensitivity: 0.68, specificity: 0.77
• Qualitative test – Sensitivity: 0.58, specificity: 0.88
failed to reach the level of specificity due to higher rate of FP d/t
infection, inflammation, hematuria, urolithiasis, instrumentation.
40

uroVysion
• FISH assay.
• Identifies fluorescently labelled DNA probes that bind to intranuclear
chromosomes
• Detects aneuploidy of chromosomes 3,7,17 and heterozygous loss of
9P21 locus in exfoliated urothelial cells
41

• Positive test
✓5 or more urinary cells with gains of 2 or more chromosomes
✓10 or more cells with gain of single chromosome
✓Heterozygous deletion of 9P21 in > 20 exfoliated cells.
• Considered as reflex test in the setting of atypical cytology >> a
negative FISH correlates with benign cytological changes.
42

• Sensitivity : 0.63
• Specificity: 0.87
• Better sensitivity in low grade tumors.
• It Constitute an accurate surveillance assay by anticipating disease
recurrence.
• Useful in monitoring patients treated with intravesical BCG.
43

BLADDER TUMOR ANTIGEN
• Detects : Human complement factor H- related protein AND complement factor H
using MA.
• FDAAPPROVED – for diagnosis and follow-up of BC
• 2 assay-1) BTA TRAK- ELISA test. SN-0.65, SP-0.74
2) BTA STAT- immunochromatin POC test. SN- 0.64%, SP-0.77
• Highly sensitive test but specificity remained lower than cytology.
• False positive due to hematuria, BPH, h/o BCG instillation, inflammation,
infection, urolithiasis, bowel interposition…
44

ImmunoCyt/uCyt+
• Combination of cytology and immunofloroscence.
• Detects exfoliated BC cells in urine by using 3 fluorescent monoclonal antibodies
targeting 3 specific antigen of BC cell.
• M344- high molecular weight CEA
• LDQ10 and 19A11 are bladder tumor cell associated mucin.
• SN- 0.78 & SP-0.78
• As a cell based assay , it is less impacted by hematuria and inflammatory
condition.
• Less frequent use in clinical care due to user dependency, interobserver variability
and limited evidence.
45

CxBladder
• Real-time reverse transcription polymerase chain reaction( RT-qPCR).
• Based on detection of 5 mRNAs with BC ( IGFBP5, HOXA13, MDK, CDK1)
and ( CXCR2) in non malignant inflammatory condition.
• Limited studies. SN-85%, SP-85%
• Able to distinguish between low grade Ta tumors and other detected urothelial
carcinoma.
46

47

INVESTIGATIONAL BIOMARKERS
• Protein based and cell based biomarkers-
- apoptosis markers
- angiogenesis markers
- proliferation and invasion
- metabolomics
• Gene based biomarkers-
- Aurora A kinase (AURKA)
- FGFR3
- microsatellite loss of heterozygosity detection
- DNA methylation
- micro RNA
48

APOPTOSIS MARKERS
• Soluble Fas ( sFas)
➢isoforms are antiapoptotic proteins produced and released by BC cells.
➢Measurable by ELISA.
➢Independent predictor of BC recurrence
• Clusterin
➢multifunctional secretory glycoprotein .
➢Measuable by ELISA.
• Survivin
➢Antiapoptotic protein exclusively expressed by malignant epithelium.
➢Dot-blot technique ( bio-Dot assay).
49

Angiogenesis markers-
VEGF:- measured by ELISA in voided urine.
Proliferation and invasion-
Telomerase
Hyaluronic acid
Fibronectin
CD44 antigen
50

51

52

53

Key points for urinary biomarkers
• Many urinary biomarkers are able to assess BC.
• The Food and Drug Administration in the USA has approved Bladder Check
(NMP22) and UroVysion for use in screening of bladder cancer
• Combination of biomarkers seems to increase their performance.
• Their clinical relevance is not obvious enough to enable their wide spread use.
• Most of them have not reached quality criteria established by guidelines.
54

CURRENT PRACTICE
• MICROHEMATURIA
- Addition of any current urine biomarkers does not preclude a
cystoscopy and does not change management
• GROSS HEMATURIA
- Negative test also not preclude cystoscopy…1
• Currently used in combination with cystoscopy as a surveillance
strategy for pt with H/O NMIBC…
55

• There are certain situations where the marker is positive but
no tumor is found on white light cystoscopy.
• This phenomenon has been observed in UroVysion FISH test
more predominantly and to moderate extent in cytology and to
lesser extent in BTA test and NMP22
• a positive UroVysion FISH test even in the absence of
confirmatory cystoscopical or cytological findings could
predict disease recurrence in 35-63% of patients within the
next 6-10 months.
• If blue light cystoscopy was used in these situations of
positive marker, ONE CAN DETECT A CANCER
• this phenomenon of anticipatory positive test should be
taken as an advantage, both in screening and surveillance.
56

STAGING
57

Definition of TNM Staging
Primary tumor (T)
Tx - Primary tumor cannot be assessed
To - No evidence of primary tumor
Ta - Non invasive papillary carcinoma
Tis - Carcinoma in situ
T1 - Tumor invades subepithelial
connective tissue
T2 - Tumor invades muscularis propria
T2a - Tumor invades superficial
muscularis propria
T2b - Tumor invades deep muscularis
propria 58

T3 - Tumor invades peri vesical tissue
T3a - Microscopically
T3b - Macroscopically
( extravesical mass)
T4 - Tumor invades any of the following
( prostatic stroma, seminal vesicles,
uterus, vagina, pelvic & abd wall)
T4a - Tumour invades prostatic stroma,
uterus, vagina
T4b – Tumour invades pelvic wall,
abdominal wall
59

Regional Lymph Nodes(N)
• Nx - Lymph nodes cannot be assessed
• N0 - No lymph node metastasis
• N1 - Single regional lymph metastasis in
true pelvis ( hypogastric, obturator,
external iliac or presacral LN)
• N2 – Multiple regional lymph metastasis
in true pelvis ( hypogastric, obturator,
external iliac or presacral LN)
• N3 - Lymph node metastasis to
common iliac lymph nodes 60

Distant Metastasis ( M)
• M0 – No distant metastasis
• M1 - Distant metastasis
• M1a- distant set of LN
• M1b- spread to 1 or more distant organ (bone, liver, lung)
61

STAGE STAGE GROUPING
0a Ta, N0, M0
0is Tis, N0, M0
1 T1, N0, M0
2 T2 a/b, N0, M0
3A T3a/b/ T4a, N0, M0
T1-4a, N1, M0
3B T1-T4a N2
N3 M0
4A T4b N0 M0
Any T , Any , M1a
4B Any T, Any N, M1b
62

• Broadly speaking NMIBC comprises stage 0 and stage 1
• MI organ confined bladder cancer - stage 2
• MI locally advanced bladder cancer - stage 3
• Metastatic disease - stage 4
• NMIBC SUBDIVISION
• Low risk- primary ( not recurrent ), low grade pappilary (Ta), solitary
tumours < 3 cm
• Intermediate risk – multiple/ recurrent large > 3 cm low grade Ta
• High risk – any high grade histologic features ( i.e. CIS or T1)
63

PROGNOSIS
• Genetic instability - hallmark of invasive urothelial cancer, but
specifically alterations of TP53, RB, and PTEN carry a very poor
prognosis
• Most significant prognostic factors –
Grade , Depth of invasion ( stage) & Presence of CIS
• Angio lymphatic invasion - poor prognostic sign
• Proliferation markers, such as MIB-1 and PCNA
found in high-grade tumors , associated with worse prognosis
64

• Non muscle invasive - good prognosis with
5 yr survival rate of 82 – 100%
• 5-year survival rate decreases with increasing stage, as follows:
Ta, T1, CIS – 82-100%
T2 – 63-83%
T3a – 67-71%
T3b – 17-57%
T4 – 0-22%
• Metastatic urothelial Cancer – poor prognosis with
5-10% survival 65

THANK YOU
66

Bladder carcinoma- urinary biomarkers diagnosis and staging

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