Urinary markers in Urothelial carcinoma(Bladder)

1. UROTHELIAL
CARCINOMA: URINARY
BIO-MARKERS
Gaurav Nahar
DNB Urology (Std.)
MMHRC, Madurai

2. INTRODUCTION
 Urothelial carcinoma: MC malignancy of urinary tract
and 2nd MC cause of death among genitourinary
tumors.
 Gross, painless hematuria- primary symptom in 85%
of patients with a newly diagnosed bladder tumor, and
microscopic hematuria occurs in virtually all patients.

3. DIAGNOSIS
 Full hematuria evaluation for bladder cancer includes
cystoscopy, urine cytology, upper-tract imaging (CT
scan of abdomen & pelvis), and a prostate-specific
antigen (PSA) blood test.
 A PSA blood test is recommended, because 10% of
patients with recurrent gross hematuria will have
prostate cancer.
 AUA guidelines for microscopic hematuria evaluation
include a cystoscopy, upper tract imaging, and urine
cytology.

4. DIAGNOSIS
 Microscopic hematuria requires a full evaluation, but
low-risk patients do not require repeat evaluations.
 High-risk individuals primarily are those with a
smoking history and should be evaluated every 6
months.

5. DIAGNOSIS
 Main diagnostic test for bladder cancer is cystoscopy
and biopsy.
 White Light Cystoscopy(rigid/flexible)- Gold
standard.
 Blue light Cystoscopy- using porphyrin(5-hexa ALA)
dye.
 Narrow Band Imaging(NBI).
 Random bladder biopsies.

6.  Urine cytology.
 Urine markers.

7. URINE CYTOLOGY
 Voided urine cytology is the standard noninvasive
method for diagnosis in the detection of bladder
carcinoma.
 Microscopic examination of voided urine or bladder-
washing specimens(barbotage) for exfoliated cancer
cells.
 Cytology is used to assess morphologic changes in
intact cells- cellular clumping, a high nuclear-to-
cytoplasmic ratio, nucleoli, and atypia.

8. URINE CYTOLOGY
 At least 100 mL of a freshly voided specimen required.
 First morning sample should not be used because of
frequent presence of cytolysis.
 Bladder wash cytology yields more tumor cells in the
sample and is more sensitive in identifying cancer,
especially for high-grade tumors, but it also yields a
higher false-positive rate than voided urine
cytology.

9. URINE CYTOLOGY
 High sensitivity in high-grade tumours but low
sensitivity in low-grade tumours.
 Useful for detection of CIS, where its sensitivity is 28-
100%.
 Useful as an adjunct to cystoscopy, when a high-grade
malignancy or CIS is present.
 Positive voided urinary cytology can indicate an
urothelial tumour anywhere in the urinary tract, from
the calyx to the ureters, bladder, and proximal urethra.
 Negative cytology does not exclude the presence of a
tumour in the urinary tract.

10. URINE CYTOLOGY
 Evaluation can be hampered by low cellular yield,
UTIs, stones, or intravesical instillations, but in
experienced hands, specificity exceeds 90%.
 Positive cytology + negative cystoscopy, it is necessary
to exclude a tumour in the upper tract (CT-urography),
CIS in the bladder (random biopsies or photodynamic
diagnosis (PDD) targeted biopsies) and tumour in
prostatic urethra (prostatic urethral biopsy).

11. URINARY MOLECULAR
MARKERS
Characteristics of a good urinary bladder cancer
marker:
 Technically simple(a point-of-care test, with readily
available results, easy to perform);
 Low cost;
 Good reliability and reproducibility;
 high specificity( to avoid unnecessary work-up
because of false-positive results) & high sensitivity (to
avoid the risk of missing a tumour);
 Ability to detect high-risk urothelial cancer before it
escapes curative treatment.

12. POTENTIAL APPLICATIONS
1. Screening of high-risk population for bladder cancer;
not recommended routinely.
2. Exploration of patients after haematuria or other
symptoms suggestive of BC (primary detection).
3. Surveillance of NMIBC(non muscle-invasive bladder
cancer).

13.  More than 30 urinary biomarkers reported for use in
bladder cancer diagnosis.
 Only a few are commercially available; remainder are
still being tested.
 Sensitivity higher and specificity lower compared to
urine cytology.
 Benign conditions and BCG influence many urinary
marker tests.

14. URINE BIOMARKERS
 Bladder tumor antigen (BTA)
assays- BTA stat & BTA TRAK
 Nuclear matrix protein-22
 NMP 52
 BLCA-4 and BLCA-1
 UroVysion (FISH)
 Cytokeratins(CK 20 & CYFRA 21-
1)
 HA and HAase
 Telomerase
 Survivin
 Quanticyt nuclear karyometry
 Fibrin degradation products
 Microsatellite analysis
 Immunocytology
(ImmunoCyt/uCyt+)
 CertNDx/FGFR3
 CxBladder
 DD23 monoclonal antibody
 Lewis X antigen
 Automated image cytometry
 Aurora kinase A
 Carcinoembryonic antigen-
related cell adhesion molecule
(CEACAM1)

16. NUCLEAR MATRIX PROTEIN-22
(NMP-22)

17. NMP-22
 a nonchromatin nuclear matrix protein used to form
cell nuclei; supports nuclear shape and organizes
DNA.
 also takes part in DNA replication, transcription, &
RNA processing.
 released from the nuclei of tumor cells after they die
and can be detected in the urine.

18. NMP-22
 NMP-22 is shed into urine and has a 20-times higher
concentration in urine of bladder cancer patients than
in noncancer controls.
 A cut-off of 10 units/mL is used to identify patients
with or without cancer.
 A lower cutoff level of 5 units/mL improves sensitivity
but significantly worsens specificity.
 Cutoff level not related to stage or grade of disease.
 False positives: active UTI or significant hematuria.

19.  Using a cutoff level of 10 units/mL, overall sensitivity
and specificity- 49% and 87%, respectively.
 The sensitivity for Ta, T1, and T2 tumors was 36%,
65%, and 88%, respectively.

20.  NMP-22 BladderChek test (Alere; Waltham, Mass) is
an in vitro immunoassay for qualitative detection of
NMP-22 in urine.
 Does not depend on intact cells and does not require
expert analysis or laboratory time.
 Provides an absolute positive or negative test result,
similar to a pregnancy test.
 Painless and noninvasive assay.
 Provides results within 30 minutes (thus allowing
performance during an office visit).
 Cost is less than half that of cytology.

21.  When combined with cystoscopy, NMP-22 test
improves the detection of recurrence in patients with a
history of bladder cancer.
 Combination of the NMP-22 test with cystoscopy
increases overall sensitivity to 99%(from initial 91%
with cystoscopy alone)
 Significantly more sensitive than urine cytology.

22. BLADDER TUMOR ANTIGEN(BTA)
-STAT & TRAK

23. BLADDER TUMOR
ANTIGEN(BTA)-STAT & TRAK
 Uses monoclonal antibodies to detect complement
factor H-related protein and complement factor H in
voided urine specimens.
 These factors are found in bladder cancer cell lines and
inhibit the complement cascade to prevent cell lysis.

24. BLADDER TUMOR
ANTIGEN(BTA)

25. BLADDER TUMOR
ANTIGEN(BTA)
 BTA STAT is a point of care qualitative assay with an
average sensitivity and specificity of 68.7% (53-89%)
and 73.7% (54-93%), respectively.
 BTA TRAK is a quantitative enzyme-linked
immunosorbent assay with similar sensitivity and
specificity of 62% (17-78%) and 73.6% (51-95%),
respectively.

26.  Specificity of both tests can be significantly decreased,
as false positives have been noted to occur in the
setting of hematuria, urolithiasis, inflammation,
recent instrumentation, other genitourinary
malignancies, and intravesical BCG therapy.

27. IMMUNOCYTOLOGY
(Immunocyt/uCyt+)
 An immunohistochemical test with 3 fluorescent
monoclonal antibodies directed at mucin-based urothelial
cell antigens found on exfoliated cells.
 Two antibodies, LDQ10 and M344, are directed against
mucins, specifically glycoproteins found on epithelial cell
surfaces in malignancy, and labeled with fluorescein.
 The other antibody is labeled Texas red and directed
against a high molecular weight glycosylated form of
carcinoembryonic antigen CEA 19A211.
 M344 and CEA 19A211 expressed in 71% and 90% of Ta-T1
tumors, respectively.

28. Immunocyt/uCyt+
 Use of Immunocyt/uCyt+ improves sensitivity at a
minimum of 15% over cytology alone.
 Improved sensitivity in low-grade tumors; improves
further with combination of cytology and
Immunocyt/uCyt+; Specificity is slightly lower than
that of cytology.

29. Positive

30. Immunocyt/uCyt+
LIMITATIONS:
 Requires processing in laboratories with properly
trained personnel.
 Requires a minimum of 500 negative cells on the slide
in order for the sample to be deemed negative.
 As is common with other protein-based assays, false
positives are common in UTIs, urolithiasis & BPH.

31. FGFR3/CertNDx

32. FGFR3/CertNDx
 Fibroblast growth factor receptor 3 (FGFR3) belongs to
a family of tyrosine kinase receptors; encoded by the
FGFR3 gene.
 Specific point mutations in various domains result in
constitutive activation of the receptor; found in
approximately 50% of urothelial carcinomas.
 Frequency of these mutations is high in low-grade
pTa tumors and low in pT1-4 tumors.
 Presence of the FGFR3 mutation is a selective marker
for favorable disease, with a low recurrence rate and
improvement in disease-specific survival.

33. FGFR3/CertNDx
 CertNDx- a multianalyte diagnostic assay used for the
evaluation of hematuria and monitoring bladder
cancer recurrence.
 Analyze urine for presence of mutant FGFR3,
quantified matrix metalloproteinase 2 (MMP-2), and
hypermethylation of TWIST1 and NID2. This allows
for the presence of 2 biomarker cutoff values.
 Unlike other assays, this is not affected by the degree
of hematuria or presence of other urinary tract
diseases.

34. CxBladder

35. CxBladder
 CxBladder (PacificEdge, Dunedin, New Zealand) is a
recently released urine-based assay consisting of 5
mRNA markers, CDC2, HOXA13, MDK, IGFBP5, and
CXCR5.
 sensitivity superior to that of NMP-22 and cytology, at
83%, with a specificity of 85%. Interestingly, specificity
for high-grade tumors was 97% while the specificity
for low-grade tumors was 69%. This offers a potential
adjunct to cystoscopy for the diagnosis of urothelial
carcinoma.

36. LEWIS BLOOD GROUP X
ANTIGEN
 Lewis blood group antigen X is usually absent from
urothelial cells in adults except for occasional umbrella
cells.
 There is increased Lewis X expression in bladder
cancers, and it is independent of secretor status,
grade, and stage.
 The sensitivity and specificity for the detection of
bladder cancer is 75% and 85%, respectively.
 There is no commercially available test to date.

37. CYTOKERATINS (CK 20 &
CYFRA 21-1)
 CK 20 and CYFRA 21.1 are
fragments of cytoskeletal
proteins; can be detected
in urine of bladder cancer
patients by either protein
or mRNA detection.
 CK 20 has a sensitivity and
specificity of 85% and 76%,
respectively.

38.  CYFRA 21.1, with a cutoff value of 4 ng/mL, found a
sensitivity and specificity of 43% and 68%,
respectively.
 Unfortunately, none of the Ta tumors were identified
at the 4 ng/mL cutoff. Decreasing the CYFRA 21.1
cutoff to 1.5 ng/mL increased Ta detection to 33%, but
the specificity dropped to an unacceptable 43%.
 Therefore it is not a useful marker in the current form,
or at least for low-grade disease.

39. FISH (UroVysion)

40. FISH (UroVysion)
 Fluorescence in-situ hybridization (FISH) identifies
fluorescently labeled DNA probes (for the centromeres
of chromosomes 3, 7, and 17 and a locus-specific probe
for 9p21) that bind to intranuclear chromosomes.
 The current commercially available probes evaluate
aneuploidy for chromosomes 3, 7, and 17 and
homozygous loss of 9p21.
 The median sensitivity and specificity of FISH analysis
is 79% and 70%, respectively.

41. FISH (UroVysion)
 FISH analysis is moderately useful for high-grade
disease and may be anticipatory of new tumor
formation.
 Approved by the US Food and Drug Administration
(FDA) in 2005 as an aid for initial diagnosis of bladder
cancer in patients with hematuria and subsequent
monitoring for tumor recurrence in patients previously
diagnosed with bladder cancer.

42. FISH (UroVysion)
 FISH is considerably more sensitive and only slightly
less specific than cytology in diagnosing urothelial
carcinoma.
 A useful initial diagnostic tool in patients suspected of
both new and recurrent bladder cancer.

43. MICROSATELLITE ANALYSIS

44. MICROSATELLITE ANALYSIS
 short DNA repeats present throughout the chromosomes;
lost in some tumor cells- multiple markers available to
identify these.
 Microsatellite analysis amplifies these repeats in the
genome that are highly polymorphic, and PCR
amplification can detect tumor-associated loss of
heterozygosity by comparing peak ratio of two alleles in
tumor DNA in urine sample with the presence of alleles in
a blood sample from the same individual.
 The sensitivity and specificity range from 72% to 97% and
80% to 100%, respectively.
 Microsatellite analysis is the most promising marker of
all.

45.  if the microsatellite analysis is persistently positive,
there was an 83% 2-year recurrence rate, but if the
analysis was persistently negative, only 22% of
patients had recurrent tumors.

46. DNA METHYLATION
ANALYSIS
 CpG dinucleotide islands cluster around promoters in an
unmethylated state to allow gene expression .
 Methylation of CpG islands shuts down the promoter, and
if the promoter in question is part of a tumor suppressor
gene then cancer can form.
 Examples of promoter methylation of CpG islands causing
epigenetic changes in urothelial cancer include the
P16/CDKN2A gene.
 Sensitivity of gene methylation - 75%.
 Methylated CpG islands can be found in normal urothelial
cells of older patients.

48. SURVIVIN
 Survivin is an antiapoptotic protein that has a high
expression in urothelial cancer.
 Survivin is found in 10% to 30% of bladder cancers and is
readily shed into urine.
 Sensitivity and specificity in the detection of urothelial
tumors is 64% to 100% and 87% to 93%, respectively.
 May be useful in predicting which patients will respond to
intravesical therapy.
 Survivin was relatively poor at detecting advanced-stage or
high-grade tumors, with a sensitivity of 71% for stage T2
tumors and 80% for high-grade cancers.

49. HYLAURONIC ACID
 Hylauronic acid controls intercellular communications
and cell replication.
 Urothelial cancer induces hylauronic acid production
from fibroblasts, and the amount correlates with the
stage of the disease.
 Sensitivity and specificity is 91% to 100% and 84% to
90%, respectively.
 Sensitivity and specificity for discriminating between
low-grade and high-grade lesions is unclear.

50. TELOMERASE
 Telomerase resides at the terminal ends of
chromosomes and duplicates random DNA repeats to
prevent cell death.
 Telomerase activity is measured in telomeric repeat
application protocol (TRAP) and is detected in 80% of
urine from patients with bladder cancer with no grade
differential.
 The sensitivity and specificity is 90% and 88%,
respectively.

52. BCLA-4
 BCLA-4 is a nuclear transcription factor present in
bladder tumors and adjacent benign areas of the
bladder, but not in benign urothelium.
 sensitivity of 89%-96% with a specificity of 100% for
bladder cancer.
 BLCA-4 has a potential role in tumor development or
progression.
 high expression level of BLCA-4 identifies patients at
high risk.

53. ROLE OF URINARY
MARKERS
 No urinary biomarker is sensitive and specific enough
to replace cystoscopy in the primary detection or
follow-up of bladder cancer.
 Adjunctive role of a non-invasive urine test performed
before follow-up cystoscopy.
 Combination of cystoscopy with urine markers, in
select situations, is appropriate for surveillance of
patients with non–muscle-invasive bladder cancer.

54. Thank
you…