Biomarkers in the

management of
non-muscle
invasive bladder
cancer (NMIBC)
Peer reviewed by
Jørgen Bjerggaard Jensen
Aarhus University Hospital, Aarhus, Denmark
Joost Boormans
Erasmus MC Cancer Institute, Rotterdam, the Netherlands
Hugh Mostafid
Royal Surrey County Hospital, Guildford, UK
Overview

Hurdles in the surveillance of

NMIBC
The role of biomarker tests in
decision making
The role of biomarker tests in
NMIBC
Hurdles in the surveillance of NMIBC
 Surveillance is key in NMIBC

Haematuria

Cystoscopy +/- Cytology

NMIBC
Up to 70% of patients experience + risk stratification
recurrence within 2 years of primary
treatment
Disease
management
Up to 10-15% eventually
progress to MIBC

Recurrence Progression MIBC

Stages and grades of NMIBC1,2

* ** **

* Low grade papillary tumour:

overall orderly appearance but with easily recognisable
variation of architectural and/or cytologic features

** High grade papillary tumour: predominantly

or totally disorderly appearance at low magnification
with both architectural and cytologic abnormalities

1. Babjuk M et al. EAU 2020 Guidelines on non-muscle-invasive Bladder Cancer (TaT1 and CIS).
2. Moch H et al. WHO Classification of Tumours of the Urinary System and Male Genital Organs (4 th edition) 2016, Lyon, France.
NMIBC risk stratification1

NMIBC

Low risk tumour Intermediate risk tumour High risk tumour

Primary, solitary, TaG1 All tumours between the category Any of the following:
(PUNLMP, LG), < 3 cm, of low- and high risk
• T1 tumour
no CIS
• HG tumour
• Carcinoma in situ
• Multiple, recurrent and
large (> 3 cm) TaLG tumours

1. Babjuk M et al. EAU 2020 Guidelines on non-muscle-invasive Bladder Cancer (TaT1 and CIS).
NMIBC recurrence and progression1

Factor Recurrence Progression Recurrence score Probability of recurrence at Probability of recurrence at

1 year, % (95% CI) 5 years, % (95% CI)
2-7 tumours 3 3
0 15 (10-19) 31 (24-37)
≥8 tumours 6 3
1-4 24 (21-26) 46 (42-49)
≥3 cm diameter 3 3
5-9 38 (35-41) 62 (58-65)
≤1 recurrence/year 2 2
10-17 61 (55-67) 78 (73-84)
>1 recurrence/year 4 2
Ta 0 0
T1 1 4
Concurrent CIS 1 6 Progression score Probability of progression at Probability of progression at
G1 0 0 1 year, % (95% CI) 5 years, % (95% CI)
G2 1 0 0 0.2 (0-0.7) 0.8 (0-1.7)
G3 2 5 2-6 1 (0.4-1.6) 6 (5-8)
Total score 0-17 0-23 7-13 5 (4-7) 17 (14-20)
14-23 17 (10-24) 45 (35-55)

1. Babjuk M et al. EAU 2020 Guidelines on non-muscle-invasive Bladder Cancer (TaT1 and CIS).
Cystoscopy and cytology:
Current guidelines for diagnosis

EAU 2020 guidelines1

Recommendations for diagnosis Strength rating
Perform cystoscopy in patients with symptoms suggestive of bladder cancer or during Strong
surveillance. It cannot be replaced by cytology or by any other non-invasive test.

Use voided urine cytology as an adjunct to cystoscopy to detect high-grade tumour. Strong

1. Babjuk M et al. EAU 2020 Guidelines on non-muscle-invasive Bladder Cancer (TaT1 and CIS).
 Cystoscopy and cytology:
Current guidelines for diagnosis (I)
Cystoscopy (usually white light)
• High sensitivity for papillary tumours: 86%1 • Lower sensitivity for carcinoma in situ (CIS):
• Moderate to high specificity: 43-98%2 55%1
High number of false positives 45% of patients with CIS are missed
• Operator-dependent assessment  risk of
bias

100
90 86% 86%
80 76%
70
55%
Sensitivity

60
50
40
30
20
10
0
Overall Low grade High grade CIS

1. Daneshmand S et al. Urol Oncol 2018; 36:361.e1-361. 2. Jocham D et al. Eur Urol 2008; 53:1138-1150.
 Cystoscopy and cytology:
Current guidelines for diagnosis (II)
Cytology
• High specificity: 93%1 • Moderate sensitivity in high-grade tumours: 54%1
 Low number of false positives  46% of high-grade tumours are not detected
• Low sensitivity in low-grade (G1/G2) tumours: 11%1
 89% of low-grade tumours are not detected
100
90 • Operator-dependent assessment with variable
80 results across institutions2
70
58%
54% • High rate of atypical (inconclusive) results3
Sensitivity

60
50 41%
40
30
20 11%
10
0
Overall Low grade High grade CIS

1. Freifeld Y et al. BJU Int 2019; 124:251-257. 2. Raitanen M-K et al. Eur Urol 2002; 41:284-289. 3. VandenBussche CJ et al. Cancer Cytopathol 2018; 126:381-389.
 Cystoscopy: Current guidelines for surveillance
EAU 2020 guidelines1
Recommendations for follow-up Strength rating
Base follow-up of TaT1 tumours and carcinoma in situ (CIS) on regular cystoscopy. Strong
Patients with low-risk Ta tumours should undergo cystoscopy at three months. If negative, Weak
subsequent cystoscopy is advised nine months later, and then yearly for five years.
Patients with high-risk tumours should undergo cystoscopy and urinary cytology at three Weak
months. If negative, subsequent cystoscopy and cytology should be repeated every three
months for a period of two years, and every six months thereafter until five years, and then
yearly.
Patients with intermediate-risk Ta tumours should have an in-between (individualised) Weak
follow-up scheme using cystoscopy.

1. Babjuk M et al. EAU 2020 Guidelines on non-muscle-invasive Bladder Cancer (TaT1 and CIS).
 The cystoscopy surveillance schedule:
EAU guidelines in practice
Diagnosis: cystoscopy

Low risk Ta tumour Intermediate risk Ta tumour High risk tumour

Follow-up Follow-up Follow-up
3 months 1 Individualised scheme 3 months 1
9 months later 1 Total # of cystoscopies 8 - >20 Every 3 months for 2 years 8
Yearly for 5 years 5 Every 6 months for 5 years 10
Total # of cystoscopies 8 Yearly ?
Total # of cystoscopies >20

The number of cytoscopies a patient has to undergo in case no recurrence happens. In case of recurrence, the follow-up start from the beginning again.
Hurdles in NMIBC management
• Moderate specificity and moderate sensitivity in CIS
Limitations of • Operator dependent, decreased sensitivity under bacillus Calmette-
cystoscopy Guerin (BCG) treatment
• Invasive procedure and reduced compliance over time
• Logistic burden: commute to and time in hospital
• Morbidity

• Moderate sensitivity in high-grade tumours

• Low sensitivity in low-grade tumours
Limitations of cytology • High rate of atypical results (10-15%)
• Risk of missing high-grade recurrence in certain populations (such
as BCG treated patients)

• Repeat cystoscopy/cytology in follow-up

• Resource burden: physicians’ and nurses’ time
Economic cost
• Long waiting times for cystoscopy appointment
• 60% of cost is attributed to surveillance and recurrence
The role of biomarker tests in decision
making
Biomarkers: What do the numbers mean?

“Proven clinical
“100%
“92% sensitivity and
sensitivity,
sensitivity, specificity for
independently
excluding low and high
of tumour stage
Ta-LG” grade bladder
and grade”
cancer”

“Clinical
studies
demonstrate a
combined 95% “A
sensitivity” breakthrough
NPV of 97%”
 Biomarkers: What do the numbers mean?

“Proven clinical
“100%
“92% sensitivity and
sensitivity,
sensitivity, specificity for
independetly of
excluding Ta- low and high
Sensitivity, specificity, negative predictive value (NPV) and positive
LG” grade bladder
tumour stage
and grade”
cancer”
predictive value (PPV) can guide the selection of the most appopriate
“Clinical biomarker test
studies
demonstrate a
combined 95% “A
sensitivity” breakthrough
NPV of 97%”
 Quality parameters for diagnostic tests (I)
TP
Sensitivity = =
TP + FN +
= the proportion of diseased persons who
True negative (TN)
receive a positive test result
False positive (FP)
 The lower the sensitivity, the more diseased patients
receive an incorrect negative test result (high false
False negative (FN) negative rate)

True positive (TP)

TN
Specificity = =
TN + FP +

= the proportion of healthy persons who

receive a negative test result

 The lower the specificity, the more unaffected patients

receive an incorrect positive test result (high false positive
rate)
 Quality parameters for diagnostic tests (II)
TN
NPV = =
TN + FN +

True negative (TN) = the proportion of negative test results that is

correct
False positive (FP)
 The lower the NPV, the higher the risk that your patient with
a negative test result is a missed diseased patient
False negative (FN)

True positive (TP)

TP
PPV = =
TP + FP +

= the proportion of positive test results that is

correct

 The lower the PPV, the more tests incorrectly labeled

unaffected patients as affected
 Quality parameters for diagnostic tests (III)
TP
Sensitivity = = The probability of a diseased
TP + FN
person having a positive test result Characteristics of a diagnostic test

Not affected by the prevalence of the

TN
Specificity = = The probability of a healthy disease in the test population
TN + FP
person having a negative test result

TN Impacted by the prevalence of the disease in the

NPV = = The probability that a person test population and by sensitivity:
TN + FN
with a negative test truly doesn't
have the disease Sensitivity Prevalence NPV

TP = The probability that a person 95% 5% 100%

PPV =
TP + FP with a positive test truly has the 0% 5% 94%
disease
95% 30% 98%
Example: changing prevalence
Scenario 1: Scenario 2:
Disease prevalence = 6% Disease prevalence = 30%
The same diagnostic test is used in both scenarios
Truth Truth
Diseased Healthy Diseased Healthy
Positive 4 (TP) 44 (FP) Positive 20 (TP) 33 (FP)
Test result

Test result
Negative 2 (FN) 50 (TN) Negative 10 (FN) 37 (TN)
TP 4 TP 20
Sensitivity = = = 67% Sensitivity = = = 67%
TP + FN 4+2 TP + FN 20 + 10
TN 50 TN 37
Specificity = = = 53% Specificity = = = 53%
TN + FP 50 + 44 TN + FP 37 + 33
TN 50 TN 37
NPV = = = 96% NPV = = = 79%
TN + FN 50 + 2 TN + FN 37 + 10
TP 4 TP 20
PPV = = = 8% PPV = = = 38%
TP + FP 4 + 44 TP + FP 20 + 33
 Example: changing prevalence
Scenario 1: Scenario 2:
Disease prevalence = 6% Disease prevalence = 30%
The same diagnostic test is used in both scenarios
Truth Truth
Diseased Healthy Diseased Healthy
The prevalence
Positive
of the disease
4 (TP) 44 (FP)
in the cohort impacts
Positive
the NPV
20 (TP)
significantly
33 (FP)
↓
Test result

Test result
Negative 2 (FN) 50 (TN) Negative 10 (FN) 37 (TN)

Therefore,
Sensitivity =
TP NPV’s
=
4of different cohorts cannot be
= 67% Sensitivity =
TP compared
=
20
= 67%
TP + FN 4+2 TP + FN 20 + 10
TN 50 TN 37
Specificity = = = 53% Specificity = = = 53%
TN + FP 50 + 44 TN + FP 37 + 33
TN 50 TN 37
NPV = = = 96% NPV = = = 79%
TN + FN 50 + 2 TN + FN 37 + 10
TP 4 TP 20
PPV = = = 8% PPV = = = 38%
TP + FP 4 + 44 TP + FP 20 + 33
Example: changing sensitivity
Scenario 1: Scenario 2:
Disease prevalence = 5% Disease prevalence = 5%
Sensitivity = 80% Sensitivity = 0%
Specificity = 80% Specificity = 80%
Truth Truth
Diseased Healthy Diseased Healthy
Positive 4 (TP) 19 (FP) Positive 0 (TP) 19 (FP)
Test result

Test result
Negative 1 (FN) 76 (TN) Negative 5 (FN) 76 (TN)
TP 4 TP 0
Sensitivity = = = 80% Sensitivity = = = 0%
TP + FN 4+1 TP + FN 0+5
TN 76 TN 76
Specificity = = = 80% Specificity = = = 80%
TN + FP 76 + 19 TN + FP 76 + 19
TN 76 TN 76
NPV = = = 99% NPV = = = 91%
TN + FN 76 + 1 TN + FN 79 + 5
TP 4 TP 0
PPV = = = 17% PPV = = = 0%
TP + FP 4 + 19 TP + FP 0 + 19
Example: changing sensitivity
Scenario 1: Scenario 2:
Disease prevalence = 5% Disease prevalence = 5%
Sensitivity = 80% Sensitivity = 0%
Specificity = 80% Specificity = 80%
Truth Truth
Diseased Healthy Diseased Healthy
Positive 4 (TP) 19 (FP) Positive 0 (TP) 19 (FP)
Besides disease prevalence,
Test result

Test result
Negative 1 (FN) 76 (TN) Negative 5 (FN) 76 (TN)
test sensitivity also has a significant impact on NPV
TP 4 TP 0
Sensitivity = = = 80% Sensitivity = = = 0%
TP + FN 4+1 TP + FN 0+5
TN 76 TN 76
Specificity = = = 80% Specificity = = = 80%
TN + FP 76 + 19 TN + FP 76 + 19
TN 76 TN 76
NPV = = = 99% NPV = = = 91%
TN + FN 76 + 1 TN + FN 79 + 5
TP 4 TP 0
PPV = = = 17% PPV = = = 0%
TP + FP 4 + 19 TP + FP 0 + 19
Diagnostic quality parameters: how to use them
Rule IN
1. A high PPV + 2. High specificity
Ensures that a patient with a Ensures that a patient with a
positive test result, is affected positive test result is affected
Depends on prevalence: if
everybody is affected, the PPV
would be 100%

For rule IN:

Combine PPV and specificity

Diagnostic quality parameters: how to use them
Rule OUT
1. A high NPV + 2. High sensitivity + 3. High specificity
Ensures that a patient with Ensures that a negative test Ensures that the rate of false
a negative test result, is result indicates a healthy positives is low, so the test is
unaffected patient useful in the clinical setting

Depends on prevalence: if
everybody is unaffected,
the NPV would be 100%

For rule OUT:

Combine NPV, sensitivity and specificity
The role of biomarker tests in NMIBC
Clinical application of biomarker tests

Negative
Surveillance
cystoscopy ± cytology

Tumour Surveillance
detection cystoscopy ±
& resection ± Re-TURBT cytology ± Instillations

Positive Treat:
TURBT ± instillations

Rule in to improve HG detection

Workup / treat:
Rule out unnecessary procedures Equivocal Biopsy/CT/TURBT/
instillations
 Comparing biomarkers: development phase

Discovery Training Validation

Research to find markers - Final marker panel - Prospective multicenter

- Algorithm analysis
- Definition of cut-off for - Representative cohort of
positive/negative result patients
- Comparison with gold
standard
 Comparing biomarkers: ease of interpretation

Human reader, complex criteria No automated output Automated, standardised

for negative/positive OR complex criteria for output
negative/positive
Urovysion
 UroVysion
Fluorescent in situ hybridisation (FISH) for the detection of urothelial cancer-related
Type of test
chromosomal abnormalities in cells in urine
• Chromosomal abnormalities in chromosome 3, 7 and 17
Markers • Loss of 9p21
Development phase

Ease of interpretation
FDA approved / CE-IVD marked
FDA approval/CE mark

• Hybridisation instrument (for instance, HYBrite from Vysis)

Technology/device • Epi-illumination fluorescence microscope
requirements • Experienced staff for adequate interpretation of the staining
Diagnosis of bladder cancer in patients with haematuria / monitoring for tumour
Clinical application
recurrence; in conjuction with standard diagnostic procedures

CE (Conformité Européenne); FDA: Food and Drug Administration; IVD: in vitro diagnostic
 Diagnosis & surveillance
Validated
UroVysion: Scientific evidence Ease of interpretation:

• Review paper1
PERFORMANCE
• 21 studies comprising 2,852
individuals
• Performance characteristics 53% 79% 80%
provided for mix of diagnosis and
surveillance
SENSITIVITY SENSITIVITY SPECIFICITY
LOW GRADE HIGH GRADE

“The broad range of sensitivity and specificity for UroVysion FISH reported in different papers is notable and may
“
not only reflect patient selection, study design, and tumour prevalence, but also technical aspects such as cutoff
definitions and experience of laboratory staff.

1. Schmitz-Dräger BJ et al. Urol Int 2015; 94:1-24.

ADXBLADDR
ADXBLADDER
Type of test Enzyme-linked immunosorbent assay (ELISA) using MCM5 antibodies

Markers MCM5 protein

Development phase

Ease of interpretation

FDA approval/CE mark Not FDA approved / Not CE marked

Technology/device
requirements Spectrophotometer or plate reader

Clinical application Diagnosis of bladder cancer in patients with haematuria / bladder cancer follow-up
 Diagnosis
ADXBLADDER: Validated
Scientific evidence for diagnosis Ease of interpretation:

• Prospective blinded, multicentre NMIBC

study1
• 856 patients presenting with
haematuria 65% 68.4% 96.4% -
• Bladder cancer prevalence: 8.6%
• Assay performed on full-void
urine prior to flexible cystoscopy SENSITIVITY SPECIFICITY NPV PPV
• Gold Standard: cystoscopy and
imaging 97.6 98.9 99.8 96.6 9799.8
100 88
86
80

Percentage
60 55 53
Sensitivity
“ ADXBLADDR has the potential to replace
“
cytology as an adjunctive test in bladder cancer
40 NPV

diagnosis 20
0
Low High CIS pTa ≥ pT1
1. Dudderidge T et al. Eur Urol Oncol 2020; 3: 42-46. grade grade
 Surveillance
ADXBLADDER: Validated
Scientific evidence for surveillance Ease of interpretation:

• Prospective blinded, multicentre OVERALL PERFORMANCE

study1
• 1431 patients with previous
diagnosis of NMIBC were 45% 71% 92% -
recruited
• Bladder cancer recurrence
prevalence: 8.6% SENSITIVITY SPECIFICITY NPV PPV
• HG recurrence prevalence: 2.6%
• Assay performed on voided
urine HIGH GRADE PERFORMANCE

73% 71% 99% 6.2%

SENSITIVITY SPECIFICITY NPV PPV

1. Roupret M et al. J Urol 2019; doi: 10.1097/JU.0000000000001084
Xpert Bladder Cancer Detection/Monitor
Xpert Bladder Cancer Detection/Monitor
Type of test Real-time PCR based mRNA expression
• ABL1 – CRH – IGF2 – UPK1B – ANXA10
Markers • Expression levels of the markers are combined to classify samples as negative or
positive using a linear model
Development phase

Ease of interpretation

FDA approval/CE mark Not FDA approved / CE-IVD marked

Technology/device Cepheid GeneXpert Instrument Systems

requirements
Clinical application Diagnosis of bladder cancer in patients with haematuria / bladder cancer follow-up
 Diagnosis
Xpert Bladder Cancer Detection: Validated
Scientific evidence for diagnosis Ease of interpretation:

• Multicentre study1 PERFORMANCE

• 895 patients without prior history
of bladder cancer 100 97 98 95 96
88 85 88
• Bladder cancer prevalence: not 78
80 76
reported
• Assay performed on urine 60 58 60
sample collected before Xpert
cystoscopy 40
40 Cytology
• Gold Standard: pathological UroVysion
confirmation 20

0
Sensitivity High Grade Specificity NPV
“Xpert Bladder Cancer Detect represents a promising
new tool for adjunctive use in combination with other
Sensitivity

diagnostic modalities to further optimise the detection

“
of bladder cancer in haematuric patients.

1. van Valenberg FJP et al. Eur Urol Suppl 2017; 16: e190.
 Surveillance
Xpert Bladder Cancer Monitor: Validated
Scientific evidence for surveillance Ease of interpretation:

• Multicentre study1 OVERALL PERFORMANCE

• 239 patients included in the
monitoring study
• Overall recurrence prevalence: 74% 80% 93% 44%
18%
• High grade recurrence
prevalence: 10% SENSITIVITY SPECIFICITY NPV PPV
• Assay performed on voided
urine samples
• Gold Standard: pathological HIGH GRADE DISEASE ONLY
confirmation
• 508 patients included in the
specificity study 83% 76% 98% 28%

SENSITIVITY SPECIFICITY NPV PPV

1. van Valenberg FJP et al. Eur Urol 2019; 75: 853-860.

Bladder EpiCheck
Bladder EpiCheck
Type of test Real-time PCR assay of methylation markers panel
Markers • 15 methylation markers
• Episcore (0-100) reflects the overall methylation levels in the urine sample
• Positive sample when Episcore ≥ 60
Development phase

Ease of interpretation

FDA approval/CE mark Pending FDA clearance / CE-IVD marked

Technology/device • RT-PCR machine

requirements • Molecular biology lab
Clinical application Bladder cancer follow-up
 Surveillance
Bladder EpiCheck: Validated
Scientific evidence for surveillance Ease of interpretation:

• Multicentre, prospective, single- All patients

arm, blinded clinical trial1,2
• 822 patients
• Recurrence prevalence: 12.2% 63% 86% 94% 45%1
• Urine sample collected before
cystoscopy
SENSITIVITY SPECIFICITY NPV PPV
• Gold Standard: pathological
confirmation

Excluding LG Ta recurrences

“Application of this test could reduce the current

burden of repeat cystoscopy and cytology tests,
86% 99%
for example, by alternating between the follow-
“
up gold standard and Bladder EpiCheck.1 SENSITIVITY NPV

1. Witjes JA et al. Eur Urol Oncol 2018; 1: 307-13. 2. Lozano F et al. EAU2019, poster 709.
 Surveillance
Bladder EpiCheck: Validated
Scientific evidence for surveillance Ease of interpretation:

• Single-centre, prospective, non- SENSITIVITY HG PAPILLARY TUMOURS

randomised, blinded clinical trial1 100 95
90 88 87
82
• 151 HG patients for follow-up 80
70 64
• Recurrence prevalence: not Sensitivity
60 54
reported NPV
50
• Urine sample collected before 40
Bladder Cytology Cystoscopy
cystoscopy/TURBT EpiCheck
• Gold Standard: pathological
confirmation or a physician’s
decision to start treatment SENSITIVITY CIS TUMOURS
100 98 98
The test might clinically improve the BCa 93
“ management in terms of reduced number of
90
80
87
76
inconclusive/suspicious reports of cytology 70
60 Sensitivity
and endoscopy, reduced number of further 60
NPV
“ 50
examinations, reduced associated patient
40
and economic burdens.1 Bladder Cytology Cystoscopy
EpiCheck
1. Racioppi M et al. EAU2020, poster 908.
Cxbladder Detect/Monitor
Cxbladder Detect/Monitor
Type of test RT-PCR based mRNA expression
Markers • IGFBP5 – HOXA13 – MDK – CDK1 – CXCR2
• Clinical information about previous bladder cancer: primary versus recurrent
urothelial cancer, time since previous tumour resection
• Cxbladder Detect score (range 0-10) of ≥0.23 indicates a high probability of
bladder cancer. A Cxbladder Monitor score of ≥3.5 warrants a clinician-directed
protocol to determine the presence of recurrent bladder cancer
Development phase
Ease of interpretation Not indicated
FDA approval/CE mark Not FDA approved / Not CE marked
Technology/device
requirements None – sample shipment to central lab
Clinical application Diagnosis of bladder cancer in patients with haematuria / bladder cancer follow-up
 Diagnosis
Cxbladder Detect: Validated
Scientific evidence for diagnosis Ease of interpretation:

• Prospective, multicentre study1 OVERALL PERFORMANCE

• 485 patients with macroscopic
haematuria
• Bladder cancer prevalence: 82% 85% - -
13.6%
• Assay performed on voided
urine samples SENSITIVITY SPECIFICITY NPV PPV
• Gold Standard:
cystoscopy/histology

1. O’Sullivan P et al. J Urol 2012; 188: 741-7.

 Surveillance
Cxbladder Monitor: Validated
Scientific evidence for surveillance Ease of interpretation:

• Prospective, multicentre study1

ALL PATIENTS
• 1,036 urine samples from 763
patients undergoing surveillance
• 493 samples used as training set 92% 32% 96% -
• 543 samples used in validation
• Recurrence prevalence: 15.1%
• Assay performed on voided SENSITIVITY SPECIFICITY NPV PPV
urine sample collected before
cystoscopy
• Gold Standard: cystoscopy with SENSITIVITY PER TUMOUR STAGE
or without CT scan/histology

89% 97% 100%

Ta CIS T1+
1. Kavalieris L et al. J Urol 2017; 197: 1419-26.
Uromonitor
Uromonitor
Type of test Sensitive multiplex competitive allele-specific discrimination real-time PCR
Markers • TERT promoter mutations [c.1-124C>T (TERTp-124) and c.1-146C>T (TERTp-146)]
• FGFR3 mutations [p.R248C (FGFR3-248) and p.S249C (FGFR3-249)]
Development phase

Ease of interpretation Not indicated

FDA approval/CE mark
Not FDA approved / CE-IVD marked

Technology/device
requirements None – sample shipment to central lab

Clinical application Bladder cancer follow-up

 Surveillance
Validation Discovery
Uromonitor: discovery results required Ease of interpretation:

• Prospective, multicentre SENSITIVITY

discovery study1 100
100
• 331 urine samples from 185 86.7
patients 80 73.5
79.4

• 122 samples of surveillance

patients 60

• Recurrence prevalence: 28% 42.9

40
• Gold standard: Uromonitor Cystoscopy Cytology Cystoscopy + Uromonitor +
cytology cystoscopy
cystoscopy/cytology/histology
SPECIFICITY
100 93.2 93.2 93.9
87.9 86.4
“Uromonitor used as an adjunct to cystoscopy
allowed achieving a 100% sensitivity and 88.6%
80

“
specificity, an important upgrade in comparison to 60
the cystoscopy and cytology combination.
40
Uromonitor Cystoscopy Cytology Cystoscopy + Uromonitor +
1. Batista R et al. Front Genet 2019; 18: 1237. cytology cystoscopy
Urodiag
Urodiag
Type of test Highly specific multiplex quantitative real-time PCR
Markers • Mutations in FGFR3 gene (S249C – R248C – G372C – Y375C)
• Level of DNA methylation of HS3ST2 – SEPTIN9 – SLIT2
Development phase

Ease of interpretation Not indicated

FDA approval/CE mark Not FDA approved / CE-IVD marked

Technology/device
requirements Not indicated

Clinical application Diagnosis of bladder cancer in patients with haematuria / bladder cancer follow-up
 Diagnosis & surveillance
Discovery
Urodiag: discovery study Validation
required Ease of interpretation:

• Prospective discovery study1 PERFORMANCE

• 158 NMIBC patients for follow-up
included, resulting in 583 urine
samples
• 158 samples from diagnosis
90.3% 65.1% 97%
• 425 samples taken during follow-up
SENSITIVITY SPECIFICITY NPV

“With a high sensitivity and high NPV, our set of

markers is useful for avoiding biopsies and
SENSITIVITY BY STAGE AND GRADE
96.4% 100% 100% 96%
decreasing the frequency of cystoscopic 100 93.6%
surveillance, thereby allowing for both patient
“
quality of life improvement and cost reduction.
50

0
pTa pT1 CIS Low grade High grade
(N=55) (N=11) (N=2) (N=47) (N=25)
1. Roperch J-P et al. BMC Cancer 2016; 16:704.
How to choose the appropriate biomarker
test?
Biomarkers for “rule IN”

Negative
Surveillance
cystoscopy ± cytology
Tumour Surveillance
detection cystoscopy ±
& resection ± Re-TURBT cytology ± Instillations

Positive Treat:
Rule in patients who have bladder cancer & TURBT ± instillations
patients with recurrence and/or progression

High specificity:
Low false positive rate  minimise the number of
unnecessary procedures Workup / treat:
High PPV: Equivocal Biopsy/CT/TURBT/
A patient with a positive test result, is affected instillations
Biomarkers for rule IN for diagnosis

UroVysion1 ADXBLADDR2 Xpert Bladder Cxbladder

Detection3 Detect4

Specificity 80% 68.4% 85% 85%

PPV NR NR NR NR

NR: not reported

1. Schmitz-Dräger BJ et al. Urol Int 2015; 94:1-24. 2. Dudderidge T et al. Eur Urol Oncol 2020; 3: 42-46. 3. van Valenberg FJP et al. Eur Urol Suppl 2017; 16: e190.
4. O’Sullivan P et al. J Urol 2012; 188: 741-7.
Biomarkers for rule IN for surveillance

ADXBLADDER1 Xpert Bladder Cxbladder Bladder

Monitor2 Monitor3 EpiCheck4,5

Specificity 71% 80% 32% 86%

PPV NR 44% NR 45%

NR: not reported

1. Roupret M et al. J Urol 2019; doi: 10.1097/JU.0000000000001084 2. van Valenberg FJP et al. Eur Urol 2019; 75: 853-860.
3. Kavalieris L et al. J Urol 2017; 197: 1419-26. 4. Witjes JA et al. Eur Urol Oncol 2018; 1: 307-13. 5. Lozano F et al. EAU2019, poster 709
Biomarkers for “rule OUT”

Negative
Surveillance
cystoscopy ± cytology
Tumour Surveillance
detection cystoscopy ±
& resection ± Re-TURBT cytology ± Instillations

Rule out unnecessary procedures Positive Treat:

TURBT ± instillations
High sensitivity:
Low false negative rate  minimise the number of
missed patients with recurrence
High specificity:
Low false positive rate  minimise the number of
unnecessary procedures Workup / treat:
Equivocal Biopsy/CT/TURBT/
High NPV: instillations
A patient with a negative test result, is unaffected
 Biomarkers for rule OUT for surveillance

 Overall

 High grade

NR: not reported

1. Schmitz-Dräger BJ et al. Urol Int 2015; 94:1-24. 2.Roupret M et al. J Urol 2019; doi: 10.1097/JU.0000000000001084. 3. Van Valenberg FJP et al. Eur Urol Suppl 2017; 16: e190.
4. Kavalieris L et al. J Urol 2017; 197: 1419-26. 5. Witjes JA et al. Eur Urol Oncol 2018; 1: 307-13. 6. Lozano F et al. EAU2019, poster 709.
Conclusion
Conclusion

• Biomarker tests used as “rule in” test need to have a high specificity
and high PPV
• Positive cytology is considered gold standard to rule-in high grade tumours,
given its high specificity (93%)
• However, 10-15% of the cytology results are “atypical”
• At the moment, none of the biomarker tests have a specificity higher than
cytology
• Biomarker tests used as “rule out” test require a high sensitivity, high
specificity and high NPV
• Bladder EpiCheck is the only biomarker test, validated for surveillance, that
provides a high sensitivity, high specificity and high NPV for ruling out HG
tumours