Bladder Cancer – Clinical T-stage
Muscularis propria Adventitia
Lamina propria Urothelium
Papilloma Tis Ta
T1
T2
T3a
T3b
T4
Local spread
High risk of recurrence and
progression – long follow up
needed
Routine management;
cystoscopy,cytology and
transurethral resection
UBC Rapid – urinary POC test?
“new” substance produced by the tumour normal substance produced by the tumour at elevated concentrations substance in causal relation with the tumour
What is a tumour marker ?
Commercially available tumor
markers in Bladder Cancer
sens (%) spec (%)
• Urinary cytology 19-70 73-100
• NMP22 45-92 54-91
• BTA 51-90 54-93
• Immunocyt 50-100 69-79
• UroVision (FISH) 69-87 89-96
• Survivin 64-94 93-100
• Cyfra 65-99 57-88
• TPA 16-80 73-100
• UBC 40-81 72-97
Tumor Markers in Bladder Cancer (ideal
test for urinary bladder cancer screening
and monitoring)
• Noninvasive, rapid, objective, high sensitivity and specificity, accurate and easy to perform and interpret
• High sensitivity in primary disease and possibility to postpone and reduce the number of cystoscopies during follow-up
• High specificity is mandatory if the test should be able to replace urinary cytology
What are cytokeratins?
Cytoskeletal proteins
– Intermediate filament proteins
– Keratin gene family - 54 distinct genes
– Cytokeratins - a family of >20 different
proteins
– Cytokeratins 8, 18 and 19 most abundant
Epithelial cell specific expression
– Overexpressed in transformed cells
Cell death mode - necrosis and/or apoptosis
B. Sundström, T. Stigbrand: Int J Biol Markers, 1993
Expression of cytokeratins in various epithelia.
Cytokeratin protein
structures
• Acidic proteins (type I, CKs 9-20, 40-56 kDa)
• Basic proteins (type II, CKs 1-8, 53-68 kDa)
• Obligate heterodimer formations
• Detection of CK fragments in circulation
Cytokeratin tumor markers
• Tumor cell activity markers • Early and distinct signals
• Management of patients with carcinomas • Prognosis, monitoring and follow-up
• Complementary to volume markers • Defined panels give increased sensitivity and prolonged
lead times
• Not organ specific
Sensitivity = x 100
Specificity = x 100
PPV = x 100
NPV = x 100
Tumor Marker Terminology
True positives
true positives + false negatives
True negatives
false positives + true negatives
True positives
true positives + false positives
True negatives
true negatives + false negatives
Cut-off - Upper reference level
Negative Positive
Ideal situation
Real situation
High specificity -
low sensitivity
High sensitivity-
low specificity
Disease absent Disease present
Non-malignant conditions
Increased urine levels can be found
in case of
• pregnancy
• renal failure
• stones
• general infections (UTI)
• catheter
• new bladder
• instrumental collected urine
Absorbent pad
Detector
reagent Anti mouse
Control line Capture antibody
Test line
Added sample
with antigen
UBC Rapid - Assay Principle
UBC Rapid - Technical Performance
Sample volume 75 ul urine
Read UBC Rapid at 10 minutes
Analytical sensitivity 5 ug/L
Cut point (OMEGA Reader) 10-14.5 ug/L
Batch-to-batch variation 5-15 %
Interference (Hb, Bil, Alb) no interfence detected
Stability (cassette) accelerated testing 24 months
Blood in urine - contamination
• Comparative analysis of available POC-
test at German market
– UBC rapid
– NMP22 BladderChek
– BTA stat
• Artificial blood (heparinized) contamination
in normal urine specimens
• Analysis of test specificity in relation to
blood contamination Lüdecke et at. Anticancer Res 2012
UBC rapid – addition of heparinized blood to
normal urine
Urine Dilution 1:10 (1), 1:200 (3), 1:800 (5), 1:6400 (8), 1:25600 (10)
Conclusions
• UBC rapid and NMP22 BladderChek are
insensitive against blood in any concentration
• BTA stat produces false positive results in
macro/gross haematuria and in micro haematuria
(up to 150 erythrocytes/µl)
• Choice of antigen is important
• Complement system proteins induce cross
reactivity (BTA)
• Clinically BTA stat is not suitable in primary
diagnostic work flow for BC Lüdecke et al. Anticancer Res 2012
Urinary bladder test – objective,
accurate, rapid and demonstrating
high sensitivity and specificity
Can UBC Rapid be applied in bladder cancer and discriminate between
urothelium bladder cancer (CIS, Ta, T1) and
muscle invasive tumors (T2-T4)
TaG1, T1G1 tumors (low-risk) and TaG3, T1G3 and CIS tumors (high-risk)
UBC rapid- POC-assay
• Detection of soluble fragments of cytokeratin 8 and 18
• Quantitative POC-assay using OMEGA Reader
• Sensitivity: 64-66%, Specificity: 71-90 %
3,3 ng/ml
15,6 ng/ml
72 ng/ml
Quantitative evaluation
UBC Rapid
• 198 Patients (151 m, 47 f) with
suspicion of urothelial carcinom
(147 Primary diagnosis, 51
Surveillance)
• Cystoscopy for confirmation
•Urine analysis :
Urinstix/Urine microscopy
Urine cytology
NMP22 Bladder Check
UBC Rapid (visual/quantitative with Ω 100 Reader)
Ritter et al. Urol Oncology 2014
Patient data • 61 Patients with bladder cancer
(39 new detected, 22 recurrences)
– 41 x Ta
– 7 x T1
– 10 x Muscle invasive (T2-T4)
– 17 x G1
– 26 x G2
– 15 x G3
– 3 x Carcinoma in situ
• Hematuria in 106 patients (52%)
• Urinary infection in 39 patients(19,7%)
• 12 Patients with Urolithiasis (6%)
• 40 Patients with BPH (20%)
Results
Sens Spec
PPV NPV AUC
Cytology 51.0 80.3
51.0 80.3 0.65
Bladder check
(NMP22)
16.3 95.3 62.5 70.5 0.55
UBC Rapid visual 37.7 88.3 58.9 76.1 0.63
UBC Rapid OMEGA
quantitative test
(Cut point 12 µg/l)
62.3 70.1 47.5 80.5 0.69
Additional information by adding
UBC Rapid test
• Detection of 12 cytology negative tumors (cut-off
12 µg/l) with UBC Rapid – including 3 high-risk
tumors (T1/G3/Cis)
• Sensitivity for cytology and UBC Rapid combined:
73,7 % for all patients, for high-risk tumors
(T1,G3,Cis) 88%
• Specificity (cytology and UBC Rapid): 58,5 %
• AUC (cytology and UBC Rapid): 0,74
Conclusions (Tübingen study)
• Cystoscopy is the mainstay for the diagnosis of
bladder cancer
• UBC Rapid demonstrates high diagnostic
sensitivity in high-risk bladder cancer patients
• Benign conditions of the urinary tract must be
considered when evaluate data as they may
cause false positive reading
• UBC Rapid is a quick assay and test result will
be availble at the patient visit
• Quantitative UBC Rapid measures a continous
parameter – suitable for risk stratification
Clinical value of UBC Rapid (Giessen study)
UBC Rapid/OMEGA Reader analysed in 178 patients;
- 68 bladder cancer (UC), 72 benign diseases (infections, stones, BPH, renal disturbances, non-UC tumors), 15 catheter patients/instrument collection of urine and 23 normals
OMEGA reader decision level ¨19 ug/L¨ (<19 neg, >19 pos)
UBC Rapid signal in normals 5.4 ug/L and in bladder cancer patients median 47 ug/L
NED patients without active disease showed negative UBC Rapid results
Lüdecke et al. 2015 (to be published)
Results from Giessen study
UBC Rapid T-stage: pTa 44 ug/L, pT1 49 ug/L, pT2-T4 136 ug/L
UBC Rapid Grade: G1 13 ug/L, G2 51 ug/L, G3 100 ug/L
UBC Rapid showed a sensitivity of 72% at a specificity of 92%,
PPV 96%, NPV 53% and accuracy 77%
UBC Rapid was positive in benign patients (decision level 19
ug/L) in 63-100% of the patients (UTI 63%, stone patients 82%,
catheter patients 83% and ileum-condoit 100%)
To reach a specificity >90% for UBC Rapid it is mandatory to
exclude benign cases in routine practice
Conclusions (Giessen study) (UBC Rapid/Omega Reader)
►UBC Rapid is a powerful diagnostic parameter for primary diagnosis and for monitoring bladder cancer patients
►Bladder cancer patients; for daily use the exclusion of benign diseases are mandatory to reduce false positives (spec>90%)
►UBC Rapid/Omega Reader combination ensures an objective reading of the POC test
►UBC Rapid/OMEGA Reader offers a prognostic interpretation of data – UBC concentrations are related to tumor stage (T) and tumor grade (G)
Multicentre-study for detection of urinary
bladder cancer using UBC Rapid
(Ecke/Arndt)
195 bladder cancer patients (108 low grade tumors –
papillary Ta; 87 high grade tumors – G3, Cis, T2-T4)
and 136 healthy controls (no history of bladder
cancer)
UBC Rapid POC test was evaluated visually and
quantitatively using OMEGA POC Reader
Statistical analysis – ROC optimization (cut-off and
AUC), sensitivities and specificities
UBC Rapid results (Ecke/Arndt)
UBC Rapid diagnostic criteria:
sensitivity - all tumors 54.8%, high grade tumors
(CIS, T1G2, T1G3, T2-T4) 72.4 % and low grade Ta-
tumors 43.7%
specificity - 93.4% at all patient evaluations
ROC-analysis for optimization of UBC Rapid cut
point (sensitivity/specificity profile) gave 10 ug/L and
AUC of 0.738
Pathological values of UBC Rapid in urine are higher
in UC-patients versus control subjects
Conclusions (Ecke/Arndt)
UBC Rapid can differentiate between
pathological values in urine and normals –
cytokeratin concentrations in bladder cancer
patients versus controls
UBC Rapid demonstrates high sensitivity for high
grade tumors at a specificty exceeding 90%
Visual analysis (strong and intermediate test
positive test bands) gave similar results as
quantitative determination
CONCLUSIONS
UBC Rapid combines proven dry-pad urine chemistry technology in cassette test format (proteomic marker)
UBC Rapid in combination with OMEGA Reader - a quantitative POC test is available
UBC Rapid demonstrates high diagnostic capacity
UBC can identify patients with primary bladder cancer and patient with recurrent tumors
UBC Rapid deliver reliable, trusted test results and offers a prognostic interpretation (stage and grade)
UBC Rapid is an alternative to urine cytology
UBC Rapid is a complementary tool to cystoscopy
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