UBC Rapid – Point of Care

Cytokeratin 8/18 assay in bladder

cancer

Roland Einarsson, PhD

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

UBC Rapid - Assay Procedure

UBC Rapid – Evaluation of results

T C T C

T C

Negative test

Positive test

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

Risk stratification – quantitative UBC Rapid

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