Materials & Methods• prospective study: February 2006 - January 2007prospective study: February 2006 - January 2007• inclusion criteria:inclusion criteria:

- patients undergoing transurethral resection (TUR-B) for newly diagnosed BCa, recurrent BCa &- patients undergoing transurethral resection (TUR-B) for newly diagnosed BCa, recurrent BCa & cystoscopically suspicious bladder lesionscystoscopically suspicious bladder lesions- BCa patients before cystectomy - BCa patients before cystectomy

• exclusion criteria:exclusion criteria:- patients with prostate cancer and other non-urothelial tumors- patients with prostate cancer and other non-urothelial tumors

• controls:controls:- BPH patients, cystitis patients, healthy volunteers- BPH patients, cystitis patients, healthy volunteers

• collection of urine and tissue samples:collection of urine and tissue samples:- for every TUR-B (prim. / sec. / tert.)- pre-operative urine samplepre-operative urine sample- intra-operative tumor tissue & “normal appearing” bladder mucosa- intra-operative tumor tissue & “normal appearing” bladder mucosa- - same procedure for recurrences (Fig.1)- controls: 1 urine sample- controls: 1 urine sample

• processing and measurement of urine and tissue samples:processing and measurement of urine and tissue samples:- collection of up to 250 ml urine, - collection of up to 250 ml urine, processed immediately after transport to the laboratory immediately after transport to the laboratory- preparation of cellular components from urine by centrifugation (10 min, 4°C, 830 x g)- preparation of cellular components from urine by centrifugation (10 min, 4°C, 830 x g)- two washing steps in ice cold PBS- two washing steps in ice cold PBS- resuspension of the cellular pellet in 700 µl RNA-stabilizing buffer (Invitek, Berlin), storage at -80°C- resuspension of the cellular pellet in 700 µl RNA-stabilizing buffer (Invitek, Berlin), storage at -80°C- collection of malignant and apparently non-malignant bladder tissue specimens during TUR-BT- collection of malignant and apparently non-malignant bladder tissue specimens during TUR-BT- immediate snap-freezing in liquid nitrogen- immediate snap-freezing in liquid nitrogen- preparation of cryosections (60-80 slices à 4 µM) and addition of 600 µl RNA-stabilizing buffer (Invitek, Berlin)- preparation of cryosections (60-80 slices à 4 µM) and addition of 600 µl RNA-stabilizing buffer (Invitek, Berlin)- isolation of total RNA by standard protocols (- isolation of total RNA by standard protocols (Spin Tissue/Cell RNA Mini KitSpin Tissue/Cell RNA Mini Kit, Invitek, Berlin), Invitek, Berlin)- cDNA-synthesis using up to 500 µg RNA, random hexamer primers (Amersham) and Superscript II Rnase H - cDNA-synthesis using up to 500 µg RNA, random hexamer primers (Amersham) and Superscript II Rnase H

Reverse Transcriptase (Invitrogen)Reverse Transcriptase (Invitrogen)- quantitative PCR for transcript levels of survivin, XIAP & Ki67 and the reference gene TBP in urine and tissue- quantitative PCR for transcript levels of survivin, XIAP & Ki67 and the reference gene TBP in urine and tissue samples using gene-specific primers and probes (LightCycler, Roche), detection limit of 10 transcript moleculessamples using gene-specific primers and probes (LightCycler, Roche), detection limit of 10 transcript molecules

• correlation of the relative expression levels (internal normalization to TBP) of survivin, XIAP & Ki67correlation of the relative expression levels (internal normalization to TBP) of survivin, XIAP & Ki67 with clinico-pathological data using SPSS 12.0 with clinico-pathological data using SPSS 12.0

• Patients (n = 128)Patients (n = 128)• age (median) = 70 yrs. (34 – 93yrs.)age (median) = 70 yrs. (34 – 93yrs.)• M : F = 76 : 52 (59,4% : 40,6%)M : F = 76 : 52 (59,4% : 40,6%)• newly diagnosed : recurrence = 103 : 25 (80,5% : 19,5%)newly diagnosed : recurrence = 103 : 25 (80,5% : 19,5%)• PSA (median; 63 pts.) = 1.36 (0.16 – 33.91)PSA (median; 63 pts.) = 1.36 (0.16 – 33.91)• tumor stage:tumor stage:

- 18 pts. NT = no tumor detectable- 18 pts. NT = no tumor detectable- 66 pts. pTa- 66 pts. pTa- 18 pts. pT1- 18 pts. pT1- 21 pts. >pT1- 21 pts. >pT1- 4 pts. pTis only; for 1pt. tumor stage n.a.- 4 pts. pTis only; for 1pt. tumor stage n.a.

• ControlsControls

• tumor grade:tumor grade:- 18 pts. NT = no tumor detectable- 18 pts. NT = no tumor detectable- 10 pts. LMP (low malignancy potential)- 10 pts. LMP (low malignancy potential)- 22 pts. low grade- 22 pts. low grade- 78 pts. high grade- 78 pts. high grade

Introduction• Bladder cancer (BCa): 4Bladder cancer (BCa): 4thth most common cancer in men & 9 most common cancer in men & 9thth leading cause of death worldwide leading cause of death worldwide• cystoscopy & urine cytology: current gold standards for diagnosis & surveillance of BCacystoscopy & urine cytology: current gold standards for diagnosis & surveillance of BCa• no ideal tumor marker for non-invasive diagnostic & surveillance at the momentno ideal tumor marker for non-invasive diagnostic & surveillance at the moment• therefore new diagnostic methods and markers are needed, e.g. BCa-related transcript markers:therefore new diagnostic methods and markers are needed, e.g. BCa-related transcript markers:

• survivin (SVV) & XIAP: inhibitor of apoptosis proteins (IAP)survivin (SVV) & XIAP: inhibitor of apoptosis proteins (IAP)• Ki67: proliferation marker, essential for cell cycle progressionKi67: proliferation marker, essential for cell cycle progression• selectively over-expressed in most human malignancies incl. BCaselectively over-expressed in most human malignancies incl. BCa• association between over-expression and higher stage & grade and with unfavorable prognosis association between over-expression and higher stage & grade and with unfavorable prognosis • possible markers (tissue and urine specimens ) and therapeutic targets for BCapossible markers (tissue and urine specimens ) and therapeutic targets for BCa

Objectives to establish methods for quantitative transcript measurements (QPCR) in urine and tissue specimensto establish methods for quantitative transcript measurements (QPCR) in urine and tissue specimens

to determine suitability of transcript levels of different BCa-related genes (survivin, Ki67 and XIAP) into determine suitability of transcript levels of different BCa-related genes (survivin, Ki67 and XIAP) in

urine samples as diagnostic, surveillance and prognostic markers of BCaurine samples as diagnostic, surveillance and prognostic markers of BCa

to validate this bio-molecular technique as additional, non-invasive diagnostic tool in BCa assessmentto validate this bio-molecular technique as additional, non-invasive diagnostic tool in BCa assessment

Fig.3 Fig.3 Transcript markers in urine specimens of BCa patients & controlsTranscript markers in urine specimens of BCa patients & controlsThe relative transcript levels of Ki67, SVV and XIAP (normalized to TBP) in urine samples of BCa patients undergoing TUR-B are shown in comparison to control groups (patients with BPH or cystitis and healthy volunteers). The “no tumor”-group comprises patients undergoing TUR-B due to the suspicion of having a BCa which could no be proven histologically. The solid lines within the boxplots represent the median transcript levels, numbers of samples per group are indicated.

For healthy controls the absent values were substituted by zero. Median values are presented.For healthy controls the absent values were substituted by zero. Median values are presented.

Fig.1 Fig.1 Course of treatment for BCa patients and sample collectionCourse of treatment for BCa patients and sample collectionPatients with newly diagnosed or recurrent BCa undergo a TUR-BPatients with newly diagnosed or recurrent BCa undergo a TUR-B... . If needed, a second scope is scheduled 4-6 weeks later. When necessary, a tertiary TUR-B or cystectomy is performed. Before each intervention, , a tertiary TUR-B or cystectomy is performed. Before each intervention, urine and tissue samples were collected when available.urine and tissue samples were collected when available.

Fig.2 Fig.2 Transcript markers in malignant and non-malignant bladder tissue specimensTranscript markers in malignant and non-malignant bladder tissue specimensDistribution of the relative transcript levels of Ki67, SVV and XIAP (normalized to TBP) in unpaired malignant and non-malignant bladder specimens collected during TUR-B. The solid lines within the boxplots represent the medians, numbers of samples per group are indicated. Ratios of the medians in the Tu and Tf groups are shown in the table, an up-regulation in these tumor tissues is observed only for Ki67 and SVV.

Median values are presented.Median values are presented.

Conclusion & Outlook

relative transcript levels of Ki67 and SVV possibly useful as BCa markers in urine samplesrelative transcript levels of Ki67 and SVV possibly useful as BCa markers in urine samples

dependence on tumor stage and grade for both markersdependence on tumor stage and grade for both markers

XIAP not suitable for discrimination XIAP not suitable for discrimination between disease/ patient groups

continuation of sample collection for better statistical calculationscontinuation of sample collection for better statistical calculations

subsequent definition of cut-off values for calculation of test performance in comparison to cytology definition of cut-off values for calculation of test performance in comparison to cytology

correlation with follow-up data correlation with follow-up data possible prediction of recurrence (SVV is well-known predictor) possible prediction of recurrence (SVV is well-known predictor)

Results• TissueTissue specimensspecimens: : RNA quality RNA quality satisfactory for reliable QPCR measurement, only few samples for reliable QPCR measurement, only few samples had negative negative

reference gene reference gene expression• Initially, , two reference genes testedtwo reference genes tested for tissue and urine specimens: for tissue and urine specimens: TBP expression (≥ 10 transcript

molecules) more consistent than HPRT • Urine specimensUrine specimens: negative correlation between reference gene expression & urinary contamination by RBCs, : negative correlation between reference gene expression & urinary contamination by RBCs,

WBCs & bacteria WBCs & bacteria up to 20% of the samples with negative reference gene up to 20% of the samples with negative reference gene expression (e.g. patients with (e.g. patients with

infection or macro-hematuria)infection or macro-hematuria)• Thus, initial collection of collection of urine one day one day after TUR-Bafter TUR-B high high frequency of negative reference gene expression• Marker validationMarker validation in tissue specimensin tissue specimens by by comparing tumor & tumor-free specimens: only : only up-regulationup-regulation (1.6- & (1.6- &

1.9-fold) of 1.9-fold) of Ki67Ki67 and SVVand SVV in tumor specimens compared to apparently tumorfree tissue samples (Fig.2), in tumor specimens compared to apparently tumorfree tissue samples (Fig.2),

enlargement of sample cohorts planned, stronger up-regulation in invasive BCa expected enlargement of sample cohorts planned, stronger up-regulation in invasive BCa expected comparison to comparison to

cystectomy specimens plannedcystectomy specimens planned• similar trends observed in urine samples: transcript levels of similar trends observed in urine samples: transcript levels of Ki67 and SVV higher in urines from BCa patientsKi67 and SVV higher in urines from BCa patients

compared to healthycompared to healthy individuals and patients with individuals and patients with cystitiscystitis, but also , but also slightly elevatedslightly elevated levels in urines from levels in urines from BPH BPH

patientspatients, , no clear differences in XIAP no clear differences in XIAP levels in the different control groups (Fig.3)levels in the different control groups (Fig.3)• dependence on tumor stage and gradedependence on tumor stage and grade observed for transcript levels of observed for transcript levels of Ki67, SVV and XIAPKi67, SVV and XIAP (Fig.4 & 5) (Fig.4 & 5)• patients without histologically proven BCa (patients without histologically proven BCa (NT groupNT group) displayed ) displayed higher transcript levelshigher transcript levels of all three markers of all three markers

compared to healthycompared to healthy individuals, comparison to cytology will be performed after completion of measurements individuals, comparison to cytology will be performed after completion of measurements

primary TUR-Bprimary TUR-B

cystectomycystectomysecondary TUR-Bsecondary TUR-B

tertiary TUR-Btertiary TUR-B cystectomycystectomy

4-6 weeks4-6 weeks

4-6 weeks4-6 weeks

recurrencerecurrence

recurrencerecurrence

Non-invasive detection of bladder cancer by measurement of Non-invasive detection of bladder cancer by measurement of tumor-related transcript markers in urinetumor-related transcript markers in urine

Catharina RippelCatharina Rippel11, Juliane Schmidt, Juliane Schmidt11, Woei-Yun Siow, Woei-Yun Siow22, Susanne Fuessel, Susanne Fuessel11, Axel Meye, Axel Meye11, Andrea Lohse, Andrea Lohse11, , Marc-Oliver GrimmMarc-Oliver Grimm11, Oliver W. Hakenberg, Oliver W. Hakenberg11, Manfred P. Wirth, Manfred P. Wirth11

11 Department of Urology, Technical University of Dresden, Germany, Department of Urology, Technical University of Dresden, Germany, 22 Dept. of Urology, National University Hospital, Singapore Dept. of Urology, National University Hospital, Singapore

Fig.5 Fig.5 Transcript markers in urine correlated with tumor gradeTranscript markers in urine correlated with tumor gradeDistribution of the relative transcript levels of Ki67, SVV and XIAP (normalized to TBP) in urine specimens collected before TUR-B correlated to tumor grade. The solid lines within the boxplots represent the median transcript levels, numbers of samples per group are indicated.

Fig.4 Fig.4 Transcript markers in urine correlated with tumor stageTranscript markers in urine correlated with tumor stageDistribution of the relative transcript levels of Ki67, SVV and XIAP (normalized to TBP) in urine specimens collected before TUR-B correlated to tumor stage. The solid lines within the boxplots represent the median transcript levels, numbers of samples per group are indicated.

Healthy NT pTa pT1 >pT1

Stage

0

5

10

15

20

Ki6

7/T

BP

13.0

19.0

44.015.0

53.0

116

77

80

99

144

50

46

105

100

24

137

131

129130

Healthy NT pTa pT1 >pT1

Stage

0

5

10

15

20

25

SV

V/T

BP

11.0

17.0

44.0

14.0 49.0

100

39

102

154167

125

47

66

41

56

91

48

88

4

70

87

131

135

136

Healthy NT pTa pT1 >pT1

Stage

0

100

200

300

400

XIA

P/T

BP

11.016.0

35.0

15.0 46.0

39

105

86

46

131160129

128

124

125

42

96

3

25

56

91

21

88

87

151

Healthy NT low grade high grade

Grade

0

5

10

15

Ki6

7/T

BP

67.0

44.0

15.0 15.0

116

46

105

100

144

80

24

61

137

131

129

130

Healthy NT low grade high grade

Grade

0

5

10

15

20

25

SVV/T

BP

63.0

44.0

14.0

13.0

25

61

154167

125

47

41

4

7066

87

48

56

37

88

91

131

135

136

Healthy NT low grade high grade

Grade

0

100

200

300

400

500

XIA

P/T

BP

57.0

35.0

15.013.0

61

131160

128

124

125

25

96

3

21

56

87

88

91

151

tumourfree tumour

Type of Tissue

0

5

10

15

20

25

30

Ki6

7 /

TB

P

65.0

36.0

tumourfree tumour

Type of Tissue

0

2

4

6

8

SV

V /

TB

P

65.0

36.0

94

35

tumourfree tumour

Type of Tissue

0

20

40

60

80

XIA

P /

TB

P

65.0 36.0

BT NT BPH Cystitis Healthy

Urine

0

3

6

9

12

15

Ki6

7/T

BP

6.0 44.0

90.0

15.0 38.0

50

90

4

116

204

46

100

95

24

60

155

184

197216

191

189

190

BT NT BPH Cystitis Healthy

Urine

0

5

10

15

20

25

SV

V/T

BP

4.0

44.0

82.0

14.0 28.0

60

152149

214

47

4

6864

83

48

41

56

37

84

87

125

167

191

195

196

BT NT BPH Cystitis Healthy

Urine

0

100

200

300

400

500

600

XIA

P/T

BP

5.0

35.0

76.0 15.0 25.0

54

42

191220

60

91

3

25

21

56

83

84

87

128

124

125

173

149

150

211

BPHBPH cystitiscystitis healthyhealthyn = 53n = 53 n = 7n = 7 n = 44n = 44

67 yrs (52-85)67 yrs (52-85) 35 yrs (19-76)35 yrs (19-76) 28.5 yrs (18-59)28.5 yrs (18-59)

PSA median 2.21 ng/mlPSA median 2.21 ng/ml

(0.28 – 13.71 ng/ml)(0.28 – 13.71 ng/ml)

M : F 1:6M : F 1:6

(14,3% : 85,7%)(14,3% : 85,7%)

M : F 13:27M : F 13:27

(32,5 % : 67.5 %)(32,5 % : 67.5 %)

unpaired tissue samplesunpaired tissue samples Ki67 / TBPKi67 / TBP SVV / TBPSVV / TBP XIAP / TBPXIAP / TBP

tumor tissue (n = 42)tumor tissue (n = 42) 3.943.94 1.321.32 15.8515.85

tumor-free tissue (n = 72)tumor-free tissue (n = 72) 1.881.88 0.740.74 15.7315.73

median Tu / median Tfmedian Tu / median Tf 2.102.10 1.781.78 1.011.01

clinical diagnosis Ki67 / TBP SVV / TBP XIAP / TBP

BCa / TUR-B (n=110)

(positive histology)2.18 2.79 44.47

NT = no tumor (n=18)

(negative histology)0.62 1.71 42.87

BPH (n=53) 0.80 1.45 36.0

cystitis (n=7) 1.65 1.23 46.24

healthy (n=44) 0 0 0