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Differential expression of mismatch repair proteins, MLH1 and MSH2, in Transitional Cell Carcinoma (TCC) of the bladder
77 IDENTIFICATION
OF URINARY
CELL CARCINOMA
BIOMARKERS
WITH SELDI MASS SPECTROMETRY
Munro Nicholas, Rogers Marga, Craven Rachel, Harnden Patricia, Eardley Ian, Banks Rosamonde,
Knowles Margaret
ICRF Clinical Centre, St James University INTRODUCTION:
Current
follow up of Transitional
Cell Carcinoma
Robust methods for the identification
techniques
for the diagnosis
and
(TCC) lack sensitivity and specificity.
of patients at high risk of progression
are
also lacking. There is therefore a need to develop novel markers for diagnostic follow-up
and
prognostic
Desorption/lonisation spectroscopy
purposes.
(SELDI)
with selective affinity capture
derived from classical chromatological PATIENTS differing
& METHODS:
stage and grade,
Surfaced
combines
Enhanced
the analytical of different
separation
power
urological
malignancies
Laser
protein chip surfaces
moieties.
healthy
controls
entities such as urinary infection, benign prostatic hypertrophy
and 40 clinical (BPH) and other
were analysed on SELDI nickel binding (IMAC3) and
anion binding (SAX2) ProteinChips.rM RESULTS:
Reproducible
differences
in protein
profiles
were seen between
urine from TCC patients and controls. At least 7 differentially were demonstrated
CONCLUSIONS: approach
These
early
results
to identify TCC biomarkers.
expression
expressed proteins
and are in the process of being identified. A 3.4 kDa marker
was identified as defensin by immunodepletion
experiments.
support
Ongoing
levels of all the individual
whilst comparing
complete
profiles using neural network software. P5
INTRODUCTION & OBJECTIVES: Genomic instability is a key factor in the multistep process of carcinogen&s. Defective MMR protein function (MMR (-) can give rise to genomic instability and has been implicated in both sporadic and hereditary colon cancer. In colorectal cancer MMR (-) turnours have a distinctive phenotype. In Transitional Cell Carcinoma (TCC) MMR loss and subsequent microsatellite instability (MSI) has been observed at varying frequencies. We investigated the expression in TCC of two MMR proteins (MSHZ and MLHI) usually responsible for MSI. MATERIAL & METHODS: 102 TCC’s were studied. All but three tumours had at least 5 years follow up (median 6 years). Sixty-nine were male and 33 were female with the mean age of 70 years old. Smoking and occupational data was known. The tumours were composed of 48 GI-2 Ta, I8 G3 Tl and 36 G2-3 T2-3. Sections from twenty-five normal ureters (from renal cell carcinoma patients) were used to establish normal urothelial expression of both MSH2 and MLHl. Five-micrometer slides from formalin fixed, paraffin embedded tumours were used. Immunohistochemistry was performed by standard methods. The primary monoclonal antibodies were Ab-I MSH2 and 1329 1A MLH I. Statistical analysis was performed by Fishers exact test, Kaplan Meier Survival curves and Log rank test. Ethics committee approval was granted. RESULTS: All normal ureteric sections showed strong expression of both proteins in all epithelial layers. Therefore, turnours were scored as having reduced expression if ~50% of cells were positive. 25 (25%) of TCC’s showed reduced MLHI or MSH2 expression. Reduced expression was strongly correlated with advanced stage (~~0.03). Differential MSH2 or MLHI expression independently predicted TCC relapse (~~0.02) for all tumours following treatment. Differential MSH2 or MLHI expression predicted recurrence of superficial TCC’s (p
the use of this proteomic
work will identify and quantify
markers
Mark’,Hamdy Freddie’
of mass
Freshly voided urine from 60 patients with TCC of 40 age-matched
Catto Jamesl. Xinarianos George ‘, Burton Julian’, Meuth
‘Institute for cancer studies, University of Sheffield, Sheffield, United Kingdom, ‘Pathology, University of Sheffield, Sheffield, United Kingdom, ?jection of Urology, University of Sheffield, Sheffield, United Kingdom
Hospital, Leeds, United Kingdom
non-invasive
78 DIFFERENTIAL EXPRESSION OF MISMATCH REPAIR PROTEINS. MLHl AND MSH2, IN TRANSITIONAL CELL CARCINOMA (TCC) OF: THE BLADDER
OF TRANSITIONAL
CONCLUSIONS:
Differential expression of MSH2 and MLHI is seen in a significant percentage of TCC’s. Reduced expression occurs at an advanced stage in TCC development. TCC’s with reduced MSH2+MLHl relapse after treatment.
expression are less likely to
NE~O-~~L~~ WC RESEAffCH Sunday, February 2411.45-13.15 hrs, Room D
80
IMPROVED SACRAL NEUROMODULATION IN THE TREATMENT OF THE HYPERACTIVE DETRUSOR: SIGNAL MODIFICATION IN AN ANIMAL MODEL Braun Peter Martin’, Seif Christophl, Cherwon Francisco’, Alken Peter’,Jtinemann Klaus-Peter* lUrology, University
University Hospital Mannheim, Hospital Kiel, Kiel, Germany
Elly’, Martinez
Mannheim,
Germany,
Portillo
*Urology,
INTRODUCTION & OBJECTIVES: A peripheral nerve evaluation test (PNE test) is carried out before the implantation of a sacral neuromodulator. The aim of the PNE test is to determine the response of the patient to this type of therapy. Published reports and our own results reveal that up to 80% of patients do not respond to PNE stimulation. We attempted to improve the efficacy of the PNE test by testing the effect of two different stimulation signals. MATERIAL & METHODS: PNE foramen electrodes were placed in S3 in 6 ITN anaesthetised GGttinger minipigs. Firstly, detrusor instabilities were induced by the intravesical instillation of formalin. A IO-minute stimulation phase with both a quasitrapezoidal (QT) signal and a rectangular signal followed. An interval of 30 min. elapsed between the two series of stimulation. The attained pressure values were registered on a urodynamic unit and evaluated as contractions and amplitudes per minute.
A NEW ANIMAL MODEL FOR PHYSIOLOGICAL INVESTIGATION OF URINARY INCONTINENCE AFTER PERIPHERAL PUDENDAL LESION Martinez Portillo Francisco JosC' , Osmonov Alken Pete?, Jiinemann Klaus-Peter1 ‘Urology, University Hospital Hospital Mannheim, Mannheim,
Kiel, Kiel, Germany
Damiar Kumbun’ , Biihler Georg’,
Germany,
*Urology,
University
INTRODUCTION & OBJECTIVES: Peripheral pudendal nerve injuries after pelvic or birth traumas lead to urine and faecal incontinence. The aim of the following study was to develop an effective microsurgical end-to-end pudendal nerve reconstruction after axotomy in rabbits. MATERIAL & METHODS: In 8 rabbits we provided the initial axotomy without nerve anastomosis (I group) after urodynamic examination of the external urethral sphincter pressure. In the II group of 8 rabbits we performed an end-to-end pudendal nerve anastomosis after axotomy. In each group the initial pressure of the external ureteral sphincter was checked after 7, 14,21,28 and 42 days postoperatively. RESULTS:
RESULTS: After formalin instillation, the average number of involuntary detrusor contractions per minute was 3.5 (iO.8) and pressure was 7.2 (+l.l). Subsequent NaCl instillation and QT stimulation reduced the contractions to 0.3 per minute (iO.3) and pressure to 0.8 (a.4) per min. Stimulation with a rectangular signal, as used in the PNE test, followed after an interval of 10 min. I .I (+O.l) contractions per min and an amplitude of 5.1 (*2.4) per min were registered with this type of stimulation. CONCLUSIONS: The acquired data clearly demonstrates that QT stimulation more effectively suppresses uncontrollable detrusor contractions in the minipig than the conventional rectangular stimulation presently applied in the PNE test. Assumably, the A-delta fibres are more effectively stimulated with QT signals. Clinical trials will verify the improved response to QT signal stimulation in PNE tests, The application of QT signal stimulation in clinical PNE tests could lead to improved diagnostics with peripheral nerve evaluation and consequently to a successful outcome in more patients with detrusor instability. European
Urology Supplements
1 (2002)
No. 1, pp. 22
CONCLUSIONS: The results of the pudendal nerve anastomosis verified recovered sphincter function, so far suggest as a reinnervation of the external ureteral muscle. Our animal model proved the possibility of nerve reconstruction by using microsurgical procedure and might be used in following investigations.
OF URINARY
CELL CARCINOMA
BIOMARKERS
WITH SELDI MASS SPECTROMETRY
Munro Nicholas, Rogers Marga, Craven Rachel, Harnden Patricia, Eardley Ian, Banks Rosamonde,
Knowles Margaret
ICRF Clinical Centre, St James University INTRODUCTION:
Current
follow up of Transitional
Cell Carcinoma
Robust methods for the identification
techniques
for the diagnosis
and
(TCC) lack sensitivity and specificity.
of patients at high risk of progression
are
also lacking. There is therefore a need to develop novel markers for diagnostic follow-up
and
prognostic
Desorption/lonisation spectroscopy
purposes.
(SELDI)
with selective affinity capture
derived from classical chromatological PATIENTS differing
& METHODS:
stage and grade,
Surfaced
combines
Enhanced
the analytical of different
separation
power
urological
malignancies
Laser
protein chip surfaces
moieties.
healthy
controls
entities such as urinary infection, benign prostatic hypertrophy
and 40 clinical (BPH) and other
were analysed on SELDI nickel binding (IMAC3) and
anion binding (SAX2) ProteinChips.rM RESULTS:
Reproducible
differences
in protein
profiles
were seen between
urine from TCC patients and controls. At least 7 differentially were demonstrated
CONCLUSIONS: approach
These
early
results
to identify TCC biomarkers.
expression
expressed proteins
and are in the process of being identified. A 3.4 kDa marker
was identified as defensin by immunodepletion
experiments.
support
Ongoing
levels of all the individual
whilst comparing
complete
profiles using neural network software. P5
INTRODUCTION & OBJECTIVES: Genomic instability is a key factor in the multistep process of carcinogen&s. Defective MMR protein function (MMR (-) can give rise to genomic instability and has been implicated in both sporadic and hereditary colon cancer. In colorectal cancer MMR (-) turnours have a distinctive phenotype. In Transitional Cell Carcinoma (TCC) MMR loss and subsequent microsatellite instability (MSI) has been observed at varying frequencies. We investigated the expression in TCC of two MMR proteins (MSHZ and MLHI) usually responsible for MSI. MATERIAL & METHODS: 102 TCC’s were studied. All but three tumours had at least 5 years follow up (median 6 years). Sixty-nine were male and 33 were female with the mean age of 70 years old. Smoking and occupational data was known. The tumours were composed of 48 GI-2 Ta, I8 G3 Tl and 36 G2-3 T2-3. Sections from twenty-five normal ureters (from renal cell carcinoma patients) were used to establish normal urothelial expression of both MSH2 and MLHl. Five-micrometer slides from formalin fixed, paraffin embedded tumours were used. Immunohistochemistry was performed by standard methods. The primary monoclonal antibodies were Ab-I MSH2 and 1329 1A MLH I. Statistical analysis was performed by Fishers exact test, Kaplan Meier Survival curves and Log rank test. Ethics committee approval was granted. RESULTS: All normal ureteric sections showed strong expression of both proteins in all epithelial layers. Therefore, turnours were scored as having reduced expression if ~50% of cells were positive. 25 (25%) of TCC’s showed reduced MLHI or MSH2 expression. Reduced expression was strongly correlated with advanced stage (~~0.03). Differential MSH2 or MLHI expression independently predicted TCC relapse (~~0.02) for all tumours following treatment. Differential MSH2 or MLHI expression predicted recurrence of superficial TCC’s (p
the use of this proteomic
work will identify and quantify
markers
Mark’,Hamdy Freddie’
of mass
Freshly voided urine from 60 patients with TCC of 40 age-matched
Catto Jamesl. Xinarianos George ‘, Burton Julian’, Meuth
‘Institute for cancer studies, University of Sheffield, Sheffield, United Kingdom, ‘Pathology, University of Sheffield, Sheffield, United Kingdom, ?jection of Urology, University of Sheffield, Sheffield, United Kingdom
Hospital, Leeds, United Kingdom
non-invasive
78 DIFFERENTIAL EXPRESSION OF MISMATCH REPAIR PROTEINS. MLHl AND MSH2, IN TRANSITIONAL CELL CARCINOMA (TCC) OF: THE BLADDER
OF TRANSITIONAL
CONCLUSIONS:
Differential expression of MSH2 and MLHI is seen in a significant percentage of TCC’s. Reduced expression occurs at an advanced stage in TCC development. TCC’s with reduced MSH2+MLHl relapse after treatment.
expression are less likely to
NE~O-~~L~~ WC RESEAffCH Sunday, February 2411.45-13.15 hrs, Room D
80
IMPROVED SACRAL NEUROMODULATION IN THE TREATMENT OF THE HYPERACTIVE DETRUSOR: SIGNAL MODIFICATION IN AN ANIMAL MODEL Braun Peter Martin’, Seif Christophl, Cherwon Francisco’, Alken Peter’,Jtinemann Klaus-Peter* lUrology, University
University Hospital Mannheim, Hospital Kiel, Kiel, Germany
Elly’, Martinez
Mannheim,
Germany,
Portillo
*Urology,
INTRODUCTION & OBJECTIVES: A peripheral nerve evaluation test (PNE test) is carried out before the implantation of a sacral neuromodulator. The aim of the PNE test is to determine the response of the patient to this type of therapy. Published reports and our own results reveal that up to 80% of patients do not respond to PNE stimulation. We attempted to improve the efficacy of the PNE test by testing the effect of two different stimulation signals. MATERIAL & METHODS: PNE foramen electrodes were placed in S3 in 6 ITN anaesthetised GGttinger minipigs. Firstly, detrusor instabilities were induced by the intravesical instillation of formalin. A IO-minute stimulation phase with both a quasitrapezoidal (QT) signal and a rectangular signal followed. An interval of 30 min. elapsed between the two series of stimulation. The attained pressure values were registered on a urodynamic unit and evaluated as contractions and amplitudes per minute.
A NEW ANIMAL MODEL FOR PHYSIOLOGICAL INVESTIGATION OF URINARY INCONTINENCE AFTER PERIPHERAL PUDENDAL LESION Martinez Portillo Francisco JosC' , Osmonov Alken Pete?, Jiinemann Klaus-Peter1 ‘Urology, University Hospital Hospital Mannheim, Mannheim,
Kiel, Kiel, Germany
Damiar Kumbun’ , Biihler Georg’,
Germany,
*Urology,
University
INTRODUCTION & OBJECTIVES: Peripheral pudendal nerve injuries after pelvic or birth traumas lead to urine and faecal incontinence. The aim of the following study was to develop an effective microsurgical end-to-end pudendal nerve reconstruction after axotomy in rabbits. MATERIAL & METHODS: In 8 rabbits we provided the initial axotomy without nerve anastomosis (I group) after urodynamic examination of the external urethral sphincter pressure. In the II group of 8 rabbits we performed an end-to-end pudendal nerve anastomosis after axotomy. In each group the initial pressure of the external ureteral sphincter was checked after 7, 14,21,28 and 42 days postoperatively. RESULTS:
RESULTS: After formalin instillation, the average number of involuntary detrusor contractions per minute was 3.5 (iO.8) and pressure was 7.2 (+l.l). Subsequent NaCl instillation and QT stimulation reduced the contractions to 0.3 per minute (iO.3) and pressure to 0.8 (a.4) per min. Stimulation with a rectangular signal, as used in the PNE test, followed after an interval of 10 min. I .I (+O.l) contractions per min and an amplitude of 5.1 (*2.4) per min were registered with this type of stimulation. CONCLUSIONS: The acquired data clearly demonstrates that QT stimulation more effectively suppresses uncontrollable detrusor contractions in the minipig than the conventional rectangular stimulation presently applied in the PNE test. Assumably, the A-delta fibres are more effectively stimulated with QT signals. Clinical trials will verify the improved response to QT signal stimulation in PNE tests, The application of QT signal stimulation in clinical PNE tests could lead to improved diagnostics with peripheral nerve evaluation and consequently to a successful outcome in more patients with detrusor instability. European
Urology Supplements
1 (2002)
No. 1, pp. 22
CONCLUSIONS: The results of the pudendal nerve anastomosis verified recovered sphincter function, so far suggest as a reinnervation of the external ureteral muscle. Our animal model proved the possibility of nerve reconstruction by using microsurgical procedure and might be used in following investigations.