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URINARY SURVIVIN IS A BIOMARKER FOR THE DIAGNOSIS OF INVASIVE BLADDER CANCER
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366
MICROSATELLITE INSTABILITY AS PREDICTOR OF SURVIVAL IN PATIENTS WITH INVASIVE UPPER URINARY TRACT TRANSITIONAL CELL CARCINOMA
URINARY SURVIVIN IS A BIOMARKER FOR THE DIAGNOSIS OF INVASIVE BLADDER CANCER
Rouprêt M.1, Fromont G.2, Cancel-Tassin G.3, Azzouzi A.R.4, Catto J.W.5, Hamdy F.C.5, Vallancien G.6, Richard F.7, Cussenot O.8 Hôpital Pitié Salpétrière, Urology, Paris, France, 2Montsouris Institute, Pathology, Paris, France, 3CeRePP-EA 3104, Genetic Research, Paris, France, 4CeRePP-EA 3104, Urology, Angers, France, 5Royal Hallamshire Hospital, Urology, Sheffield, United Kingdom, 6Montsouris Institute, Urology, Paris, France, 7Pitié, Urology, Paris, France, CeRePP-EA 3104, Urology, Paris, France 1
INTRODUCTION & OBJECTIVES: Invasive upper urinary tract transitional cell carcinoma (UUT-TCC) has a poor prognosis (overall survival (OS) <50% at 5 years). Our purpose was to establish whether high microsatellite instability (MSI) (present in almost 20% of cases) and loss of MSH2 protein expression (sometimes used to predict MSI status) are prognostic factors of OS. MATERIAL & METHODS: The files of 80 patients who underwent nephroureterectomy for invasive UUT-TCC(³pT2) between 1990 and 2002 were reviewed. The following data were collated: age at diagnosis, prior history of cancer, tobacco consumption, tumour stage and grade, and disease progression. MSI was determined by polymerase chain reaction/fragment analysis and MSH2 protein expression by immunohistochemistry on retrieved tumour tissue. RESULTS: Median age was 71.5 years. The male-to-female ratio was 2.8.High MSI and loss of MSH2 expression were encountered in the tumours of 14/80 (17%) and 21/80 (26%) patients, respectively. High MSI was significantly associated with patients with a better prognosis (T2/T3/N0-M0) (p=0.02). Mean OS was 22.5 ±18 months (range 6-78). In univariate analyses, age, stage, tumour grade, high MSI and loss of MSH2 expression were related to better OS (37 ±22 months, p=0.003; 34 ±22 months, p=0.02). Only stage, age and high MSI were prognostic factors in a multivariate analysis (p < 0.05). CONCLUSIONS: MSI and expression of MSH2 are useful prognostic factors in invasive UUT-TCC but, besides age stage, only MSI is an independent factor. High MSI indicates a better prognosis especially in patients under 71 with T2/T3/N0-M0 tumours.
Campos-Fernandes J.L.1, Descotes F.2, Decaussin M.3, André J.2, Paparel P.4, Collin-chavagnac D.2, Boisson R.C.2, Perrin P.4, Devonec M.4, Ruffion A.4 1 Hospices Civils de Lyon, Urology Lyon Sud, Pierre Bénite, France, 2Hospices Civils de Lyon, Biologie des Tumeurs, Lyons, France, 3Hospices Civils de Lyon, Anatomo-pathologie Lyon-Sud, Lyons, France, 4Hospices Civils de Lyon, Urology Lyon Sud, Lyons, France
INTRODUCTION & OBJECTIVES: Survivin is a 16.5 kDa protein involved in the inhibition of apoptosis and cell division. Survivin is expressed in embryonic tissues but not in normal adult tissues. Survivin expression has been reported in human cancers associated with clinical tumour progression. The aim of this study was to assess the diagnosis performance of urinary survivin in patients with a known diagnosis of bladder urothelial tumour. MATERIAL & METHODS: This is a monocentric prospective study. From 05/2004 to 09/2005, the urines of 65 patients with bladder tumours were collected at the time of the diagnosis. Immediately after collection, the urines were centrifuged and the supernatant was aliquoted and stored at -80°C until use. Survivin was measured by the enzyme immunometric assay kit (TiterZyme™EIA, Gentaur). Since the normalisation was required, the results were expressed in ng/mmol of urinary creatinin. RESULTS: Three groups of patients were compared: pTa G1-G2 (n=36), pTa-T1 G3 (n=21) and > pT1 (n=8). In 12 patients, no level of survivin was detectable (4 pTa G1-G2 (11%), 7 pTa-T1 G3 (33%) and 1 > pT1 (13%)). The results are shown in figure 1 for the remaining patients. Only two of the 32 patients (6%) in the group pTa G1-G2 had a urinary survivin level higher than 20 ng/mmol whereas 5 on 7 patients (71%) with an invasive carcinoma had a level higher to this value.
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CONCLUSIONS: Urinary survivin was significantly higher in patients with invasive carcinoma. Our data indicate that a high urinary survivin level could be related to the urothelial tumour invasion. These data need to be prospectively confirmed on a higher number of patients.
367 DNA METHYLATION ALTERATIONS IN BLADDER CANCER Neuhausen A., Florl A.R., Grimm M.O., Schulz W.A. Heinrich-heine University, Department of Urology, Düsseldorf, Germany INTRODUCTION & OBJECTIVES: Detection of altered DNA methylation appears to be highly useful for the detection and classification of many cancer types, e.g. prostate carcinoma. In bladder cancer, several genes have been reported to be hypermethylated at high frequency and global hypomethylation to be widespread. However, until now, hypermethylation and hypomethylation have never been investigated in the same set of tumours. MATERIAL & METHODS: High molecular weight DNA was extracted from macrodissected carcinoma tissues (5 ≤pT1, 19 pT2, 36 pT3, 12 pT4) from 72 patients treated by cystectomy, for whom a follow-up (average 70 months) was available. Hypermethylation in 6 genes (APC, CDKN2A/p16, DAPK, RARB2, RASSF1A, SFRP1) was investigated by methylation-specific PCR. Global hypomethylation was quantitatively determined by Southern blot analysis of LINE-1 sequences. RESULTS: Overall frequencies of hypermethylation were APC: 49%, CDKN2A/p16: 3% (plus 16% deletions), DAPK: 32%, RARB2: 18%, RASSF1A: 39%, SFRP1: 63%. Significant global hypomethylation was found in 87%. RARB2 hypermethylation was highly significantly (p<0.001) related to tumour stage, and in particular to lymph node involvement. After correction for multiple testing, none of the other hypermethylation events was associated with increased tumour stage or grade, patient age, gender, or survival. DNA hypomethylation tended to increase with stage, and the minority of cases lacking this change had longer recurrence-free and tumour-specific survival. CONCLUSIONS: DNA methylation alterations in bladder cancer appear to differ fundamentally from those in prostate carcinoma where several genes are co-ordinately and consistently hypermethylated. Detection of bladder cancer by hypermethylation assays will therefore have to rely on several genes including APC, RASSF1A, and SFRP1. Hypomethylation seems the most promising marker for bladder cancer detection, but its use in clinical practice presupposes the development of simpler assays. The extent of hypomethylation and individual hypermethylation events like that of RARB2 could be useful for prognostic purposes.
Eur Urol Suppl 2006;5(2):114
368 GENETIC PROFILING OF UROTHELIAL CARCINOMAS OF THE UPPER URINARY TRACT Junker K.1, Jentsch B.1, Stöhr R.2, Burger M.2, Hartmann A.3, Schubert J.1 Friedrich Schiller University, Department of Urology, Jena, Germany, 2University Regensburg, Department of Urology, Regensburg, Germany, 3University Regensburg, Institute of Pathology, Regensburg, Germany
1
INTRODUCTION & OBJECTIVES: Urothelial carcinomas (UC) of the upper urinary tract (UUT) do not differ morphologically from that of the bladder. Therefore, a similar pathological pathway is supposed. In contrast to bladder cancer only a little is known about genetic alterations in urothelial tumours of the UUT. In earlier studies it was shown, that tumours of the UUT show frequently microsatellite instability (MSI) and can arise as consequence of the HNPCC syndrome. The aim of this study was to define specific genetic alterations of UUT tumours compared to bladder cancer. Furthermore, genetic alterations were compared between tumours with and without MSI. MATERIAL & METHODS: DNA was isolated after microdissection from 38 UC of the renal pelvis and ureter with known MSI status. Comparative genomic hybridisation (CGH) was performed according to standard protocols. Briefly, DOP-PCR was carried out to amplify the whole genome DNA. Normaland tumour DNA was labelled with digoxigenin-dUTP and biotin-dUTP, respectively in a second PCR. After detection of fluorescence signals using avidin-FITC and anti-digoxigenin-Rhodamine, 15 metaphases were analysed in each case using a computer system from Metasystems. RESULTS: Genetic alterations were detected in 23 of 38 tumours. The mean number of alterations per tumour was 5.7. Most frequently, losses of chromosomes 9 (56%), 8p (21%), 10 (21%), 11 (30%) and gains of chromosomes 8q (43%), 5 (26%), 6 (26%), 7 (26%) occurred. In tumours with MSI, more alterations per tumour were detected (6.1 vs. 5.3). Losses of chromosome 9 regions were found more often in tumours without MSI (69% vs. 50%). In addition, gain of chromosomes 3 and 7 and loss of chromosome 11 were detected more frequently in these tumours. Tumours with MSI showed more often losses of chromosomes 3 and 13 as well as gains of chromosomes 6 and 8q. CONCLUSIONS: Tumours of the UUT represent similar genetic alterations compared to bladder tumours. However, frequency of genetic alterations differed between tumours with and without MSI, although typical alterations of urothelial tumours like loss of chromosomes 9 and 8p as well as gains of 8q occurred in both groups. These data raise the possibility that tumours of the UUT with a mutator phenotype develop through a different carcinogenic pathway than microsatellite-stable urothelial carcinomas of the urinary tract.
366
MICROSATELLITE INSTABILITY AS PREDICTOR OF SURVIVAL IN PATIENTS WITH INVASIVE UPPER URINARY TRACT TRANSITIONAL CELL CARCINOMA
URINARY SURVIVIN IS A BIOMARKER FOR THE DIAGNOSIS OF INVASIVE BLADDER CANCER
Rouprêt M.1, Fromont G.2, Cancel-Tassin G.3, Azzouzi A.R.4, Catto J.W.5, Hamdy F.C.5, Vallancien G.6, Richard F.7, Cussenot O.8 Hôpital Pitié Salpétrière, Urology, Paris, France, 2Montsouris Institute, Pathology, Paris, France, 3CeRePP-EA 3104, Genetic Research, Paris, France, 4CeRePP-EA 3104, Urology, Angers, France, 5Royal Hallamshire Hospital, Urology, Sheffield, United Kingdom, 6Montsouris Institute, Urology, Paris, France, 7Pitié, Urology, Paris, France, CeRePP-EA 3104, Urology, Paris, France 1
INTRODUCTION & OBJECTIVES: Invasive upper urinary tract transitional cell carcinoma (UUT-TCC) has a poor prognosis (overall survival (OS) <50% at 5 years). Our purpose was to establish whether high microsatellite instability (MSI) (present in almost 20% of cases) and loss of MSH2 protein expression (sometimes used to predict MSI status) are prognostic factors of OS. MATERIAL & METHODS: The files of 80 patients who underwent nephroureterectomy for invasive UUT-TCC(³pT2) between 1990 and 2002 were reviewed. The following data were collated: age at diagnosis, prior history of cancer, tobacco consumption, tumour stage and grade, and disease progression. MSI was determined by polymerase chain reaction/fragment analysis and MSH2 protein expression by immunohistochemistry on retrieved tumour tissue. RESULTS: Median age was 71.5 years. The male-to-female ratio was 2.8.High MSI and loss of MSH2 expression were encountered in the tumours of 14/80 (17%) and 21/80 (26%) patients, respectively. High MSI was significantly associated with patients with a better prognosis (T2/T3/N0-M0) (p=0.02). Mean OS was 22.5 ±18 months (range 6-78). In univariate analyses, age, stage, tumour grade, high MSI and loss of MSH2 expression were related to better OS (37 ±22 months, p=0.003; 34 ±22 months, p=0.02). Only stage, age and high MSI were prognostic factors in a multivariate analysis (p < 0.05). CONCLUSIONS: MSI and expression of MSH2 are useful prognostic factors in invasive UUT-TCC but, besides age stage, only MSI is an independent factor. High MSI indicates a better prognosis especially in patients under 71 with T2/T3/N0-M0 tumours.
Campos-Fernandes J.L.1, Descotes F.2, Decaussin M.3, André J.2, Paparel P.4, Collin-chavagnac D.2, Boisson R.C.2, Perrin P.4, Devonec M.4, Ruffion A.4 1 Hospices Civils de Lyon, Urology Lyon Sud, Pierre Bénite, France, 2Hospices Civils de Lyon, Biologie des Tumeurs, Lyons, France, 3Hospices Civils de Lyon, Anatomo-pathologie Lyon-Sud, Lyons, France, 4Hospices Civils de Lyon, Urology Lyon Sud, Lyons, France
INTRODUCTION & OBJECTIVES: Survivin is a 16.5 kDa protein involved in the inhibition of apoptosis and cell division. Survivin is expressed in embryonic tissues but not in normal adult tissues. Survivin expression has been reported in human cancers associated with clinical tumour progression. The aim of this study was to assess the diagnosis performance of urinary survivin in patients with a known diagnosis of bladder urothelial tumour. MATERIAL & METHODS: This is a monocentric prospective study. From 05/2004 to 09/2005, the urines of 65 patients with bladder tumours were collected at the time of the diagnosis. Immediately after collection, the urines were centrifuged and the supernatant was aliquoted and stored at -80°C until use. Survivin was measured by the enzyme immunometric assay kit (TiterZyme™EIA, Gentaur). Since the normalisation was required, the results were expressed in ng/mmol of urinary creatinin. RESULTS: Three groups of patients were compared: pTa G1-G2 (n=36), pTa-T1 G3 (n=21) and > pT1 (n=8). In 12 patients, no level of survivin was detectable (4 pTa G1-G2 (11%), 7 pTa-T1 G3 (33%) and 1 > pT1 (13%)). The results are shown in figure 1 for the remaining patients. Only two of the 32 patients (6%) in the group pTa G1-G2 had a urinary survivin level higher than 20 ng/mmol whereas 5 on 7 patients (71%) with an invasive carcinoma had a level higher to this value.
.
CONCLUSIONS: Urinary survivin was significantly higher in patients with invasive carcinoma. Our data indicate that a high urinary survivin level could be related to the urothelial tumour invasion. These data need to be prospectively confirmed on a higher number of patients.
367 DNA METHYLATION ALTERATIONS IN BLADDER CANCER Neuhausen A., Florl A.R., Grimm M.O., Schulz W.A. Heinrich-heine University, Department of Urology, Düsseldorf, Germany INTRODUCTION & OBJECTIVES: Detection of altered DNA methylation appears to be highly useful for the detection and classification of many cancer types, e.g. prostate carcinoma. In bladder cancer, several genes have been reported to be hypermethylated at high frequency and global hypomethylation to be widespread. However, until now, hypermethylation and hypomethylation have never been investigated in the same set of tumours. MATERIAL & METHODS: High molecular weight DNA was extracted from macrodissected carcinoma tissues (5 ≤pT1, 19 pT2, 36 pT3, 12 pT4) from 72 patients treated by cystectomy, for whom a follow-up (average 70 months) was available. Hypermethylation in 6 genes (APC, CDKN2A/p16, DAPK, RARB2, RASSF1A, SFRP1) was investigated by methylation-specific PCR. Global hypomethylation was quantitatively determined by Southern blot analysis of LINE-1 sequences. RESULTS: Overall frequencies of hypermethylation were APC: 49%, CDKN2A/p16: 3% (plus 16% deletions), DAPK: 32%, RARB2: 18%, RASSF1A: 39%, SFRP1: 63%. Significant global hypomethylation was found in 87%. RARB2 hypermethylation was highly significantly (p<0.001) related to tumour stage, and in particular to lymph node involvement. After correction for multiple testing, none of the other hypermethylation events was associated with increased tumour stage or grade, patient age, gender, or survival. DNA hypomethylation tended to increase with stage, and the minority of cases lacking this change had longer recurrence-free and tumour-specific survival. CONCLUSIONS: DNA methylation alterations in bladder cancer appear to differ fundamentally from those in prostate carcinoma where several genes are co-ordinately and consistently hypermethylated. Detection of bladder cancer by hypermethylation assays will therefore have to rely on several genes including APC, RASSF1A, and SFRP1. Hypomethylation seems the most promising marker for bladder cancer detection, but its use in clinical practice presupposes the development of simpler assays. The extent of hypomethylation and individual hypermethylation events like that of RARB2 could be useful for prognostic purposes.
Eur Urol Suppl 2006;5(2):114
368 GENETIC PROFILING OF UROTHELIAL CARCINOMAS OF THE UPPER URINARY TRACT Junker K.1, Jentsch B.1, Stöhr R.2, Burger M.2, Hartmann A.3, Schubert J.1 Friedrich Schiller University, Department of Urology, Jena, Germany, 2University Regensburg, Department of Urology, Regensburg, Germany, 3University Regensburg, Institute of Pathology, Regensburg, Germany
1
INTRODUCTION & OBJECTIVES: Urothelial carcinomas (UC) of the upper urinary tract (UUT) do not differ morphologically from that of the bladder. Therefore, a similar pathological pathway is supposed. In contrast to bladder cancer only a little is known about genetic alterations in urothelial tumours of the UUT. In earlier studies it was shown, that tumours of the UUT show frequently microsatellite instability (MSI) and can arise as consequence of the HNPCC syndrome. The aim of this study was to define specific genetic alterations of UUT tumours compared to bladder cancer. Furthermore, genetic alterations were compared between tumours with and without MSI. MATERIAL & METHODS: DNA was isolated after microdissection from 38 UC of the renal pelvis and ureter with known MSI status. Comparative genomic hybridisation (CGH) was performed according to standard protocols. Briefly, DOP-PCR was carried out to amplify the whole genome DNA. Normaland tumour DNA was labelled with digoxigenin-dUTP and biotin-dUTP, respectively in a second PCR. After detection of fluorescence signals using avidin-FITC and anti-digoxigenin-Rhodamine, 15 metaphases were analysed in each case using a computer system from Metasystems. RESULTS: Genetic alterations were detected in 23 of 38 tumours. The mean number of alterations per tumour was 5.7. Most frequently, losses of chromosomes 9 (56%), 8p (21%), 10 (21%), 11 (30%) and gains of chromosomes 8q (43%), 5 (26%), 6 (26%), 7 (26%) occurred. In tumours with MSI, more alterations per tumour were detected (6.1 vs. 5.3). Losses of chromosome 9 regions were found more often in tumours without MSI (69% vs. 50%). In addition, gain of chromosomes 3 and 7 and loss of chromosome 11 were detected more frequently in these tumours. Tumours with MSI showed more often losses of chromosomes 3 and 13 as well as gains of chromosomes 6 and 8q. CONCLUSIONS: Tumours of the UUT represent similar genetic alterations compared to bladder tumours. However, frequency of genetic alterations differed between tumours with and without MSI, although typical alterations of urothelial tumours like loss of chromosomes 9 and 8p as well as gains of 8q occurred in both groups. These data raise the possibility that tumours of the UUT with a mutator phenotype develop through a different carcinogenic pathway than microsatellite-stable urothelial carcinomas of the urinary tract.