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493 THE FUNCTION OF FOS-RELATED ANTIGEN-1 (FRA-1) IN TRANSITIONAL CELL CARCINOMA (TCC) OF THE BLADDER
The function of Fos-related antigen-1 (Fra-1) in Transitional Cell Carcinoma (TCC) of the Bladder
493
Vickery R., Richard S., Eugene T., Kilian M. University of Leicester, Urology Group, Department of Cancer Studies and Molecular Medicine, Leicester, United Kingdom Introduction & Objectives: In England and Wales, over 5,000 patients die from bladder cancer per annum. Despite treatment, the 5-year survival rate of organconfined, muscle-invasive bladder cancer is still poor at approximately 40%. Accordingly, there is great interest in developing novel treatments to improve survival rates and quality of life. Fra-1, a member of the Fos family of proteins, is a component of the Activator Protein-1 (AP-1) transcription factor complex and has been implicated in several human cancers. Recently, an anti-Fra-1 DNA vaccine was successfully used to inhibit growth and metastasis of breast and lung cancers in a mouse model. We have previously demonstrated, using immunohistochemistry, that Fra-1 is present in the majority of muscle-invasive bladder cancers. The objectives of this study were i) to correlate Fra-1 levels with cell phenotype in a panel of bladder cancer cell lines and ii) to determine the effect of modulating Fra-1 levels on phenotype and cell motility Material & Methods: Expression of Fra-1, vimentin, and E-cadherin was assessed in HT1376, J82, RT112, RT4, T24, and UMUC3 cell lines using Western Blot analysis. Cell motility was assessed using wound healing assays. Expression of Fra-1 was experimentally modulated in bladder cancer cell lines by transfection of cells with an expression vector coding a stabilised form of Fra-1 (HA-Fra-1Δ3) or with si-RNA to Fra-1. The effects were analysed using immunocytostaining, Western Blot analysis, and wound healing assays. Results: J82, T24, and UMUC3 cells were positive for Fra-1 and demonstrated mesenchymal cell phenotype and increased cell motility. Knock-down of Fra-1 resulted in a change in phenotype with increased stress fibre formation, and decreased cell motility. Conversely, enforced Fra-1 expression resulted in a loss of stress fibres and enhanced cell motility. Conclusions: Fra-1 has a significant role in bladder cancer, acting as a regulator of phenotype and cell motility. As such, Fra-1 is a potential novel target in the treatment of bladder cancer patients. Si-RNA is an efficacious method of Fra-1 inhibition and may form the basis of a potential future therapeutic approach.
495
Prognostic implications of lymphangiogenesis in invasive transitional cell carcinoma of the bladder
Fernández M.1, Bolenz C.1, Trojan L.1, Steidler A.1, Weiss C.2, Alken P.1, Grobholz R.3, Michel M.S.1
494
Detection and molecular staging of bladder neoplasia based on peripheral blood expression of collagenases (mmp-2, mmp-9) Pascual-Mateo C., Ferruelo A., Rodriguez-Barbero J., Berenguer A., Angulo J. Hospital Getafe, Urology, Madrid, Spain
Introduction & Objectives: Molecular staging of bladder cancer based on the detection of specific genes mRNA has been inconclusive. We have evaluated the relevance of the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in peripheral blood both to segregate controls and patients with bladder cancer and to characterize bladder neoplasms. Material & Methods: Total RNA was extracted from circulating blood cells in a series of 42 individuals (11 healthy controls and 31 patients with bladder cancer) treated in our institution. Real time RT-PCR was performed using specific primers for MMP-2, MMP-9 and ribosomal 18S. The quatification value of MMP-2 and MMP-9 mRNA was described as each value relative to 18S mRNA (DDCt method) and results were compared with data obtained from conventional pathologic examination and staging. ELISA was also performed to measure the level of MMP-2 and MMP-9 proteins in the serum. Endpoint of the study was to evaluate the ability of each marker to predict presence of bladder cancer and assess clinical stage of the tumour. Results: Mean MMP-2 protein level was similar among healthy controls and patients, but significantly higher in infiltrating tumours (T2-T4) than in superficial disease (Ta-T1) (169.5 vs. 196.68 ng/ml, p=0.05). On the other hand, MMP-9 was higher in patients with cancer than controls (2.62 vs 13.73 ng/ml, p<0.05), and slightly higher in infiltrating disease compared with superficial lesions (16.19 vs 11.10 ng/ml, p=0.15). Normalized levels of mRNA MMP-2 and MMP-9 were respectively 2.7+0.6 and 1.82+0.6 times higher in patients with cancer than controls (p<0.05). Regarding clinical stage, levels of MMP-2 were significantly higher in T4 (8.44+2 times), T3 (7.7+1.6 times), T2 (4.45+1.1) and T1 (2.2+0.5) with respects to Ta. MMP-2 was significantly higher in patients with T4 (13.6+3.3 times) and T3 (2.3+0.5) lesions, but neither with T2 (1.15+0.25) nor T1 (1.43+0.2) when compared to those with noninvasive Ta. Among patients with bladder neoplasia a significant increase was observed in the expression of MMP-2 (5.22+0.9 times) and MMP-9 (9.6+1.3) when comparing metastatic and non-metastatic disease. Conclusions: Both non-invasive bladder tumour recognition and molecular staging of bladder cancer can be possible with the study of expression of collagenases MMP-2 and MMP-9. Real time RT-PCR based detection of mRNA in circulating blood appears very promising. The ability to distinguish metastatic disease is higher for MMP-9 while MMP-2 discriminates better all levels of tumour invasion.
496
Relevance of urinary interferon-gamma in the prognosis of superficial bladder cancer treated with BCG immunotherapy Zoumpos I.1, Touloupidis S.2, Giakoumelos A.1, Galaktidou T.3, Kortsaris A.4, Simopoulos K.5, Katsikas V.1
Aristotle University of Thessaloniki, 2nd Dept. Urology, Thessaloniki, Greece, 2Democritus Thrace University, Dept. Urology, Alexandroupolis, Greece, 3Theageneion Hospital, Dept. Virology, Thessaloniki, Greece, 4Democritus Thrace University, Dept. Biochemistry, Alexandroupolis, Greece, 5Democritus Thrace University, 2nd Dept. Surgery, Alexandroupolis, Greece
University Hospital Mannheim, Department of Urology, Mannheim, Germany, University Hospital Mannheim, Department of Medical Statistics, Mannheim, Germany, 3University Hospital Mannheim, Department of Pathology, Mannheim, Germany
1
Introduction & Objectives: Discovery of lymphatic endothelial cell (LEC) markers has recently focused attention on lymphangiogenesis in cancer research. The potential role of lymph vessel density (LVD) as prognostic factor as well as the functional significance of tumoral lymph vessels has not been investigated in invasive bladder transitional cell carcinoma (TCC) to date. Aim was to quantify LVD and to evaluate association of lymphatics with clinicopathological parameters and survival in bladder TCC. Furthermore, proliferation of LEC’s was evaluated in order to assess the functional significance of tumoral lymph vessels.
Introduction & Objectives: Interferon gamma, a cytokine produced by activated T-lymphocytes and natural killer cells, is regarded by many researchers as a good marker of local immune (Th1) activation of the bladder wall after intravesical instillations of BCG. Measurement of the soluble fraction of interferon-gamma in the urine can therefore provide indirect evidence of immune response that is critical for the effect of BCG to be exerted. The present study, investigated the use of soluble interferon-gamma in the urine as a possible prognostic marker for BCG-therapy of superficial bladder cancer.
1
2
Material & Methods: Data of 108 patients with muscle-invasive bladder TCC who underwent radical cystectomy from 1998 to 2005 were reviewed retrospectively. Sections were analyzed immunohistochemically by D2-40, a lymph vessel specific endothelial marker. Lymph vessel count was performed in 3 areas: intratumoral, peritumoral and normal tissue. Proliferating LEC’s were assessed with doublestaining for D2-40 and Ki-67. Results: Peritumoral lymph vessels were observed in 105 sections (97,2%), identification of intratumoral vessels was possible in 65 specimens (60,2%). Doublestaining clearly distinguished D2-40-positive lymph vessels from neighboring D2-40-negative blood vessels, which in turn were stained by the endothelial marker CD-34. Peritumoral LVD (PTLVD) (mean 9,25 +/- 5,66) was significantly higher than intratumoral LVD (ITLVD) (mean 3,51 +/- 5,1; p<0,0001) as well as than in normal tissue (mean 6,51 +/- 4,58; p<0,0001). Higher intratumoral LVD (ITLVD) correlated significantly with poor histological differentiation (p=0,01). Higher PTLVD had also an association with higher histological grade (p=0,06) in addition to a significant association with presence of lymph node metastasis (p=0,0004). Higher pathological T stage (p<0,0001), a poorer histological grade (p=0,04) and the presence of lymph node metastasis (p<0,0001) were the only significant prognostic factors for reduced disease-specific survival. LVD had no significant influence on survival. Intratumoral and peritumoral vessels showed proliferating LEC’s in all examined samples in a variable proportion. Conclusions: Lymphangiogenesis occurs in invasive bladder TCC. To our knowledge, our study is the first to investigate and confirm a strong correlation of higher PTLVD with presence of lymph nodes in clinically localized invasive bladder TCC. According to these results, an increased PTLVD may facilitate dissemination to locoregional lymph nodes. The present study is also the first to suggest the existence of proliferating lymph vessels in bladder TCC. The potential implementation of lymphangiogenesis as a prognostic factor as well as a therapeutical target, as already established for angiogenesis, requires further studies in order to determine if our information will contribute to improve outcomes in bladder TCC.
Eur Urol Suppl 2007;6(2):146
Material & Methods: 51 patients with newly diagnosed superficial bladder cancer were included in the study between 2000 and 2004. Mean age of the patients was 65.5 years and median follow-up was 43 weeks (range: 11-108 years). Patients received 6-weekly instillations of BCG (Tice-strain, 5x108 CFU). A urinary ELISA kit (CytELISA human IFNg, Cytimmune, AMS Biotechnology, Oxon, UK) was utilized for IFN-gamma measurement 6 hours post-instillation. Following completion of the induction cycle of BCG patients were subjected to cystoscopy and cytology. Statistical analysis included logistic regression analysis using SPSS software. Results: Urinary levels of interferon-gamma according to response to treatment are presented in Table 1. Table 1. Response
Mean value (pg/ml)
SD
p
IFN-γ week 1
Yes No
11,5127 14,5232
24,17847 34,80789
0,720
IFN-γ week 2
Yes No
13,5985 10,1888
21,68320 19,46929
0,558
IFN-γ week 3
Yes No
14,5088 6,2236
21,25360 9,57764
0,079
IFN-γ week 4
Yes No
18,3269 10,4504
22,30716 15,98589
0,153
IFN-γ week 5
Yes No
27,7192 9,5308
33,07618 18,42786
0,019*
IFN-γ week 6
Yes No
36,4638 6,1608
41,88771 10,90515
0,001*
There was a statistically significant difference in urinary interferon-gamma in favor of responders at the 5th and 6th instillation (p<0,05). Comparing urinary interferon-gamma levels by stage and grade resulted in no significant differences. Regression analysis showed that with an accuracy of 70,6%, urinary IFN-gamma levels at the 6th instillation can predict response to treatment. Conclusions: Urinary levels of soluble interferon-gamma can help predict the outcome of intravesical BCG instillations. More important is the 6th instillation, the last in the induction cycle. Measurement of IFN-gamma in the urine is feasible, utilizing a simple ELISA kit that is commercially available in undialyzed urine without correction to creatinine clearance. The combination of this marker of the local immune response with other tumor markers may further increase the yield of its utility in bladder cancer.
493
Vickery R., Richard S., Eugene T., Kilian M. University of Leicester, Urology Group, Department of Cancer Studies and Molecular Medicine, Leicester, United Kingdom Introduction & Objectives: In England and Wales, over 5,000 patients die from bladder cancer per annum. Despite treatment, the 5-year survival rate of organconfined, muscle-invasive bladder cancer is still poor at approximately 40%. Accordingly, there is great interest in developing novel treatments to improve survival rates and quality of life. Fra-1, a member of the Fos family of proteins, is a component of the Activator Protein-1 (AP-1) transcription factor complex and has been implicated in several human cancers. Recently, an anti-Fra-1 DNA vaccine was successfully used to inhibit growth and metastasis of breast and lung cancers in a mouse model. We have previously demonstrated, using immunohistochemistry, that Fra-1 is present in the majority of muscle-invasive bladder cancers. The objectives of this study were i) to correlate Fra-1 levels with cell phenotype in a panel of bladder cancer cell lines and ii) to determine the effect of modulating Fra-1 levels on phenotype and cell motility Material & Methods: Expression of Fra-1, vimentin, and E-cadherin was assessed in HT1376, J82, RT112, RT4, T24, and UMUC3 cell lines using Western Blot analysis. Cell motility was assessed using wound healing assays. Expression of Fra-1 was experimentally modulated in bladder cancer cell lines by transfection of cells with an expression vector coding a stabilised form of Fra-1 (HA-Fra-1Δ3) or with si-RNA to Fra-1. The effects were analysed using immunocytostaining, Western Blot analysis, and wound healing assays. Results: J82, T24, and UMUC3 cells were positive for Fra-1 and demonstrated mesenchymal cell phenotype and increased cell motility. Knock-down of Fra-1 resulted in a change in phenotype with increased stress fibre formation, and decreased cell motility. Conversely, enforced Fra-1 expression resulted in a loss of stress fibres and enhanced cell motility. Conclusions: Fra-1 has a significant role in bladder cancer, acting as a regulator of phenotype and cell motility. As such, Fra-1 is a potential novel target in the treatment of bladder cancer patients. Si-RNA is an efficacious method of Fra-1 inhibition and may form the basis of a potential future therapeutic approach.
495
Prognostic implications of lymphangiogenesis in invasive transitional cell carcinoma of the bladder
Fernández M.1, Bolenz C.1, Trojan L.1, Steidler A.1, Weiss C.2, Alken P.1, Grobholz R.3, Michel M.S.1
494
Detection and molecular staging of bladder neoplasia based on peripheral blood expression of collagenases (mmp-2, mmp-9) Pascual-Mateo C., Ferruelo A., Rodriguez-Barbero J., Berenguer A., Angulo J. Hospital Getafe, Urology, Madrid, Spain
Introduction & Objectives: Molecular staging of bladder cancer based on the detection of specific genes mRNA has been inconclusive. We have evaluated the relevance of the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in peripheral blood both to segregate controls and patients with bladder cancer and to characterize bladder neoplasms. Material & Methods: Total RNA was extracted from circulating blood cells in a series of 42 individuals (11 healthy controls and 31 patients with bladder cancer) treated in our institution. Real time RT-PCR was performed using specific primers for MMP-2, MMP-9 and ribosomal 18S. The quatification value of MMP-2 and MMP-9 mRNA was described as each value relative to 18S mRNA (DDCt method) and results were compared with data obtained from conventional pathologic examination and staging. ELISA was also performed to measure the level of MMP-2 and MMP-9 proteins in the serum. Endpoint of the study was to evaluate the ability of each marker to predict presence of bladder cancer and assess clinical stage of the tumour. Results: Mean MMP-2 protein level was similar among healthy controls and patients, but significantly higher in infiltrating tumours (T2-T4) than in superficial disease (Ta-T1) (169.5 vs. 196.68 ng/ml, p=0.05). On the other hand, MMP-9 was higher in patients with cancer than controls (2.62 vs 13.73 ng/ml, p<0.05), and slightly higher in infiltrating disease compared with superficial lesions (16.19 vs 11.10 ng/ml, p=0.15). Normalized levels of mRNA MMP-2 and MMP-9 were respectively 2.7+0.6 and 1.82+0.6 times higher in patients with cancer than controls (p<0.05). Regarding clinical stage, levels of MMP-2 were significantly higher in T4 (8.44+2 times), T3 (7.7+1.6 times), T2 (4.45+1.1) and T1 (2.2+0.5) with respects to Ta. MMP-2 was significantly higher in patients with T4 (13.6+3.3 times) and T3 (2.3+0.5) lesions, but neither with T2 (1.15+0.25) nor T1 (1.43+0.2) when compared to those with noninvasive Ta. Among patients with bladder neoplasia a significant increase was observed in the expression of MMP-2 (5.22+0.9 times) and MMP-9 (9.6+1.3) when comparing metastatic and non-metastatic disease. Conclusions: Both non-invasive bladder tumour recognition and molecular staging of bladder cancer can be possible with the study of expression of collagenases MMP-2 and MMP-9. Real time RT-PCR based detection of mRNA in circulating blood appears very promising. The ability to distinguish metastatic disease is higher for MMP-9 while MMP-2 discriminates better all levels of tumour invasion.
496
Relevance of urinary interferon-gamma in the prognosis of superficial bladder cancer treated with BCG immunotherapy Zoumpos I.1, Touloupidis S.2, Giakoumelos A.1, Galaktidou T.3, Kortsaris A.4, Simopoulos K.5, Katsikas V.1
Aristotle University of Thessaloniki, 2nd Dept. Urology, Thessaloniki, Greece, 2Democritus Thrace University, Dept. Urology, Alexandroupolis, Greece, 3Theageneion Hospital, Dept. Virology, Thessaloniki, Greece, 4Democritus Thrace University, Dept. Biochemistry, Alexandroupolis, Greece, 5Democritus Thrace University, 2nd Dept. Surgery, Alexandroupolis, Greece
University Hospital Mannheim, Department of Urology, Mannheim, Germany, University Hospital Mannheim, Department of Medical Statistics, Mannheim, Germany, 3University Hospital Mannheim, Department of Pathology, Mannheim, Germany
1
Introduction & Objectives: Discovery of lymphatic endothelial cell (LEC) markers has recently focused attention on lymphangiogenesis in cancer research. The potential role of lymph vessel density (LVD) as prognostic factor as well as the functional significance of tumoral lymph vessels has not been investigated in invasive bladder transitional cell carcinoma (TCC) to date. Aim was to quantify LVD and to evaluate association of lymphatics with clinicopathological parameters and survival in bladder TCC. Furthermore, proliferation of LEC’s was evaluated in order to assess the functional significance of tumoral lymph vessels.
Introduction & Objectives: Interferon gamma, a cytokine produced by activated T-lymphocytes and natural killer cells, is regarded by many researchers as a good marker of local immune (Th1) activation of the bladder wall after intravesical instillations of BCG. Measurement of the soluble fraction of interferon-gamma in the urine can therefore provide indirect evidence of immune response that is critical for the effect of BCG to be exerted. The present study, investigated the use of soluble interferon-gamma in the urine as a possible prognostic marker for BCG-therapy of superficial bladder cancer.
1
2
Material & Methods: Data of 108 patients with muscle-invasive bladder TCC who underwent radical cystectomy from 1998 to 2005 were reviewed retrospectively. Sections were analyzed immunohistochemically by D2-40, a lymph vessel specific endothelial marker. Lymph vessel count was performed in 3 areas: intratumoral, peritumoral and normal tissue. Proliferating LEC’s were assessed with doublestaining for D2-40 and Ki-67. Results: Peritumoral lymph vessels were observed in 105 sections (97,2%), identification of intratumoral vessels was possible in 65 specimens (60,2%). Doublestaining clearly distinguished D2-40-positive lymph vessels from neighboring D2-40-negative blood vessels, which in turn were stained by the endothelial marker CD-34. Peritumoral LVD (PTLVD) (mean 9,25 +/- 5,66) was significantly higher than intratumoral LVD (ITLVD) (mean 3,51 +/- 5,1; p<0,0001) as well as than in normal tissue (mean 6,51 +/- 4,58; p<0,0001). Higher intratumoral LVD (ITLVD) correlated significantly with poor histological differentiation (p=0,01). Higher PTLVD had also an association with higher histological grade (p=0,06) in addition to a significant association with presence of lymph node metastasis (p=0,0004). Higher pathological T stage (p<0,0001), a poorer histological grade (p=0,04) and the presence of lymph node metastasis (p<0,0001) were the only significant prognostic factors for reduced disease-specific survival. LVD had no significant influence on survival. Intratumoral and peritumoral vessels showed proliferating LEC’s in all examined samples in a variable proportion. Conclusions: Lymphangiogenesis occurs in invasive bladder TCC. To our knowledge, our study is the first to investigate and confirm a strong correlation of higher PTLVD with presence of lymph nodes in clinically localized invasive bladder TCC. According to these results, an increased PTLVD may facilitate dissemination to locoregional lymph nodes. The present study is also the first to suggest the existence of proliferating lymph vessels in bladder TCC. The potential implementation of lymphangiogenesis as a prognostic factor as well as a therapeutical target, as already established for angiogenesis, requires further studies in order to determine if our information will contribute to improve outcomes in bladder TCC.
Eur Urol Suppl 2007;6(2):146
Material & Methods: 51 patients with newly diagnosed superficial bladder cancer were included in the study between 2000 and 2004. Mean age of the patients was 65.5 years and median follow-up was 43 weeks (range: 11-108 years). Patients received 6-weekly instillations of BCG (Tice-strain, 5x108 CFU). A urinary ELISA kit (CytELISA human IFNg, Cytimmune, AMS Biotechnology, Oxon, UK) was utilized for IFN-gamma measurement 6 hours post-instillation. Following completion of the induction cycle of BCG patients were subjected to cystoscopy and cytology. Statistical analysis included logistic regression analysis using SPSS software. Results: Urinary levels of interferon-gamma according to response to treatment are presented in Table 1. Table 1. Response
Mean value (pg/ml)
SD
p
IFN-γ week 1
Yes No
11,5127 14,5232
24,17847 34,80789
0,720
IFN-γ week 2
Yes No
13,5985 10,1888
21,68320 19,46929
0,558
IFN-γ week 3
Yes No
14,5088 6,2236
21,25360 9,57764
0,079
IFN-γ week 4
Yes No
18,3269 10,4504
22,30716 15,98589
0,153
IFN-γ week 5
Yes No
27,7192 9,5308
33,07618 18,42786
0,019*
IFN-γ week 6
Yes No
36,4638 6,1608
41,88771 10,90515
0,001*
There was a statistically significant difference in urinary interferon-gamma in favor of responders at the 5th and 6th instillation (p<0,05). Comparing urinary interferon-gamma levels by stage and grade resulted in no significant differences. Regression analysis showed that with an accuracy of 70,6%, urinary IFN-gamma levels at the 6th instillation can predict response to treatment. Conclusions: Urinary levels of soluble interferon-gamma can help predict the outcome of intravesical BCG instillations. More important is the 6th instillation, the last in the induction cycle. Measurement of IFN-gamma in the urine is feasible, utilizing a simple ELISA kit that is commercially available in undialyzed urine without correction to creatinine clearance. The combination of this marker of the local immune response with other tumor markers may further increase the yield of its utility in bladder cancer.