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EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY MEMBERS (EGFR AND HER2) ARE PROGNOSTIC MARKERS AND POTENTIAL THERAPEUTIC TARGETS IN PROSTATE CANCER
P34 NOVEL TARGETS FOR DIAGNOSIS AND THERAPY OF PROSTATE CANCER Thursday, 6 April, 14.00-15.30, Room Concorde 1 / Level 4 553 EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY MEMBERS (EGFR AND HER2) ARE PROGNOSTIC MARKERS AND POTENTIAL THERAPEUTIC TARGETS IN PROSTATE CANCER Schlomm T.1, Steuber T.1, Haese A.1, Isbarn H.1, Erbersdobler A.2, Simon R.2, Graefen M.1, Sauter G.2, Huland H.1 1
University Medical Centre Hamburg Eppendorf, Urology, Hamburg, Germany, 2University
Medical Centre Hamburg Eppendorf, Pathology, Hamburg, Germany INTRODUCTION & OBJECTIVES: EGFR (HER1) and HER2 are therapeutic targets for gene specific drugs (Iressa, Tarceva, Herceptin). Treatment success depends on the presence of gene alterations including gene mutations, copy number changes, or protein overexpression. At present, treatment has been approved only in NSCLC (Iressa, Tarceva) and breast cancer (Herceptin), but increasing evidence suggests there may be other candidate tumour types as well. Given the high incidence of prostate cancer in western societies, our study aimed on the comprehensive analysis of EGFR and HER2 alterations in a high number of prostate cancers from a single centre. MATERIAL & METHODS: A tissue microarray (TMA) was constructed from 3459 radical prostatectomy specimens operated at the Department of Urology, Medical Centre HamburgEppendorf between 1992 and 2004. Clinical follow-up data were available for 2385 patients. EGFR and HER2 status were analysed by means of immunohistochemistry (IHC), fluorescence in situ hybridisation (FISH), and sequence analysis (EGFR only). IHC and FISH analysis was scored by an observer blinded for tumour stage and clinical outcome.
554 PEROXISOME PROLIFERATOR ACTIVATOR-RECEPTOR-γ IS A NEW TARGET IN THE TREATMENT OF HUMAN PROSTATE CANCER Hayama T., Matsuyama M., Tsuchida K., Takemoto Y., Nakatani T., Yoshimura R. Osaka City University Graduate School of Medicine, Department of Urology, Osaka, Japan INTRODUCTION & OBJECTIVES: Peroxisome proliferator activator-receptor (PPAR) is member of the nuclear receptors super-family of ligand-activated transcriptional factor such as steroids, thyroid hormone, vitamin D3 and retinoic acid. PPAR have three subtypes (α, β, and-γ). Recent studies have demonstrated that PPAR-γ is expressed in various cancer tissues and its ligand induces growth arrest of these cancer cells through apoptosis. In this study, we examined the expression of PPAR-γ in prostate cancer (PC) as well as the effect of PPAR-γ ligand in PC cell. MATERIAL & METHODS: Tumour specimens were obtained from 156 patients with PC, 15 with prostatic intraepithelial neoplasia, 20 with benign prostatic hyperplasia, who underwent biopsy due to PSA increase, and 12 patients with normal prostate tissues who underwent total cystectomy due to bladder cancer. PPAR-γ expression was examined by RT-PCR and immunohistochemistry. Effect of PPAR-γ ligand on PC cell growth was examined by MTT assay, and HOECHST staining was used to determine whether or not the PPAR-γ ligand induce apoptosis.
RESULTS: Receptor status was successfully analysed in 2656 (EGFR) and 2720 (HER2) tissue samples. Different levels of protein expression were found in 17,5% (EGFR) and 20.8% (HER2) of cases. EGFR and HER2 alterations were strongly linked to tumour recurrence (p<0.0001), high Gleason grade (p<0.0001), and advanced stage cancers (p<0.03). In addition, EGFR alterations, but not HER2 changes, were associated with positive surgical margins. Coexpression of EGFR and HER2 was found in 21% of EGFR/HER2 positive tumours and was linked to a particularly poor prognosis. CONCLUSIONS: EGFR and HER2 alterations are frequent in advanced prostate cancer, and strongly linked to patient prognosis. Our data would justify clinical trials using anti EGFR/ HER2 drugs in patients with advanced prostate cancer.
RESULTS: PPAR-γ expression in PC and prostatic intraepithelial neoplasia were significantly stronger than that in benign prostatic hyperplasia and normal prostate. Furthermore, PPAR-γ expression was significantly stronger in the low differentiation carcinoma. PPAR-γ ligand caused marked reduction of PC cell in a concentration-dependent and time-dependent manner. Furthermore, PPAR-γ ligand caused marked reduction of PC cell through apoptosis. CONCLUSIONS: These results demonstrated that the generated PPAR-γ in PC cell might play an important role in the carcinogenesis. PPAR-γ may become a new target in the treatment of PC.
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556
SUPERIOR ANTI-TUMOUR IMMUNE RESPONSE OF DENDRITIC CELLS FUSED WITH PROSTATE CANCER CELLS COMPARED WITH TUMOUR LYSATE-PULSED DENDRITIC CELLS
ENHANCED EFFICACY OF RADIATION SENSITIVITY BY CONTROLLED GENE DELIVERY OF PTEN EXPRESSION VECTOR CONJUGATED WITH CATIONISED GELATIN IN PROSTATE CANCER CELLS
Lee S.B.1, Jun S.Y.1, Yoo C.1, Park J.1, Park J.Y.2, Kim H.S.3, Kim C.S.1 1
Asan Medical Center, Urology, Seoul, South Korea, 2Kangneung Asan Hospital, Urology,
Kangneung, South Korea, 3Chungnam National University Hospital, Urology, Chungnam, South Korea INTRODUCTION & OBJECTIVES: Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells and methods for delivering tumour antigens into DCs are one of the major concerns of intensive investigation in DC-based tumour vaccines. Fusion of DCs with tumour cells is an effective approach for delivering tumour antigens to DCs, and DC-tumour fusion cells are potent stimulators of immune response against tumour cells. We compared anti-tumour immune response of the DC-tumour fusion cells versus the tumour lysate-pulsed DCs in hormone refractory prostate cancer cell lines. MATERIAL & METHODS: DCs were generated from the peripheral blood mononuclear cells of donors in the presence of GM-CSF and IL-4. Radiation-induced apototic prostate cancer cell line, DU145 and PC3, were fused with DCs at a ratio of 1:1 using polyethylene glycol (PEG). These hybrids were cocultured with autologous T cells to evaluate the proliferation of T cells and were analysed for their biological ability to produce cytokines and to induce tumourspecific cytotoxic T response. These anti-tumour immunity of hybrid DCs was compared with that of DCs pulsed with freeze-thawed tumour lysate. RESULTS: The DCs were successfully fused with the allogeneic prostate cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen presenting cell because they induced significant allogeneic T cells proliferation. Cytotoxic T cells stimulated by DU145-DC or PC3-DC hybrids were functionally active in killing DU145 or PC3 cells in cytotoxicity assay and their activity was more excellent in DC-tumour hybrid. In cytokine production assays, IL-12 level produced by DC-tumour hybrid is significantly higher than that of DCs pulsed with tumour lysate. Also, the generation of INF-γ by tumour specific T cells in DC hybrid group is superior to that of DCs pulsed with tumour lysate. CONCLUSIONS: Our results suggest that the DCs fused with tumour cells produce more potent anti-tumour immunity than the tumour lysate-pulsed DCs.
Tomioka A., Takada S., Tanaka M., Hirao Y. Nara Medical University, Urology, Kashihara, Japan INTRODUCTION & OBJECTIVES: We have demonstrated that an adenoviral gene therapy of PTEN can effectively treat bladder and prostate cancers, and can be effectively treated tumours which exhibit drug or radiation resistance associated with expression of phosphorylated Akt in combination with chemotherapy or radiotherapy. PTEN is well known as a tumour suppressor gene and has a phosphatase activity in the phosphatidylinositol 3’-kinase mediated signal transduction pathway and inhibits the activation of Akt, a serine-threonine kinase involved in proliferative and anti-apoptotic pathways. These days, using virus vector for cancer gene therapy is controversial, and non-viral gene transfer is a future promising procedure but several problems need to be cleared, such as transduction efficacy. MATERIAL & METHODS: We have developed non-viral compound conjugated with cationised gelatin microsphere and plasmid DNA, which is a new type of gene transfer drug and designed to release plasmid DNA and last the gene expression continuously for a long period in vivo. In this study, we originally generated the GelaTen, which is a conjugate with cationised gelatin microsphere (2 mg) and PTEN expression vector (100 μg), and examined the efficacy of GelaTen as a combination therapy with radiation in prostate cancer. RESULTS: Single direct injection of GelaTen into established subcutaneous bcl-2overexpressing PC3 prostate cancer tumours (PTEN deleted, up-regulation of phosphorylated Akt and Bcl-2) in nude mice, which reached approximately 5-7mm in diameter, resulted in significantly decreased growth compared to the conjugate with β-gal plasmid (control) or PBS treated tumours. Immunohistochemical analysis showed that tumours inducted with GelaTen expressed PTEN and exhibited decreased amounts of phosphorylated Akt, whereas tumours treated with CTL or PBS were negative for PTEN and diffusely positive for phosphorylated Akt. Since PTEN downregulates phosphorylated Akt and Bcl-2 and increases sensitivity to radiation, we explored combination therapy with GelaTen and radiation in vivo. Combination therapy with GelaTen and 5 Gy irradiation (5 days after GelaTen injection) improved the in vivo efficacy of tumour growth compared to the GelaTen monotherapy alone in these tumours. CONCLUSIONS: These data demonstrate that PTEN gene therapy with gene drug GelaTen can effectively treat prostate cancers that have genomic alterations in PTEN. Furthermore, tumours that exhibit radiation resistance associated with expression of phosphorylated Akt and Bcl-2 can be effectively treated with GelaTen and radiotherapy.
Eur Urol Suppl 2006;5(2):161
University Medical Centre Hamburg Eppendorf, Urology, Hamburg, Germany, 2University
Medical Centre Hamburg Eppendorf, Pathology, Hamburg, Germany INTRODUCTION & OBJECTIVES: EGFR (HER1) and HER2 are therapeutic targets for gene specific drugs (Iressa, Tarceva, Herceptin). Treatment success depends on the presence of gene alterations including gene mutations, copy number changes, or protein overexpression. At present, treatment has been approved only in NSCLC (Iressa, Tarceva) and breast cancer (Herceptin), but increasing evidence suggests there may be other candidate tumour types as well. Given the high incidence of prostate cancer in western societies, our study aimed on the comprehensive analysis of EGFR and HER2 alterations in a high number of prostate cancers from a single centre. MATERIAL & METHODS: A tissue microarray (TMA) was constructed from 3459 radical prostatectomy specimens operated at the Department of Urology, Medical Centre HamburgEppendorf between 1992 and 2004. Clinical follow-up data were available for 2385 patients. EGFR and HER2 status were analysed by means of immunohistochemistry (IHC), fluorescence in situ hybridisation (FISH), and sequence analysis (EGFR only). IHC and FISH analysis was scored by an observer blinded for tumour stage and clinical outcome.
554 PEROXISOME PROLIFERATOR ACTIVATOR-RECEPTOR-γ IS A NEW TARGET IN THE TREATMENT OF HUMAN PROSTATE CANCER Hayama T., Matsuyama M., Tsuchida K., Takemoto Y., Nakatani T., Yoshimura R. Osaka City University Graduate School of Medicine, Department of Urology, Osaka, Japan INTRODUCTION & OBJECTIVES: Peroxisome proliferator activator-receptor (PPAR) is member of the nuclear receptors super-family of ligand-activated transcriptional factor such as steroids, thyroid hormone, vitamin D3 and retinoic acid. PPAR have three subtypes (α, β, and-γ). Recent studies have demonstrated that PPAR-γ is expressed in various cancer tissues and its ligand induces growth arrest of these cancer cells through apoptosis. In this study, we examined the expression of PPAR-γ in prostate cancer (PC) as well as the effect of PPAR-γ ligand in PC cell. MATERIAL & METHODS: Tumour specimens were obtained from 156 patients with PC, 15 with prostatic intraepithelial neoplasia, 20 with benign prostatic hyperplasia, who underwent biopsy due to PSA increase, and 12 patients with normal prostate tissues who underwent total cystectomy due to bladder cancer. PPAR-γ expression was examined by RT-PCR and immunohistochemistry. Effect of PPAR-γ ligand on PC cell growth was examined by MTT assay, and HOECHST staining was used to determine whether or not the PPAR-γ ligand induce apoptosis.
RESULTS: Receptor status was successfully analysed in 2656 (EGFR) and 2720 (HER2) tissue samples. Different levels of protein expression were found in 17,5% (EGFR) and 20.8% (HER2) of cases. EGFR and HER2 alterations were strongly linked to tumour recurrence (p<0.0001), high Gleason grade (p<0.0001), and advanced stage cancers (p<0.03). In addition, EGFR alterations, but not HER2 changes, were associated with positive surgical margins. Coexpression of EGFR and HER2 was found in 21% of EGFR/HER2 positive tumours and was linked to a particularly poor prognosis. CONCLUSIONS: EGFR and HER2 alterations are frequent in advanced prostate cancer, and strongly linked to patient prognosis. Our data would justify clinical trials using anti EGFR/ HER2 drugs in patients with advanced prostate cancer.
RESULTS: PPAR-γ expression in PC and prostatic intraepithelial neoplasia were significantly stronger than that in benign prostatic hyperplasia and normal prostate. Furthermore, PPAR-γ expression was significantly stronger in the low differentiation carcinoma. PPAR-γ ligand caused marked reduction of PC cell in a concentration-dependent and time-dependent manner. Furthermore, PPAR-γ ligand caused marked reduction of PC cell through apoptosis. CONCLUSIONS: These results demonstrated that the generated PPAR-γ in PC cell might play an important role in the carcinogenesis. PPAR-γ may become a new target in the treatment of PC.
555
556
SUPERIOR ANTI-TUMOUR IMMUNE RESPONSE OF DENDRITIC CELLS FUSED WITH PROSTATE CANCER CELLS COMPARED WITH TUMOUR LYSATE-PULSED DENDRITIC CELLS
ENHANCED EFFICACY OF RADIATION SENSITIVITY BY CONTROLLED GENE DELIVERY OF PTEN EXPRESSION VECTOR CONJUGATED WITH CATIONISED GELATIN IN PROSTATE CANCER CELLS
Lee S.B.1, Jun S.Y.1, Yoo C.1, Park J.1, Park J.Y.2, Kim H.S.3, Kim C.S.1 1
Asan Medical Center, Urology, Seoul, South Korea, 2Kangneung Asan Hospital, Urology,
Kangneung, South Korea, 3Chungnam National University Hospital, Urology, Chungnam, South Korea INTRODUCTION & OBJECTIVES: Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells and methods for delivering tumour antigens into DCs are one of the major concerns of intensive investigation in DC-based tumour vaccines. Fusion of DCs with tumour cells is an effective approach for delivering tumour antigens to DCs, and DC-tumour fusion cells are potent stimulators of immune response against tumour cells. We compared anti-tumour immune response of the DC-tumour fusion cells versus the tumour lysate-pulsed DCs in hormone refractory prostate cancer cell lines. MATERIAL & METHODS: DCs were generated from the peripheral blood mononuclear cells of donors in the presence of GM-CSF and IL-4. Radiation-induced apototic prostate cancer cell line, DU145 and PC3, were fused with DCs at a ratio of 1:1 using polyethylene glycol (PEG). These hybrids were cocultured with autologous T cells to evaluate the proliferation of T cells and were analysed for their biological ability to produce cytokines and to induce tumourspecific cytotoxic T response. These anti-tumour immunity of hybrid DCs was compared with that of DCs pulsed with freeze-thawed tumour lysate. RESULTS: The DCs were successfully fused with the allogeneic prostate cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen presenting cell because they induced significant allogeneic T cells proliferation. Cytotoxic T cells stimulated by DU145-DC or PC3-DC hybrids were functionally active in killing DU145 or PC3 cells in cytotoxicity assay and their activity was more excellent in DC-tumour hybrid. In cytokine production assays, IL-12 level produced by DC-tumour hybrid is significantly higher than that of DCs pulsed with tumour lysate. Also, the generation of INF-γ by tumour specific T cells in DC hybrid group is superior to that of DCs pulsed with tumour lysate. CONCLUSIONS: Our results suggest that the DCs fused with tumour cells produce more potent anti-tumour immunity than the tumour lysate-pulsed DCs.
Tomioka A., Takada S., Tanaka M., Hirao Y. Nara Medical University, Urology, Kashihara, Japan INTRODUCTION & OBJECTIVES: We have demonstrated that an adenoviral gene therapy of PTEN can effectively treat bladder and prostate cancers, and can be effectively treated tumours which exhibit drug or radiation resistance associated with expression of phosphorylated Akt in combination with chemotherapy or radiotherapy. PTEN is well known as a tumour suppressor gene and has a phosphatase activity in the phosphatidylinositol 3’-kinase mediated signal transduction pathway and inhibits the activation of Akt, a serine-threonine kinase involved in proliferative and anti-apoptotic pathways. These days, using virus vector for cancer gene therapy is controversial, and non-viral gene transfer is a future promising procedure but several problems need to be cleared, such as transduction efficacy. MATERIAL & METHODS: We have developed non-viral compound conjugated with cationised gelatin microsphere and plasmid DNA, which is a new type of gene transfer drug and designed to release plasmid DNA and last the gene expression continuously for a long period in vivo. In this study, we originally generated the GelaTen, which is a conjugate with cationised gelatin microsphere (2 mg) and PTEN expression vector (100 μg), and examined the efficacy of GelaTen as a combination therapy with radiation in prostate cancer. RESULTS: Single direct injection of GelaTen into established subcutaneous bcl-2overexpressing PC3 prostate cancer tumours (PTEN deleted, up-regulation of phosphorylated Akt and Bcl-2) in nude mice, which reached approximately 5-7mm in diameter, resulted in significantly decreased growth compared to the conjugate with β-gal plasmid (control) or PBS treated tumours. Immunohistochemical analysis showed that tumours inducted with GelaTen expressed PTEN and exhibited decreased amounts of phosphorylated Akt, whereas tumours treated with CTL or PBS were negative for PTEN and diffusely positive for phosphorylated Akt. Since PTEN downregulates phosphorylated Akt and Bcl-2 and increases sensitivity to radiation, we explored combination therapy with GelaTen and radiation in vivo. Combination therapy with GelaTen and 5 Gy irradiation (5 days after GelaTen injection) improved the in vivo efficacy of tumour growth compared to the GelaTen monotherapy alone in these tumours. CONCLUSIONS: These data demonstrate that PTEN gene therapy with gene drug GelaTen can effectively treat prostate cancers that have genomic alterations in PTEN. Furthermore, tumours that exhibit radiation resistance associated with expression of phosphorylated Akt and Bcl-2 can be effectively treated with GelaTen and radiotherapy.
Eur Urol Suppl 2006;5(2):161