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THERAPEUTIC EFFICACY OF MIDKINE PROMOTER-BASED CONDITIONALLY REPLICATIVE ADENOVIRUS VECTOR FOR TARGETTING THE MIDKINE-EXPRESSING HUMAN BLADDER CANCER CELLS
374
THE JOURNAL OF UROLOGY®
generation oncolytic virus that was derived from HSV-1 (Baco-1). For in vitro studies, we infected 5637 human bladder cancer cells and MBT-2 murine bladder cancer cells with Baco-1 or FusOn-H2, respectively, and the percentage of viable cells was calculated to determine the killing activity. For in vivo evaluation, we established bladder tumor at the orthotopic site in C3H/He mice using the murine MBT-2 cells. Baco-1 or FusOn-H2 was then instilled into the bladder through the urethra respectively. Tumor volume and weight were recorded by the end of the experiment. Animal spleens were also collected to determine if any anti-tumor immunity was elicited during virotherapy in this syngeneic bladder cancer model. RESULTS: Infection of oncolytic HSVs in both cell lines led to progressive killing of tumor cells. By 72 hours after infection, FusOn-H2, when given at the initial dose of 0.1 plaque forming unit/cell, almost completely eradicated the tumor cells. Two instillations of the oncolytic HSVs into bladder of tumor-bearing mice completely eradicated the tumor in 81.8 % of mice. The remaining tumor in the FusOn-H2 treated group was visibly smaller than those of Baco-1-treated group, though QRWVWDWLVWLFDOO\VLJQL¿FDQW S! 7KHUHVXOWVRIWXPRUVSHFL¿F&7/ activity assay showed that tumor destruction by oncolytic viruses in vivo, especially by the HSV-2-based FusOn-H2, induced potent anti-tumor immune responses. CONCLUSIONS: Oncolytic virus derived from HSV-2 has potent anti-tumor activity against bladder cancer. Oncolytic effect of WKLVYLUXVLQYLYRLQGXFHVWXPRUVSHFL¿FFHOOXODULPPXQLW\WKDWIXUWKHU enhances the overall anti-tumor activity. Translating this novel virotherapy into the clinic could present an alternative intravesical therapy strategy for patients with bladder cancer. Source of Funding: Dr. Seth P. Lerner: Funds of Scott 'HSDUWPHQWRI8URORJ\%D\ORU&ROOHJHRI0HGLFLQH'U;LDROLX=KDQJ Owens Foundation.
1087 THERAPEUTIC EFFICACY OF MIDKINE PROMOTER-BASED CONDITIONALLY REPLICATIVE ADENOVIRUS VECTOR FOR TARGETTING THE MIDKINE-EXPRESSING HUMAN BLADDER CANCER CELLS Shuji Terao*, Toshiro Shirakawa, Kazushi Tanaka, Atsushi Takenaka, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh. Nishinomiya, Japan, and Kobe, Japan. INTRODUCTION AND OBJECTIVE: Conditionally replicative adenovirus (CRAD) is now widely used and has two major advantages for cancer gene therapy. The virus kills tumor cells selectively because WKHH[SUHVVLRQRI(DJHQHLVFRQWUROOHGE\WXPRUVSHFL¿FSURPRWHU$QG VHOHFWLYHUHSOLFDWLRQOHDGWRDPSOL¿FDWLRQRILWVRQFRO\WLFHIIHFWV0LGNLQH is overexpressed in many cancers including bladder cancer. Importantly, the expression of midkine in normal adult tissues is extremely restricted. 7KLVWXPRUVSHFL¿FH[SUHVVLRQRI0.VXJJHVWVWKHSRWHQWLDOXWLOLW\RI0. promoter for the trearment of bladder cancer. In this study, we attempted to develop a novel therapeutic strategy against human bladder cancer using MK promoter regulated CRAD. METHODS: We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative PCR, MK promoter activity, post plasmid transfection, by a luciferase assay, and WKHWUDQVGXFWLRQHI¿FLHQF\E\FRWUDQVIHFWLRQZLWKWKHF\WRPHJDORYLUXV &09 ȕJDOSODVPLG$IWHUZHFRQVWUDFWHGWKHUHSOLFDWLRQFRPSHWHQW adenovirus vector containing adenoviral E1a gene controlled by MK SURPRWHU$G0.(DZHDVVHVVHGWKHFHOOW\SHVSHFL¿FUHSOLFDWLRQRI Ad-MK-E1a in the above cells by measuring the E1a DNA copy number by real-time PCR and the cell growth inhibition due to this virus by the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with PBS or $G&09/DF=RU$G0.(D RESULTS: The MK mRNA expression level and MK promoterdriven luciferase activity were relatively higher and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells (P < 0.0312). Following Ad-MK-E1a infection, the E1a DNA copy QXPEHULQFUHDVHGPRUHVLJQL¿FDQWO\LQWKH..DQG$ FHOOV than in the T24 and H891 cells (P < 0.001). At a multiplicity of infection
Vol. 179, No. 4, Supplement, Monday, May 19, 2008
02, RI $G0.(D VLJQL¿FDQWO\ LQKLELWHG .. DQG FHOO growth (P < 0.001). In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth (p<0.025). CONCLUSIONS: In our study, we demonstrated the antitumor effect of Ad-MK-E1a in human bladder cancer model overexpressing MK mRNA. This novel therapeutic approach has the potential to treat patients with bladder cancer that is resistant to conventional therapies. Source of Funding: None
Kidney Cancer: Evaluation and Treatment (IV) Moderated Poster Session 38 Monday, May 19, 2008
3:30 - 5:30 pm
1088 A STUDY ON THE WIDTH OF SURGICAL MARGIN IN THE NEPHRON-SPARING SURGERY FOR RENAL CELL CARCINOMA Seong Jin Jeong, Kwang Taek Kim, Jae Seung Jeong, Byung Kyu Han, Jeong Hyun Kim, Ki Hyuk Moon, Sung Kyu Hong, SeokSoo Byun*, Sang Eun Lee. Seongnam, Republic of Korea, Seoul, Republic of Korea, and Chuncheon, Republic of Korea. INTRODUCTION AND OBJECTIVE: Eelective nephronsparing surgery (NSS) has shown satisfactory oncologic outcomes as UDGLFDOQHSKUHFWRP\LQVPDOOVL]HUHQDOWXPRUV,Q166WKHZLGWKRI healthy surgical margin has been classically proposed to be 1-2 cm, but recently, there is the opinion that several mm is safe. The aims of this study is to assess the width of healthy surgical margin microscopically and evaluate its prognostic value. METHODS: We retrospectively reviewed 98 consecutive NSS for renal cell carcinoma (RCC) in 96 patients followed-up for >1 year after surgery. Data were collected from medical records and through telephone LQWHUYLHZVDQGWKHZLGWKRIKHDOWK\VXUJLFDOPDUJLQZDVGH¿QHGDVWKH minimum thickness of normal parenchyma adjacent to the tumor. The mean follow-up was 46.8 months (13-115). RESULTS: The mean age of the patients was 51.7, and 79.2% of the patients were male. Of the patients, 89.8% and 10.2%, respectively, received elective and imperative surgeries. The location of tumor was the upper, mid and lower pole, respectively, in 33.7%, DQGRIWKHSDWLHQWVDQGWKHPHDQWXPRUVL]HZDVFP (1.0-6.1). Tumors were generally resected using a cold scissor in the pedicle clamping state to better assess the boundary between the tumors and healthy tissue. The mean ischemic time was 22.6 minutes. )UR]HQELRSV\ZDVSHUIRUPHGLQ SDWLHQWV DQGDVSDWLHQWV RIWKHPVKRZHGSRVLWLYHDGGLWLRQDOIUR]HQELRSVLHVZHUHSHUIRUPHGDQG the results were negative. In pathologic examination, 88% were found WREHFOHDUFHOOW\SHDQGVKRZHGSHULUHQDOIDWLQ¿OWUDWLRQ7KHPHDQ width of surgical margin was 2.9mm (0.5-15.0), and 28.6% were below 1mm, 40.8% 1.1-3.0mm, 22.4% 3.1-5.0mm, and 8.2% over 5.1mm, and the width decreased over time (3.7mm vs 2.3mm, p=0.003). A total of 3 patients showed a positive surgical margin, and 2 of them were those WKDWVKRZHGSRVLWLYHLQIUR]HQELRSV\DQGKDGDGGLWLRQDOELRSVLHV7KH RWKHUSDWLHQWGLGQRWKDYHIUR]HQELRSV\DQGGLGQRWVKRZDQ\HYLGHQFH of locoregional or metastatic relapse during 57 months’ follow-up. All patients were free of locoregional or metastatic relapse at last follow-up except 2. One was VHL disease and the other with negative 2mm-width surgical margin showed enlarged abdominal lymph node at a follow-up of 38 months. CONCLUSIONS: In our NSS series, the rate of margin positivity was 3.1% and the mean width of healthy surgical margin was 2.9mm. The width of healthy surgical margin is considered irrelevant to RQFRORJLFSURJQRVLVLQ166IRUVPDOOVL]H5&& Source of Funding: None
THE JOURNAL OF UROLOGY®
generation oncolytic virus that was derived from HSV-1 (Baco-1). For in vitro studies, we infected 5637 human bladder cancer cells and MBT-2 murine bladder cancer cells with Baco-1 or FusOn-H2, respectively, and the percentage of viable cells was calculated to determine the killing activity. For in vivo evaluation, we established bladder tumor at the orthotopic site in C3H/He mice using the murine MBT-2 cells. Baco-1 or FusOn-H2 was then instilled into the bladder through the urethra respectively. Tumor volume and weight were recorded by the end of the experiment. Animal spleens were also collected to determine if any anti-tumor immunity was elicited during virotherapy in this syngeneic bladder cancer model. RESULTS: Infection of oncolytic HSVs in both cell lines led to progressive killing of tumor cells. By 72 hours after infection, FusOn-H2, when given at the initial dose of 0.1 plaque forming unit/cell, almost completely eradicated the tumor cells. Two instillations of the oncolytic HSVs into bladder of tumor-bearing mice completely eradicated the tumor in 81.8 % of mice. The remaining tumor in the FusOn-H2 treated group was visibly smaller than those of Baco-1-treated group, though QRWVWDWLVWLFDOO\VLJQL¿FDQW S! 7KHUHVXOWVRIWXPRUVSHFL¿F&7/ activity assay showed that tumor destruction by oncolytic viruses in vivo, especially by the HSV-2-based FusOn-H2, induced potent anti-tumor immune responses. CONCLUSIONS: Oncolytic virus derived from HSV-2 has potent anti-tumor activity against bladder cancer. Oncolytic effect of WKLVYLUXVLQYLYRLQGXFHVWXPRUVSHFL¿FFHOOXODULPPXQLW\WKDWIXUWKHU enhances the overall anti-tumor activity. Translating this novel virotherapy into the clinic could present an alternative intravesical therapy strategy for patients with bladder cancer. Source of Funding: Dr. Seth P. Lerner: Funds of Scott 'HSDUWPHQWRI8URORJ\%D\ORU&ROOHJHRI0HGLFLQH'U;LDROLX=KDQJ Owens Foundation.
1087 THERAPEUTIC EFFICACY OF MIDKINE PROMOTER-BASED CONDITIONALLY REPLICATIVE ADENOVIRUS VECTOR FOR TARGETTING THE MIDKINE-EXPRESSING HUMAN BLADDER CANCER CELLS Shuji Terao*, Toshiro Shirakawa, Kazushi Tanaka, Atsushi Takenaka, Sadao Kamidono, Masato Fujisawa, Akinobu Gotoh. Nishinomiya, Japan, and Kobe, Japan. INTRODUCTION AND OBJECTIVE: Conditionally replicative adenovirus (CRAD) is now widely used and has two major advantages for cancer gene therapy. The virus kills tumor cells selectively because WKHH[SUHVVLRQRI(DJHQHLVFRQWUROOHGE\WXPRUVSHFL¿FSURPRWHU$QG VHOHFWLYHUHSOLFDWLRQOHDGWRDPSOL¿FDWLRQRILWVRQFRO\WLFHIIHFWV0LGNLQH is overexpressed in many cancers including bladder cancer. Importantly, the expression of midkine in normal adult tissues is extremely restricted. 7KLVWXPRUVSHFL¿FH[SUHVVLRQRI0.VXJJHVWVWKHSRWHQWLDOXWLOLW\RI0. promoter for the trearment of bladder cancer. In this study, we attempted to develop a novel therapeutic strategy against human bladder cancer using MK promoter regulated CRAD. METHODS: We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative PCR, MK promoter activity, post plasmid transfection, by a luciferase assay, and WKHWUDQVGXFWLRQHI¿FLHQF\E\FRWUDQVIHFWLRQZLWKWKHF\WRPHJDORYLUXV &09 ȕJDOSODVPLG$IWHUZHFRQVWUDFWHGWKHUHSOLFDWLRQFRPSHWHQW adenovirus vector containing adenoviral E1a gene controlled by MK SURPRWHU$G0.(DZHDVVHVVHGWKHFHOOW\SHVSHFL¿FUHSOLFDWLRQRI Ad-MK-E1a in the above cells by measuring the E1a DNA copy number by real-time PCR and the cell growth inhibition due to this virus by the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with PBS or $G&09/DF=RU$G0.(D RESULTS: The MK mRNA expression level and MK promoterdriven luciferase activity were relatively higher and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells (P < 0.0312). Following Ad-MK-E1a infection, the E1a DNA copy QXPEHULQFUHDVHGPRUHVLJQL¿FDQWO\LQWKH..DQG$ FHOOV than in the T24 and H891 cells (P < 0.001). At a multiplicity of infection
Vol. 179, No. 4, Supplement, Monday, May 19, 2008
02, RI $G0.(D VLJQL¿FDQWO\ LQKLELWHG .. DQG FHOO growth (P < 0.001). In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth (p<0.025). CONCLUSIONS: In our study, we demonstrated the antitumor effect of Ad-MK-E1a in human bladder cancer model overexpressing MK mRNA. This novel therapeutic approach has the potential to treat patients with bladder cancer that is resistant to conventional therapies. Source of Funding: None
Kidney Cancer: Evaluation and Treatment (IV) Moderated Poster Session 38 Monday, May 19, 2008
3:30 - 5:30 pm
1088 A STUDY ON THE WIDTH OF SURGICAL MARGIN IN THE NEPHRON-SPARING SURGERY FOR RENAL CELL CARCINOMA Seong Jin Jeong, Kwang Taek Kim, Jae Seung Jeong, Byung Kyu Han, Jeong Hyun Kim, Ki Hyuk Moon, Sung Kyu Hong, SeokSoo Byun*, Sang Eun Lee. Seongnam, Republic of Korea, Seoul, Republic of Korea, and Chuncheon, Republic of Korea. INTRODUCTION AND OBJECTIVE: Eelective nephronsparing surgery (NSS) has shown satisfactory oncologic outcomes as UDGLFDOQHSKUHFWRP\LQVPDOOVL]HUHQDOWXPRUV,Q166WKHZLGWKRI healthy surgical margin has been classically proposed to be 1-2 cm, but recently, there is the opinion that several mm is safe. The aims of this study is to assess the width of healthy surgical margin microscopically and evaluate its prognostic value. METHODS: We retrospectively reviewed 98 consecutive NSS for renal cell carcinoma (RCC) in 96 patients followed-up for >1 year after surgery. Data were collected from medical records and through telephone LQWHUYLHZVDQGWKHZLGWKRIKHDOWK\VXUJLFDOPDUJLQZDVGH¿QHGDVWKH minimum thickness of normal parenchyma adjacent to the tumor. The mean follow-up was 46.8 months (13-115). RESULTS: The mean age of the patients was 51.7, and 79.2% of the patients were male. Of the patients, 89.8% and 10.2%, respectively, received elective and imperative surgeries. The location of tumor was the upper, mid and lower pole, respectively, in 33.7%, DQGRIWKHSDWLHQWVDQGWKHPHDQWXPRUVL]HZDVFP (1.0-6.1). Tumors were generally resected using a cold scissor in the pedicle clamping state to better assess the boundary between the tumors and healthy tissue. The mean ischemic time was 22.6 minutes. )UR]HQELRSV\ZDVSHUIRUPHGLQ SDWLHQWV DQGDVSDWLHQWV RIWKHPVKRZHGSRVLWLYHDGGLWLRQDOIUR]HQELRSVLHVZHUHSHUIRUPHGDQG the results were negative. In pathologic examination, 88% were found WREHFOHDUFHOOW\SHDQGVKRZHGSHULUHQDOIDWLQ¿OWUDWLRQ7KHPHDQ width of surgical margin was 2.9mm (0.5-15.0), and 28.6% were below 1mm, 40.8% 1.1-3.0mm, 22.4% 3.1-5.0mm, and 8.2% over 5.1mm, and the width decreased over time (3.7mm vs 2.3mm, p=0.003). A total of 3 patients showed a positive surgical margin, and 2 of them were those WKDWVKRZHGSRVLWLYHLQIUR]HQELRSV\DQGKDGDGGLWLRQDOELRSVLHV7KH RWKHUSDWLHQWGLGQRWKDYHIUR]HQELRSV\DQGGLGQRWVKRZDQ\HYLGHQFH of locoregional or metastatic relapse during 57 months’ follow-up. All patients were free of locoregional or metastatic relapse at last follow-up except 2. One was VHL disease and the other with negative 2mm-width surgical margin showed enlarged abdominal lymph node at a follow-up of 38 months. CONCLUSIONS: In our NSS series, the rate of margin positivity was 3.1% and the mean width of healthy surgical margin was 2.9mm. The width of healthy surgical margin is considered irrelevant to RQFRORJLFSURJQRVLVLQ166IRUVPDOOVL]H5&& Source of Funding: None