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1057 POST-CHEMOTHERAPY AKR1B10 EXPRESSION CORRELATES WITH DISEASE FREE SURVIVAL IN MUSCLE-INVASIVE BLADDER CANCER
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THE JOURNAL OF UROLOGY姞
METHODS: Quantitative PDEF expressions of 16 human pair bladder specimens (bladder cancer and adjacent normal bladder mucosa) or bladder carcinoma cells (RT-4, HT1376 and T24) were assessed using real-time reverse transcription-polymerase chain reaction (RT-qPCR) or immunoblot assays. Stably overexpressed PDEF were achieved by transfecting human PDEF overexpression vector into bladder carcinoma cells using electroporation. In vitro cell proliferation and invasion were determined by 3H-thymidine incorporation assay and matrigel invasion assay. In vivo tumorigenesis was determined by xenograft animal model. The modulation of PDEF on EMT associated genes was determined by immunoblot assays. RESULTS: The highly differentiation bladder papilloma RT-4 cells expressed significantly higher PDEF levels than bladder carcinoma HT1376 and T24 cells. Comparison between normal urothelium and bladder tumor has identified significant decrease of PDEF in the tumor part of the pair bladder tissues. Overexpression of PDEF enhanced gene expression of B-cell translocation gene 2 (BTG2), N-myc downstream regulated gene 1 (NDRG1), and maspin, which dramatically attenuated cell proliferation and invasion. Xenograph animal studies indicated that overexpression of PDEF in HT1376 cells blocked tumorigenesis in vivo. Overexpression of PDEF enhanced E-cadherin but decreased N-cadherin, snail-1, snail-2, and twist-1 protein expression in T24 cells. CONCLUSIONS: Expression of PDEF gene may be related to the extent of bladder neoplasia. A co-regulatory expression is found between PDEF and EMT associated genes. Results suggest that induction of BTG2, NDRG1, and maspin gene expression by PDEF, could account for the function of PDEF for anti-proliferation, antiinvasion, and anti-tumorigenesis in bladder carcinoma cells.
Vol. 187, No. 4S, Supplement, Monday, May 21, 2012
after definitive therapy for muscle-invasive bladder cancer. Further study is warranted to elucidate its clinical significance.
Source of Funding: CMRPG381683,CMRPG392141
1057 POST-CHEMOTHERAPY AKR1B10 EXPRESSION CORRELATES WITH DISEASE FREE SURVIVAL IN MUSCLE-INVASIVE BLADDER CANCER Yasuhiro Hashimoto*, Takuya Koie, Hayato Yamamoto, Atsushi Imai, Shingo Hatakeyama, Takahiro Yoneyama, Noritaka Kamimura, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: AKR1B10 is a member of the aldo-keto reductase superfamily of NAD(P)H-dependent oxidoreductases. AKR1B10 is considered to contribute to cell proliferation and chemoresistance. In the present study, we examined whether AKR1B10 expression correlates with disease free survival in bladder cancer specimens. METHODS: The cohort includes consecutive 57 patients with muscle-invasive bladder cancer who received neoadjuvant chemotherapy followed by radical cystectomy. All patients received two cycles of neoadjuvant chemotherapy with gemcitabine plus carboplatin. Bladder cancer specimens were obtained at pre- and post-neoadjuvant chemotherapy, which were subjected to quantitative real-time PCR and immunohistochemistry to evaluate AKR1B10 expression. Intensity scoring for immunohistochemistry was categorized using a 4-graded scoring system according to a previously published method, with positive cells for each specific marker expressed as a percentage of the total number of cells as follows: 0-10% ⫽ 0; 11%-30% ⫽ 1; 31%-70% ⫽ 2; 71%-100% ⫽ 3. RESULTS: AKR1B10 mRNA expression was significantly higher in the post-chemotherapy groups than in the pre-chemotherapy groups (p ⬍ 0.001). The average immunohistochemical intensity score in the pre-chemotherapy group was 0.83 ⫾ 1.08, compared to a significantly higher average intensity score of 2.03 ⫾ 1.03 in the post-chemotherapy group (p ⬍ 0.001) Disease free survival of the post-chemotherapy AKR1B10(⫹) patients (61.2%) was significantly lower than that of AKR1B10(-) patients (100%). (log-rank test: p ⫽ 0.039). CONCLUSIONS: Although the present study is small and preliminary, post-chemotherapy AKR1B10 expression may have a predictive potential for response of chemotherapy and disease recurrence
Source of Funding: None
1058 DIFFERENCES IN DNA METHYLATION PATTERN OF PRIMARY BLADDER TUMOURS CORRELATE WITH THEIR METASTATIC POTENTIAL Beatrice Stubendorff, Jena, Germany; Eva Dudziec, James Catto, Sheffiled, United Kingdom; Jimsgene Sanjmyatav, Mieczeslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, Kerstin Junker*, Jena, Germany INTRODUCTION AND OBJECTIVES: The prognosis of patients with metastatic bladder cancer is poor with a 5-year survival rate of only 6%. Early prediction of the metastatic risk in primary tumors can lead to improvements in prognosis and therapy. Currently, there are no parameters available that enable an individual risk assessment for patients with metastatic bladder cancer. Therefore, identification of new reliable prognostic markers for early prediction of tumor spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. METHODS: Genomic DNA was isolated from 23 invasive bladder tumour tissues with and without lymph node metastases. For enrichment of methylated fragments genomic DNA was incubated with 5-Methylcytosin antibody. Input and IP were labelled with Cy3 and Cy5.
THE JOURNAL OF UROLOGY姞
METHODS: Quantitative PDEF expressions of 16 human pair bladder specimens (bladder cancer and adjacent normal bladder mucosa) or bladder carcinoma cells (RT-4, HT1376 and T24) were assessed using real-time reverse transcription-polymerase chain reaction (RT-qPCR) or immunoblot assays. Stably overexpressed PDEF were achieved by transfecting human PDEF overexpression vector into bladder carcinoma cells using electroporation. In vitro cell proliferation and invasion were determined by 3H-thymidine incorporation assay and matrigel invasion assay. In vivo tumorigenesis was determined by xenograft animal model. The modulation of PDEF on EMT associated genes was determined by immunoblot assays. RESULTS: The highly differentiation bladder papilloma RT-4 cells expressed significantly higher PDEF levels than bladder carcinoma HT1376 and T24 cells. Comparison between normal urothelium and bladder tumor has identified significant decrease of PDEF in the tumor part of the pair bladder tissues. Overexpression of PDEF enhanced gene expression of B-cell translocation gene 2 (BTG2), N-myc downstream regulated gene 1 (NDRG1), and maspin, which dramatically attenuated cell proliferation and invasion. Xenograph animal studies indicated that overexpression of PDEF in HT1376 cells blocked tumorigenesis in vivo. Overexpression of PDEF enhanced E-cadherin but decreased N-cadherin, snail-1, snail-2, and twist-1 protein expression in T24 cells. CONCLUSIONS: Expression of PDEF gene may be related to the extent of bladder neoplasia. A co-regulatory expression is found between PDEF and EMT associated genes. Results suggest that induction of BTG2, NDRG1, and maspin gene expression by PDEF, could account for the function of PDEF for anti-proliferation, antiinvasion, and anti-tumorigenesis in bladder carcinoma cells.
Vol. 187, No. 4S, Supplement, Monday, May 21, 2012
after definitive therapy for muscle-invasive bladder cancer. Further study is warranted to elucidate its clinical significance.
Source of Funding: CMRPG381683,CMRPG392141
1057 POST-CHEMOTHERAPY AKR1B10 EXPRESSION CORRELATES WITH DISEASE FREE SURVIVAL IN MUSCLE-INVASIVE BLADDER CANCER Yasuhiro Hashimoto*, Takuya Koie, Hayato Yamamoto, Atsushi Imai, Shingo Hatakeyama, Takahiro Yoneyama, Noritaka Kamimura, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: AKR1B10 is a member of the aldo-keto reductase superfamily of NAD(P)H-dependent oxidoreductases. AKR1B10 is considered to contribute to cell proliferation and chemoresistance. In the present study, we examined whether AKR1B10 expression correlates with disease free survival in bladder cancer specimens. METHODS: The cohort includes consecutive 57 patients with muscle-invasive bladder cancer who received neoadjuvant chemotherapy followed by radical cystectomy. All patients received two cycles of neoadjuvant chemotherapy with gemcitabine plus carboplatin. Bladder cancer specimens were obtained at pre- and post-neoadjuvant chemotherapy, which were subjected to quantitative real-time PCR and immunohistochemistry to evaluate AKR1B10 expression. Intensity scoring for immunohistochemistry was categorized using a 4-graded scoring system according to a previously published method, with positive cells for each specific marker expressed as a percentage of the total number of cells as follows: 0-10% ⫽ 0; 11%-30% ⫽ 1; 31%-70% ⫽ 2; 71%-100% ⫽ 3. RESULTS: AKR1B10 mRNA expression was significantly higher in the post-chemotherapy groups than in the pre-chemotherapy groups (p ⬍ 0.001). The average immunohistochemical intensity score in the pre-chemotherapy group was 0.83 ⫾ 1.08, compared to a significantly higher average intensity score of 2.03 ⫾ 1.03 in the post-chemotherapy group (p ⬍ 0.001) Disease free survival of the post-chemotherapy AKR1B10(⫹) patients (61.2%) was significantly lower than that of AKR1B10(-) patients (100%). (log-rank test: p ⫽ 0.039). CONCLUSIONS: Although the present study is small and preliminary, post-chemotherapy AKR1B10 expression may have a predictive potential for response of chemotherapy and disease recurrence
Source of Funding: None
1058 DIFFERENCES IN DNA METHYLATION PATTERN OF PRIMARY BLADDER TUMOURS CORRELATE WITH THEIR METASTATIC POTENTIAL Beatrice Stubendorff, Jena, Germany; Eva Dudziec, James Catto, Sheffiled, United Kingdom; Jimsgene Sanjmyatav, Mieczeslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, Kerstin Junker*, Jena, Germany INTRODUCTION AND OBJECTIVES: The prognosis of patients with metastatic bladder cancer is poor with a 5-year survival rate of only 6%. Early prediction of the metastatic risk in primary tumors can lead to improvements in prognosis and therapy. Currently, there are no parameters available that enable an individual risk assessment for patients with metastatic bladder cancer. Therefore, identification of new reliable prognostic markers for early prediction of tumor spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. METHODS: Genomic DNA was isolated from 23 invasive bladder tumour tissues with and without lymph node metastases. For enrichment of methylated fragments genomic DNA was incubated with 5-Methylcytosin antibody. Input and IP were labelled with Cy3 and Cy5.