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EXPRESSION OF MULTIPLE BIOMARKERS IS ASSOCIATED WITH LOCALLY ADVANCED UROTHELIAL CARCINOMA OF THE BLADDER IN A PROSPECTIVE EVALUATION
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THE JOURNAL OF UROLOGY®
pelvic lymphadenectomy (median follow-up: 61.6 months). RESULTS: The expression of p53 was altered in 25% of patients, p21 in 46%, pRB in 39%, p27 in 35%, Survivin in 49%, and KI67 in 34%. In multivariable analyses, p53, p27, and KI67 were independently associated with UCB recurrence (HR 3.66; p=0.033; HR 3.76, p=0.048; and HR 3.96, p=0.021, respectively) and UCB-specific mortality (HR 5.25; p=0.016; HR 3.68, p=0.043; and HR 6.23, p=0.009, respectively). The combination of these three biomarkers stratified patients into statistically significantly different risk groups for disease recurrence (p<0.001) and UCB-specific mortality (p<0.001). Addition of number of altered markers increased the concordance indices of the base model that included grade, lymph node status, lymphovascular invasion, and concomitant carcinoma in-situ for disease recurrence and UCB-specific survival from 54.7% to 71.7% and from 64.3% to 77.5%, respectively. CONCLUSIONS: Assessment of p53, p27, and KI67 in UCB specimens improves the prediction of recurrence-free and UCB-specific survival in patients with pT1 disease at RC. These markers may help stratify the heterogeneous population of pT1 patients into risk groups that can be used to guide clinical decision-making regarding observation versus adjuvant therapy. Source of Funding: National Institute of Health; T32 training grant (T32CA082088)
1043 EXPRESSION OF MULTIPLE BIOMARKERS IS ASSOCIATED WITH LOCALLY ADVANCED UROTHELIAL CARCINOMA OF THE BLADDER IN A PROSPECTIVE EVALUATION Christian Bolenz*, Dallas, TX; Shahrokh F Shariat, New York, NY; Travis Edwards, Ganesh V Raj, Raheela Ashfaq, Arthur I Sagalowsky, Yair Lotan, Dallas, TX INTRODUCTION AND OBJECTIVES: Retrospective studies have shown that molecular markers can improve risk stratification of patients with urothelial carcinoma of the bladder (UCB). In a phased, systematic evaluation of markers, we have identified a panel of biomarkers (cyclin E1, p53, p21, p27 and pRB/Ki-67) associated with clinical outcomes in patients treated with radical cystectomy (RC) for UCB. In the next phase, we initiated a prospective protocol to evaluate the utility of these biomarkers in improving clinical decision-making for patients with high-grade UCB. METHODS: 120 patients [median age 70 (range 35-90)] treated with transurethral resection (TUR; n=74) or RC (n=46) for high-grade UCB were prospectively evaluated starting in January 2007. Standardized immunohistochemical staining and scoring was performed. A prognostic score (PS; a2 altered biomarkers=Favorable; >2 altered biomarkers=Unfavorable) was defined and correlated with available data. RESULTS: Expression of at least one biomarker was altered in 96.7% of patients. Cyclin E1, p53, p21, p27 and pRB/Ki-67 expressions were altered in 11 (9.2%), 70 (58.3%), 19 (15.8%), 48 (40.0%) and 101 (84.2%) specimens, respectively. 39 patients (32.5%) had an unfavorable PS. When analyzed individually, only altered p21 and p27 were significantly associated with locally advanced tumor stages (apT2 vs. pT3/pT4; p=0.014 and p<0.001). An unfavorable PS was associated with advancing tumor stage (p<0.001) and locally advanced UCB (apT2 vs. pT3/pT4; p<0.001). In the subgroup of patients who had both TUR and then underwent RC (n=23), an unfavorable PS at TUR was associated with locally advanced tumor stage at RC (p=0.026), pathologic upstaging (p=0.002) and the presence of LVI (p=0.008) in the RC specimen. Conversely, 93.3% of patients with a favorable PS at TUR had the same stage at RC. CONCLUSIONS: The preliminary analysis of our ongoing prospective trial suggests that a panel of five biomarkers improves our prediction of patients likely to be upstaged at RC. An unfavorable PS at TUR may identify patients who are most likely to benefit from neo-adjuvant chemotherapy before RC. More robust endpoints such as recurrence and survival will be assessed after further accrual and longer follow-up. Source of Funding: None
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
1044 DISCOVERY OF THE METHYLATION OF THE METASTASIS SUPPRESSOR GENE, KISS-1, IN BLADDER CANCER Nerea Mendez, Esteban Orenes-Piñero, Marta Gaspar, Marta Fierro, Madrid, Spain; Thorsten Ecke, Bad Saarow, Germany; Marta Gil, Joan Palou, Joaquin Bellmunt, Barcelona, Spain; Carlos CordonCardo, New York, NY; Antonio Lopez-Beltran, Cordoba, Spain; Marta Sanchez-Carbayo*, Madrid, Spain INTRODUCTION AND OBJECTIVES: In an attempt to uncover the mechanisms by which the metastasis suppressor gene, KiSS1 is lost in bladder progression, we tested the hypothesis of epigenetic silencing. An enriched 5’-CpG islands was identified around the transcription start site of KiSS1, supporting its susceptibility to be epigenetically modified by hypermethylation. To the best of our knowledge, KiSS1 had not been reported to be epigenetically altered in bladder cancer or any other tumor type. In this report, the impact and clinical relevance of KiSS1 methylation along bladder cancer progression was evaluated using in vitro strategies as well as on tissue specimens. METHODS: The methylation status of KiSS1 was analyzed by two polymerase chain reaction (PCR) analysis strategies of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil. First, genomic sequencing of both strands of KiSS1 promoter was analyzed after bisulfite treatment of genomic DNA. A second strategy used PCR with primers specific for either the methylated or the modified unmethylated DNA (MS-PCR). The epigenetic silencing of KiSS1 by hypermethylation was tested in bladder cancer cells (n=13) before and after azacytidine treatment. The methylation status of KiSS1 promoter was then evaluated by MS-PCR in a large series of 524 bladder tumors. KiSS1 transcript expression were analyzed by RTPCR on bladder tumors (n=177) for which KiSS1 methylation was assessed by MS-PCR. RESULTS: KiSS1 hypermethylation was frequent in the bladder cancer cells analyzed and associated with low gene expression by RTPCR. KiSS-1 expression was increased in vitro by the demethylating agent azacytidine in methylated cells. KiSS1 was found to be frequently methylated in a large series of 524 bladder tumors (68.1%). KiSS1 methylation was significantly associated with tumor stage (P<0.001) and tumor grade (P<0.001). Interestingly, a significant association was found between transcript levels by quantitative RT-PCR in bladder tumors and increasing tumor stage (p<0.001). Moreover, the presence of KiSS1 methylation was associated with low transcript expression (p<0.001). CONCLUSIONS: KiSS1 was identified to be epigenetically modified in bladder cancer. The association of KiSS1 methylation with cancer progression suggests the utility of incorporating KiSS1 methylation assessment in the clinical management of patients affected by uroepithelial neoplasias. Source of Funding: None
1045 COMPARISON OF 2002 TNM NODAL STATUS WITH LYMPH NODE DENSITY IN NODE-POSITIVE PATIENTS AFTER RADICAL CYSTECTOMY FOR BLADDER CANCER: ANALYSIS BY THE NUMBER OF LYMPH NODES REMOVED Taekmin Kwon*, Dalsan You, Jinsung Park, In Gab Jeong, Cheryn Song, Jun Hyuk Hong, Hanjong Ahn, Choung-Soo Kim, Seoul, Republic of Korea INTRODUCTION AND OBJECTIVES: Because 2002 TNM pathologic nodal (pN) status was established in patients from whom a relatively small number of lymph nodes had been removed, it is necessary to validate this staging system in current clinical practice, in which the removal of more lymph nodes is recommended during radical cystectomy and pelvic lymphadenectomy. We assessed the ability of lymph node density (LND) and pN status to predict disease-specific survival (DSS) in node-positive patients after radical cystectomy for bladder cancer and investigated whether these factors were affected by the number of lymph nodes removed during pelvic lymphadenectomy.
THE JOURNAL OF UROLOGY®
pelvic lymphadenectomy (median follow-up: 61.6 months). RESULTS: The expression of p53 was altered in 25% of patients, p21 in 46%, pRB in 39%, p27 in 35%, Survivin in 49%, and KI67 in 34%. In multivariable analyses, p53, p27, and KI67 were independently associated with UCB recurrence (HR 3.66; p=0.033; HR 3.76, p=0.048; and HR 3.96, p=0.021, respectively) and UCB-specific mortality (HR 5.25; p=0.016; HR 3.68, p=0.043; and HR 6.23, p=0.009, respectively). The combination of these three biomarkers stratified patients into statistically significantly different risk groups for disease recurrence (p<0.001) and UCB-specific mortality (p<0.001). Addition of number of altered markers increased the concordance indices of the base model that included grade, lymph node status, lymphovascular invasion, and concomitant carcinoma in-situ for disease recurrence and UCB-specific survival from 54.7% to 71.7% and from 64.3% to 77.5%, respectively. CONCLUSIONS: Assessment of p53, p27, and KI67 in UCB specimens improves the prediction of recurrence-free and UCB-specific survival in patients with pT1 disease at RC. These markers may help stratify the heterogeneous population of pT1 patients into risk groups that can be used to guide clinical decision-making regarding observation versus adjuvant therapy. Source of Funding: National Institute of Health; T32 training grant (T32CA082088)
1043 EXPRESSION OF MULTIPLE BIOMARKERS IS ASSOCIATED WITH LOCALLY ADVANCED UROTHELIAL CARCINOMA OF THE BLADDER IN A PROSPECTIVE EVALUATION Christian Bolenz*, Dallas, TX; Shahrokh F Shariat, New York, NY; Travis Edwards, Ganesh V Raj, Raheela Ashfaq, Arthur I Sagalowsky, Yair Lotan, Dallas, TX INTRODUCTION AND OBJECTIVES: Retrospective studies have shown that molecular markers can improve risk stratification of patients with urothelial carcinoma of the bladder (UCB). In a phased, systematic evaluation of markers, we have identified a panel of biomarkers (cyclin E1, p53, p21, p27 and pRB/Ki-67) associated with clinical outcomes in patients treated with radical cystectomy (RC) for UCB. In the next phase, we initiated a prospective protocol to evaluate the utility of these biomarkers in improving clinical decision-making for patients with high-grade UCB. METHODS: 120 patients [median age 70 (range 35-90)] treated with transurethral resection (TUR; n=74) or RC (n=46) for high-grade UCB were prospectively evaluated starting in January 2007. Standardized immunohistochemical staining and scoring was performed. A prognostic score (PS; a2 altered biomarkers=Favorable; >2 altered biomarkers=Unfavorable) was defined and correlated with available data. RESULTS: Expression of at least one biomarker was altered in 96.7% of patients. Cyclin E1, p53, p21, p27 and pRB/Ki-67 expressions were altered in 11 (9.2%), 70 (58.3%), 19 (15.8%), 48 (40.0%) and 101 (84.2%) specimens, respectively. 39 patients (32.5%) had an unfavorable PS. When analyzed individually, only altered p21 and p27 were significantly associated with locally advanced tumor stages (apT2 vs. pT3/pT4; p=0.014 and p<0.001). An unfavorable PS was associated with advancing tumor stage (p<0.001) and locally advanced UCB (apT2 vs. pT3/pT4; p<0.001). In the subgroup of patients who had both TUR and then underwent RC (n=23), an unfavorable PS at TUR was associated with locally advanced tumor stage at RC (p=0.026), pathologic upstaging (p=0.002) and the presence of LVI (p=0.008) in the RC specimen. Conversely, 93.3% of patients with a favorable PS at TUR had the same stage at RC. CONCLUSIONS: The preliminary analysis of our ongoing prospective trial suggests that a panel of five biomarkers improves our prediction of patients likely to be upstaged at RC. An unfavorable PS at TUR may identify patients who are most likely to benefit from neo-adjuvant chemotherapy before RC. More robust endpoints such as recurrence and survival will be assessed after further accrual and longer follow-up. Source of Funding: None
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
1044 DISCOVERY OF THE METHYLATION OF THE METASTASIS SUPPRESSOR GENE, KISS-1, IN BLADDER CANCER Nerea Mendez, Esteban Orenes-Piñero, Marta Gaspar, Marta Fierro, Madrid, Spain; Thorsten Ecke, Bad Saarow, Germany; Marta Gil, Joan Palou, Joaquin Bellmunt, Barcelona, Spain; Carlos CordonCardo, New York, NY; Antonio Lopez-Beltran, Cordoba, Spain; Marta Sanchez-Carbayo*, Madrid, Spain INTRODUCTION AND OBJECTIVES: In an attempt to uncover the mechanisms by which the metastasis suppressor gene, KiSS1 is lost in bladder progression, we tested the hypothesis of epigenetic silencing. An enriched 5’-CpG islands was identified around the transcription start site of KiSS1, supporting its susceptibility to be epigenetically modified by hypermethylation. To the best of our knowledge, KiSS1 had not been reported to be epigenetically altered in bladder cancer or any other tumor type. In this report, the impact and clinical relevance of KiSS1 methylation along bladder cancer progression was evaluated using in vitro strategies as well as on tissue specimens. METHODS: The methylation status of KiSS1 was analyzed by two polymerase chain reaction (PCR) analysis strategies of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil. First, genomic sequencing of both strands of KiSS1 promoter was analyzed after bisulfite treatment of genomic DNA. A second strategy used PCR with primers specific for either the methylated or the modified unmethylated DNA (MS-PCR). The epigenetic silencing of KiSS1 by hypermethylation was tested in bladder cancer cells (n=13) before and after azacytidine treatment. The methylation status of KiSS1 promoter was then evaluated by MS-PCR in a large series of 524 bladder tumors. KiSS1 transcript expression were analyzed by RTPCR on bladder tumors (n=177) for which KiSS1 methylation was assessed by MS-PCR. RESULTS: KiSS1 hypermethylation was frequent in the bladder cancer cells analyzed and associated with low gene expression by RTPCR. KiSS-1 expression was increased in vitro by the demethylating agent azacytidine in methylated cells. KiSS1 was found to be frequently methylated in a large series of 524 bladder tumors (68.1%). KiSS1 methylation was significantly associated with tumor stage (P<0.001) and tumor grade (P<0.001). Interestingly, a significant association was found between transcript levels by quantitative RT-PCR in bladder tumors and increasing tumor stage (p<0.001). Moreover, the presence of KiSS1 methylation was associated with low transcript expression (p<0.001). CONCLUSIONS: KiSS1 was identified to be epigenetically modified in bladder cancer. The association of KiSS1 methylation with cancer progression suggests the utility of incorporating KiSS1 methylation assessment in the clinical management of patients affected by uroepithelial neoplasias. Source of Funding: None
1045 COMPARISON OF 2002 TNM NODAL STATUS WITH LYMPH NODE DENSITY IN NODE-POSITIVE PATIENTS AFTER RADICAL CYSTECTOMY FOR BLADDER CANCER: ANALYSIS BY THE NUMBER OF LYMPH NODES REMOVED Taekmin Kwon*, Dalsan You, Jinsung Park, In Gab Jeong, Cheryn Song, Jun Hyuk Hong, Hanjong Ahn, Choung-Soo Kim, Seoul, Republic of Korea INTRODUCTION AND OBJECTIVES: Because 2002 TNM pathologic nodal (pN) status was established in patients from whom a relatively small number of lymph nodes had been removed, it is necessary to validate this staging system in current clinical practice, in which the removal of more lymph nodes is recommended during radical cystectomy and pelvic lymphadenectomy. We assessed the ability of lymph node density (LND) and pN status to predict disease-specific survival (DSS) in node-positive patients after radical cystectomy for bladder cancer and investigated whether these factors were affected by the number of lymph nodes removed during pelvic lymphadenectomy.