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1139 MICRORNAS DETECTION AS URINE MARKERS IN PATIENTS WITH UROTHELIAL CARCINOMA
Vol. 183, No. 4, Supplement, Monday, May 31, 2010
In addition, we also found 138 loci that were hypermethylated not only in tumors but also in adjacent normal appearing tissue when compared to normal urothelium from bladder cancer-free patients, indicating the presence of an epigenetic field defect in bladder cancer patients. These loci may provide markers for the identification of individuals at risk for developing bladder cancer, as they are only methylated in bladders with cancer. CONCLUSIONS: Taken together our data reveal distinct methylation patterns between non-invasive and invasive bladder tumors and an epigenetic field defect in bladders with cancer. Therefore aberrant DNA methylation provides markers that can detect the presence of non-invasive and invasive tumors, and detect likelihood of recurrence after resection, as well as predict high risk patients. Source of Funding: RO1 (NCI)
THE JOURNAL OF UROLOGY姞
e441
Source of Funding: The Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B) 20390427 and (C) 20591861, 2008.
1140 IDENTIFICATION OF OCCULT LYMPH NODE DISEMINATION IN PATIENTS WITH BLADDER CANCER USING FORMALIN-FIXED PARAFFIN-EMBEDDED SAMPLES. Cristina Gazquez*, Lourdes Mengual, Maria J Ribal, Mercedes Marin-Aguilera, Pedro L Fernandez, Antonio Alcaraz, Barcelona, Spain
1139 MICRORNAS DETECTION AS URINE MARKERS IN PATIENTS WITH UROTHELIAL CARCINOMA Hideki Enokida*, Takeshi Chiyomaru, Kazumori Kawakami, Kazuya Kawahara, Kagoshima, Japan; Masahiko Inahara, Satoko Kojima, Chiba, Japan; Kenryu Nishiyama, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Currently, tumor marker for urothelial carcinoma (UC) depends on urine cytology because of its high sensitivity (around 95%) despite of its low sensitivity (around 30%). Bladder tumor antigen (BTA) and nuclear matrix protein (NMP)-22 are are not widely used because of their low specificity for distinguishing UC from non-malignant diseases. MicroRNAs are small noncoding RNAs of about 22 nucleotides in length, that function as negative regulators of gene expression through antisense complimentarily to specific messenger RNAs. Recent studies suggest that aberrant microRNA expression contribute to the development of various human malignancies. Our previous miRNA screening of UC demonstrated that miR-96 and miR-183 were the top 2 up-regulated miRNAs among 667 miRNAs examined. We hypothesize that these microRNAs are detectable in urine and useful biomarkers for distinguishing UCfrom non-UC patients. METHODS: Total RNA of urine was extracted from 54 UCs, 15 urinary tract infections (UTIs) and 27 healthy controls (HCs). A realtime PCR based experiment was used to evaluate expression levels of the miRNAs. The expression level of each miRNA was calculated by a following formula: expression level ⫽ 2 (40-Ct). RESULTS: The expression levels of miR-96 and miR-183 in urine from UCs were significantly higher than that from UTIs or HCs (each p ⬍ 0.0001). ROC curve analysis revealed that miR-96 and miR-183 have good sensitivity (70.4 and 74.1%, respectively) and specificity (90.5 and 71.4%, respectively) to distinguish UC from non-UC patient. We found significant difference in miR-96 expressions between invasive (⬎ pTa) and non-invasive (pTa) UCs (p ⫽ 0.0271), but a trend in miR-183 (p ⫽ 0.0508). There were significant differences in miR-96 or miR-183 expressions between the samples from pre- and post operation (p ⬍ 0.05). To our surprise, miR-96 was positively detected in 11 of 20 UC patients who had a negative diagnosis by urine cytology. When we combined microRNA analysis with urine cytology in our cohort, the sensitivity and the specificity reached to 78% and 90.5%, respectively for UC detection. CONCLUSIONS: Our data suggest that detection of miR-96 and miR-183 may serve as potential biomarkers for UC. Diagnostic accuracy for urothelial carcinoma can be improved by a combination of urine cytology and the microRNAs detection.
INTRODUCTION AND OBJECTIVES: The aim of this study was to test two potential mRNA markers of bladder urothelial cell carcinoma (UCC) in lymph nodes (LN) and to compare the performance of these markers with patients’ clinical outcome. METHODS: A total of 189 and 35 formalin-fixed paraffin-embedded (FFPE) LN samples from 90 UCC patients and 35 controls, respectively, were collected from 1993 to 2005 (median follow-up of 100 months). According to previous results of our group, two genes, FXYD3 and KRT20, were selected to study UCC dissemination in LN. RNA was extracted from 3 to 16-year-old FFPE LN samples. Total RNA was reverse transcribed, preamplified and finally evaluated by quantitative real time PCR (qRT-PCR). The resulting gene expression values were statistically compared with histological results and patients’ clinical outcome. RESULTS: We found that the combination of FXYD3 and KRT20 genes yielded a 100% sensitivity and specificity differentiating LN with UCC dissemination from controls. The expression of both genes allowed the identification of UCC in 5.7% of patients with previous histologically negative LN. Half of these patients died because of the bladder cancer. CONCLUSIONS: Using the combination of two molecular markers it was possible to improve the sensitivity of histological analysis to detect occult LN dissemination. Even though half of the histologically negative and qRT-PCR positive LN patients did not recurred after a median follow-up of 100 months, it remains possible that lymphadenectomy had a curative effect in these patients. Thus, the analysis of these two molecular markers could be a complementary technique to the routine histological analysis. Source of Funding: None
1141 NOVEL ZEB1 EXPRESSION LINKED TO ALTERED MICRORNA EXPRESSION PROFILES IN BLADDER TUMOR PROGRESSION Patrick Kenney, Niall Harty*, Matthew Wszolek, Kimberly Rieger-Christ, Burlington, MA; Brasil Silva Neto, Porto Alegre, Brazil; Justin Gould, Ron Zeheb, Burlington, MA; Massimo Loda, Boston, MA; John Libertino, Ian Summerhayes, Burlington, MA INTRODUCTION AND OBJECTIVES: Epithelial-mesenchymaltransition (EMT) is a process that has been demonstrated as an underlying event in late-stage bladder tumorigenesis accompanied by increased invasive potential. Presently, we do not know all the players that trigger invasion in bladder cancer although numerous transcriptional regulators have been implicated in this process in alternative cancers. In this study we assessed the role of ZEB1 in bladder cancer against the background of a changing microRNA expression profile linked to the acquisition of an invasive phenotype. METHODS: Total RNA was extracted from formalin-fixed, paraffin-embedded bladder tumor tissue. Expression level of specific miR-
In addition, we also found 138 loci that were hypermethylated not only in tumors but also in adjacent normal appearing tissue when compared to normal urothelium from bladder cancer-free patients, indicating the presence of an epigenetic field defect in bladder cancer patients. These loci may provide markers for the identification of individuals at risk for developing bladder cancer, as they are only methylated in bladders with cancer. CONCLUSIONS: Taken together our data reveal distinct methylation patterns between non-invasive and invasive bladder tumors and an epigenetic field defect in bladders with cancer. Therefore aberrant DNA methylation provides markers that can detect the presence of non-invasive and invasive tumors, and detect likelihood of recurrence after resection, as well as predict high risk patients. Source of Funding: RO1 (NCI)
THE JOURNAL OF UROLOGY姞
e441
Source of Funding: The Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B) 20390427 and (C) 20591861, 2008.
1140 IDENTIFICATION OF OCCULT LYMPH NODE DISEMINATION IN PATIENTS WITH BLADDER CANCER USING FORMALIN-FIXED PARAFFIN-EMBEDDED SAMPLES. Cristina Gazquez*, Lourdes Mengual, Maria J Ribal, Mercedes Marin-Aguilera, Pedro L Fernandez, Antonio Alcaraz, Barcelona, Spain
1139 MICRORNAS DETECTION AS URINE MARKERS IN PATIENTS WITH UROTHELIAL CARCINOMA Hideki Enokida*, Takeshi Chiyomaru, Kazumori Kawakami, Kazuya Kawahara, Kagoshima, Japan; Masahiko Inahara, Satoko Kojima, Chiba, Japan; Kenryu Nishiyama, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Currently, tumor marker for urothelial carcinoma (UC) depends on urine cytology because of its high sensitivity (around 95%) despite of its low sensitivity (around 30%). Bladder tumor antigen (BTA) and nuclear matrix protein (NMP)-22 are are not widely used because of their low specificity for distinguishing UC from non-malignant diseases. MicroRNAs are small noncoding RNAs of about 22 nucleotides in length, that function as negative regulators of gene expression through antisense complimentarily to specific messenger RNAs. Recent studies suggest that aberrant microRNA expression contribute to the development of various human malignancies. Our previous miRNA screening of UC demonstrated that miR-96 and miR-183 were the top 2 up-regulated miRNAs among 667 miRNAs examined. We hypothesize that these microRNAs are detectable in urine and useful biomarkers for distinguishing UCfrom non-UC patients. METHODS: Total RNA of urine was extracted from 54 UCs, 15 urinary tract infections (UTIs) and 27 healthy controls (HCs). A realtime PCR based experiment was used to evaluate expression levels of the miRNAs. The expression level of each miRNA was calculated by a following formula: expression level ⫽ 2 (40-Ct). RESULTS: The expression levels of miR-96 and miR-183 in urine from UCs were significantly higher than that from UTIs or HCs (each p ⬍ 0.0001). ROC curve analysis revealed that miR-96 and miR-183 have good sensitivity (70.4 and 74.1%, respectively) and specificity (90.5 and 71.4%, respectively) to distinguish UC from non-UC patient. We found significant difference in miR-96 expressions between invasive (⬎ pTa) and non-invasive (pTa) UCs (p ⫽ 0.0271), but a trend in miR-183 (p ⫽ 0.0508). There were significant differences in miR-96 or miR-183 expressions between the samples from pre- and post operation (p ⬍ 0.05). To our surprise, miR-96 was positively detected in 11 of 20 UC patients who had a negative diagnosis by urine cytology. When we combined microRNA analysis with urine cytology in our cohort, the sensitivity and the specificity reached to 78% and 90.5%, respectively for UC detection. CONCLUSIONS: Our data suggest that detection of miR-96 and miR-183 may serve as potential biomarkers for UC. Diagnostic accuracy for urothelial carcinoma can be improved by a combination of urine cytology and the microRNAs detection.
INTRODUCTION AND OBJECTIVES: The aim of this study was to test two potential mRNA markers of bladder urothelial cell carcinoma (UCC) in lymph nodes (LN) and to compare the performance of these markers with patients’ clinical outcome. METHODS: A total of 189 and 35 formalin-fixed paraffin-embedded (FFPE) LN samples from 90 UCC patients and 35 controls, respectively, were collected from 1993 to 2005 (median follow-up of 100 months). According to previous results of our group, two genes, FXYD3 and KRT20, were selected to study UCC dissemination in LN. RNA was extracted from 3 to 16-year-old FFPE LN samples. Total RNA was reverse transcribed, preamplified and finally evaluated by quantitative real time PCR (qRT-PCR). The resulting gene expression values were statistically compared with histological results and patients’ clinical outcome. RESULTS: We found that the combination of FXYD3 and KRT20 genes yielded a 100% sensitivity and specificity differentiating LN with UCC dissemination from controls. The expression of both genes allowed the identification of UCC in 5.7% of patients with previous histologically negative LN. Half of these patients died because of the bladder cancer. CONCLUSIONS: Using the combination of two molecular markers it was possible to improve the sensitivity of histological analysis to detect occult LN dissemination. Even though half of the histologically negative and qRT-PCR positive LN patients did not recurred after a median follow-up of 100 months, it remains possible that lymphadenectomy had a curative effect in these patients. Thus, the analysis of these two molecular markers could be a complementary technique to the routine histological analysis. Source of Funding: None
1141 NOVEL ZEB1 EXPRESSION LINKED TO ALTERED MICRORNA EXPRESSION PROFILES IN BLADDER TUMOR PROGRESSION Patrick Kenney, Niall Harty*, Matthew Wszolek, Kimberly Rieger-Christ, Burlington, MA; Brasil Silva Neto, Porto Alegre, Brazil; Justin Gould, Ron Zeheb, Burlington, MA; Massimo Loda, Boston, MA; John Libertino, Ian Summerhayes, Burlington, MA INTRODUCTION AND OBJECTIVES: Epithelial-mesenchymaltransition (EMT) is a process that has been demonstrated as an underlying event in late-stage bladder tumorigenesis accompanied by increased invasive potential. Presently, we do not know all the players that trigger invasion in bladder cancer although numerous transcriptional regulators have been implicated in this process in alternative cancers. In this study we assessed the role of ZEB1 in bladder cancer against the background of a changing microRNA expression profile linked to the acquisition of an invasive phenotype. METHODS: Total RNA was extracted from formalin-fixed, paraffin-embedded bladder tumor tissue. Expression level of specific miR-