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MP-15.08: Detection of TMPRSS2–ERG Fusion Gene in Urine and Blood of Prostate Cancer Patients
MODERATED POSTER SESSIONS
MP-15.07 Comprehensive Analyses of Genetic Predisposition for Prostate Cancer in Japanese Men Kaku H1, Huang P1, Sakai A2, Watanabe M1, Chen J1, Saika T1, Nasu Y1, Shimizu K2, Kumon H1 1 Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Department of Molecular Genetics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Introduction and Objective: Single nucleotide polymorphism (SNP) arrays provide a high-resolution platform for describing several types of genetic changes simultaneously. With the resolution of these arrays increasing exponentially, they are becoming powerful tools for describing the genetic events underlying cancer. Materials and Methods: We have genotyped more than 50 non-synonymous Missense-SNPs of cancer-related genes in 162 prostate cancer patients and 135 matched healthy males in Japan. Results: Twelve SNPs of 10 genes were significantly associated with the incidence of prostate cancer. These genes included 2 of DNA-repair genes, 5 of tumor suppressor genes, 1 each of metabolizing enzyme gene, chromosome-segregation gene and apoptosis regulator gene. Cancer-association of these 9 of 12 SNPs was a novel finding and 9 high-risk and 2 protective associations were detected. The odds ratio (OR) of the associations ranged between 0.42 – 12.2, and an average statistic power by the Cochran-Armitage formula was 96% and 88% at the 0.05 and 0.01 significance level, respectively. Conclusions: Thus, our novel way of combining the risk factors by using the statistically significant SNPs would be useful for predicting prostate cancer predisposition in Japanese. We believe that it contributes for prevention and early detection of the disease in clinical field. This strategy can be also applied for other malignancies and for other ethnic populations by choosing proper SNPs combinations. MP-15.08 Detection of TMPRSS2–ERG Fusion Gene in Urine and Blood of Prostate Cancer Patients Kalogeropoulos T3, Velaeti S1, Arvanitakis T3, Dimitriadis E1, Kontogianni-Katsarou K4, Apostolaki A2, Gozen A5, Teber D5, Petraki K4, Pandis N1 1 Department of Genetics, “Saint Savvas” Anticancer Hospital, Athens, Greece; 2Pa-
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thology Department, “Saint Savvas” Anticancer Hospital, Athens, Greece; 3Urology Department, “Saint Savvas” Anticancer Hospital, Athens, Greece; 4Pathology Department, “Evangelismos” General Hospital, Athens, Greece; 5Urology Department SLK Kliniken, Heilbronn, Germany Introduction and Objective: Since prostate cells can be detected in the urine and in blood of men with prostate cancer, urine or blood based diagnostic tests have been tested using prostate cancer cell specific markers. Reports in the last years have shown that PCa frequently over-express the ETS family members ETV1 and ERG and that as many as 70% of such PCa have chromosomal rearrangements that lead to the fusion of the 5’ end of the androgen regulated serine protease TMPRSS2 (21q22.2) to the 3’ end of either ERG (21q22.3) or ETV1 (7p21.3), ETV4. The ability to detect these fusion genes in urine/blood of PCa patients was also examined as an aid in diagnosis. In this study we tested the ability to detect the fusion genes in urine and blood specimens of PCa patients employing a nested RT-PCR and direct sequencing approach. Materials and Methods: Urine and blood samples were collected from each patient following a digital rectal exam before either needle biopsy or radical prostatectomy. Total RNA was isolated using an RNA extraction kit (Macherey-Nagel) according to the manufacturer’s instructions. The RNA samples were processed for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen) and random hexamers. NestedPCR and Real-Time PCR approaches were used for TMPRSS2-ERG and ETV1 detection, as previously described. Results: Transcripts for TMPRSS2-ERG were found in 4 of the 15 urine samples, whereas among the 19 blood samples, 3 were found positive for the same fusion transcript. Both urine and blood tested positive in only one case. In all other fusion positive cases, chimeric transcripts detected either in urine or the blood. No association was found with disease stage and Gleason scores. Conclusions: In previous studies the detection of fusion genes in urine sediments was found to aid non-invasive diagnosis. Here we show that TMPRSS2-ERG fusion gene can be detected in urine and blood samples of prostate cancer patients with the Nested PCR approach. The correlation of blood fusion gene detection with the disease course should further investigated.
MP-15.09 Functional Analysis of a Novel Soft Tissue Sarcoma Metastasis-Associated Molecule in Prostate Cancer Kajita Y1,2, Furu M2, Nagayama S3, Nishiyama H1, Nakamura E1, Ogawa O1, Toguchida J2 1 Department of Urology, Graduate School of Medicine, Kyoto, Japan; 2Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; 3Department of Surgery, Kyoto University, Kyoto, Japan Introduction and Objective: To identify a molecular target for therapy in spindle soft tissue sarcoma, we performed cDNA microarray using 65 clinical sarcoma samples. When disease-related death and occurrence of distant metastases were defined as endpoints, we found a novel molecule, hereafter named C7059, was significantly up-regulated in event-positive groups. This time we investigated whether C7059 is also associated with metastatic progression in prostate cancer, as well as in soft tissue sarcoma. Materials and Methods: We checked C7059 expression in prostate cancer cell lines and created PC-3 cells in which C7059 was knocked down by RNAi. Matrigel chambers were used to see their invasive ability in vitro study. To evaluate their invasiveness in vivo, these cells were inoculated in testes of six week old nude mice (N⫽8) and, after an observation period of two months, mice were sacrificed to measure the volume of the tumor at the primary and the metastatic sites. We also performed an immunohistochemical study with C7059 in prostate biopsy specimens (N⫽47) to evaluate the relationship between C7059 expression and clinical state of disease retrospectively. Results: Among prostate cancer cell lines, highly invasive PC-3 and DU145 were positive, whereas less invasive LNCaP was negative in C7059 expression. Knockingdown its expression in PC-3 cells showed reduced invasiveness in Matrigel invasion assay. Also in vivo study using nude mice, these cells showed decreased tumor volume both at the primary and the metastatic sites compared to the control. An Immunohistochemical study in prostate biopsy specimens revealed slight, but gradually increasing ratio of C7059 positive staining according to the stage progression; 2/6 (33.3%), 5/9 (35.7%) and 13/23 (55.6%) cases were C7059 positive in stage B, C and D. As a conclusion, C7059 is a novel molecule that is associated with metastatic progression in pros-
UROLOGY 74 (Supplment 4A), October 2009
MP-15.07 Comprehensive Analyses of Genetic Predisposition for Prostate Cancer in Japanese Men Kaku H1, Huang P1, Sakai A2, Watanabe M1, Chen J1, Saika T1, Nasu Y1, Shimizu K2, Kumon H1 1 Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Department of Molecular Genetics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Introduction and Objective: Single nucleotide polymorphism (SNP) arrays provide a high-resolution platform for describing several types of genetic changes simultaneously. With the resolution of these arrays increasing exponentially, they are becoming powerful tools for describing the genetic events underlying cancer. Materials and Methods: We have genotyped more than 50 non-synonymous Missense-SNPs of cancer-related genes in 162 prostate cancer patients and 135 matched healthy males in Japan. Results: Twelve SNPs of 10 genes were significantly associated with the incidence of prostate cancer. These genes included 2 of DNA-repair genes, 5 of tumor suppressor genes, 1 each of metabolizing enzyme gene, chromosome-segregation gene and apoptosis regulator gene. Cancer-association of these 9 of 12 SNPs was a novel finding and 9 high-risk and 2 protective associations were detected. The odds ratio (OR) of the associations ranged between 0.42 – 12.2, and an average statistic power by the Cochran-Armitage formula was 96% and 88% at the 0.05 and 0.01 significance level, respectively. Conclusions: Thus, our novel way of combining the risk factors by using the statistically significant SNPs would be useful for predicting prostate cancer predisposition in Japanese. We believe that it contributes for prevention and early detection of the disease in clinical field. This strategy can be also applied for other malignancies and for other ethnic populations by choosing proper SNPs combinations. MP-15.08 Detection of TMPRSS2–ERG Fusion Gene in Urine and Blood of Prostate Cancer Patients Kalogeropoulos T3, Velaeti S1, Arvanitakis T3, Dimitriadis E1, Kontogianni-Katsarou K4, Apostolaki A2, Gozen A5, Teber D5, Petraki K4, Pandis N1 1 Department of Genetics, “Saint Savvas” Anticancer Hospital, Athens, Greece; 2Pa-
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thology Department, “Saint Savvas” Anticancer Hospital, Athens, Greece; 3Urology Department, “Saint Savvas” Anticancer Hospital, Athens, Greece; 4Pathology Department, “Evangelismos” General Hospital, Athens, Greece; 5Urology Department SLK Kliniken, Heilbronn, Germany Introduction and Objective: Since prostate cells can be detected in the urine and in blood of men with prostate cancer, urine or blood based diagnostic tests have been tested using prostate cancer cell specific markers. Reports in the last years have shown that PCa frequently over-express the ETS family members ETV1 and ERG and that as many as 70% of such PCa have chromosomal rearrangements that lead to the fusion of the 5’ end of the androgen regulated serine protease TMPRSS2 (21q22.2) to the 3’ end of either ERG (21q22.3) or ETV1 (7p21.3), ETV4. The ability to detect these fusion genes in urine/blood of PCa patients was also examined as an aid in diagnosis. In this study we tested the ability to detect the fusion genes in urine and blood specimens of PCa patients employing a nested RT-PCR and direct sequencing approach. Materials and Methods: Urine and blood samples were collected from each patient following a digital rectal exam before either needle biopsy or radical prostatectomy. Total RNA was isolated using an RNA extraction kit (Macherey-Nagel) according to the manufacturer’s instructions. The RNA samples were processed for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen) and random hexamers. NestedPCR and Real-Time PCR approaches were used for TMPRSS2-ERG and ETV1 detection, as previously described. Results: Transcripts for TMPRSS2-ERG were found in 4 of the 15 urine samples, whereas among the 19 blood samples, 3 were found positive for the same fusion transcript. Both urine and blood tested positive in only one case. In all other fusion positive cases, chimeric transcripts detected either in urine or the blood. No association was found with disease stage and Gleason scores. Conclusions: In previous studies the detection of fusion genes in urine sediments was found to aid non-invasive diagnosis. Here we show that TMPRSS2-ERG fusion gene can be detected in urine and blood samples of prostate cancer patients with the Nested PCR approach. The correlation of blood fusion gene detection with the disease course should further investigated.
MP-15.09 Functional Analysis of a Novel Soft Tissue Sarcoma Metastasis-Associated Molecule in Prostate Cancer Kajita Y1,2, Furu M2, Nagayama S3, Nishiyama H1, Nakamura E1, Ogawa O1, Toguchida J2 1 Department of Urology, Graduate School of Medicine, Kyoto, Japan; 2Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; 3Department of Surgery, Kyoto University, Kyoto, Japan Introduction and Objective: To identify a molecular target for therapy in spindle soft tissue sarcoma, we performed cDNA microarray using 65 clinical sarcoma samples. When disease-related death and occurrence of distant metastases were defined as endpoints, we found a novel molecule, hereafter named C7059, was significantly up-regulated in event-positive groups. This time we investigated whether C7059 is also associated with metastatic progression in prostate cancer, as well as in soft tissue sarcoma. Materials and Methods: We checked C7059 expression in prostate cancer cell lines and created PC-3 cells in which C7059 was knocked down by RNAi. Matrigel chambers were used to see their invasive ability in vitro study. To evaluate their invasiveness in vivo, these cells were inoculated in testes of six week old nude mice (N⫽8) and, after an observation period of two months, mice were sacrificed to measure the volume of the tumor at the primary and the metastatic sites. We also performed an immunohistochemical study with C7059 in prostate biopsy specimens (N⫽47) to evaluate the relationship between C7059 expression and clinical state of disease retrospectively. Results: Among prostate cancer cell lines, highly invasive PC-3 and DU145 were positive, whereas less invasive LNCaP was negative in C7059 expression. Knockingdown its expression in PC-3 cells showed reduced invasiveness in Matrigel invasion assay. Also in vivo study using nude mice, these cells showed decreased tumor volume both at the primary and the metastatic sites compared to the control. An Immunohistochemical study in prostate biopsy specimens revealed slight, but gradually increasing ratio of C7059 positive staining according to the stage progression; 2/6 (33.3%), 5/9 (35.7%) and 13/23 (55.6%) cases were C7059 positive in stage B, C and D. As a conclusion, C7059 is a novel molecule that is associated with metastatic progression in pros-
UROLOGY 74 (Supplment 4A), October 2009